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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
227 Genetics of Inherited Eye Disease
Monday, May 06, 2013 8:30 AM-10:15 AM
Exhibit Hall Poster Session
Program #/Board # Range: 1307-1360/A0001-A0054
Organizing Section: Genetics
Program Number: 1307 Poster Board Number: A0001
Presentation Time: 8:30 AM - 10:15 AM
Overview of genetic causes and identification of novel loci for
retinal dystrophies in Pakistan
Muhammad I. Khan1, 2, Maleeha Azam1, 2, Muhammad Ajmal1, 3, Rob
W. Collin1, 4, Raheel Qamar2, 3, Anneke I. Den Hollander1, 5, Frans P.
Cremers1, 4. 1Human Genetics, Radboud University Nijmegen
Medical Centre, Nijmegen, Netherlands; 2Biosciences, COMSATS
Institute of Information Technology, Islamabad, Pakistan; 3Shifa
College of Medicine, Shifa Tameer-e-Millat University, Islamabad,
Pakistan; 4Nijmegen Centre for Molecular Life Sciences, Radboud
University Nijmegen Medical Centre, Nijmegen, Netherlands;
5
Ophthalmology, Radboud University Nijmegen Medical Centre,
Nijmegen, Netherlands.
Purpose: Inherited retinal dystrophies (iRD) are clinically and
genetically heterogeneous disorders. In Pakistan the frequency of
iRD is estimated to be 1 in 800 patients. For different iRD, 185 genes
have been identified to date. Among these genes, 119 are linked to
non-syndromic forms of the disease. In this study we aimed to
elucidate the genetic causes of 17 iRD families and to provide a
comprehensive genetic overview of all iRD that have been studied in
Pakistan. The customary consanguineous nuptials in Pakistan
underlies the frequent occurrence of autosomal recessive inherited
disorders, including RD, and therefore homozygosity mapping has
been the method of choice to map disease genes and loci.
Methods: DNAs of selected members from 17 arRD families were
genotyped on single nucleotide polymorphism microarrays and the
data were analyzed using an online homozygosity mapper tool.
Sanger sequencing was carried out for genes previously associated
with iRDs residing in the homozygous regions. In addition all
published genetic data of Pakistani families with syndromic and nonsyndromic iRDs was compiled.
Results: From a cohort of 41 randomly collected Pakistani iRD
families, most of which show retinitis pigmentosa (RP), results for 17
are described in this study. In 8 families causal genetic defects have
been identified while in another 9 consanguineous arRP families,
novel genetic loci are implicated. A total of 65 studies were identified
through a literature search that described genetic data of 156 iRD
families of Pakistani origin.
Conclusions: Taking into consideration our previously published
studies, we could genetically solve 32 of 41 (78%) families from our
iRD cohort. Keeping in mind that a proportion of the elusive variants
may reside deep intronic, we estimate that that >90% of genes
underlying non-syndromic iRD has been identified. The inventory of
all Pakistani iRD mutation data showed that Pakistani families with
arRD have been instrumental in finding ten iRD-associated genes and
have enabled the recognition of novel genotype-phenotype
correlations for three genes. Ninety percent of mutations causing nonsyndromic RD and all mutations causing syndromic forms in
Pakistani families are population specific. We propose a costeffective genetic test in which 50% of the most prevalent mutations
can be tested by sequence analysis of 14 different amplicons in 10
different genes.
Commercial Relationships: Muhammad I. Khan, None; Maleeha
Azam, None; Muhammad Ajmal, None; Rob W. Collin, Radboud
University Medical Centre (P); Raheel Qamar, None; Anneke I.
Den Hollander, None; Frans P. Cremers, None
Program Number: 1308 Poster Board Number: A0002
Presentation Time: 8:30 AM - 10:15 AM
Genome-Wide Association Analysis of Canine Retinal Dysplasia
and Vitreous Degeneration
Saija Ahonen1, 2, Hannes Lohi1, 2. 1Basic Veterinary Biosciences,
University of Helsinki, Helsinki, Finland; 2Research Programs Unit,
Molecular Neurology, University of Helsinki, Helsinki, Finland.
Purpose: Purebred dogs suffer from several hereditary vision
disorders, which resembles corresponding disorders in human
patients. Our study has focused on the genetics of retinal dysplasia
and vitreous degeneration in two different dog breeds, American
Cocker Spaniel and Italian Greyhound, respectively. Both disorders
are known to be inherited in the studied breeds, but the genetic
background has remained unknown. Both disorders may cause vision
deterioration and secondary complications may include glaucoma,
lens luxation and retinal detachment, which in turn may lead to a
complete loss of vision.
Methods: We have established a large sample cohort for multifocal
retinal dysplasia (MRD) in American Cocker Spaniel (ACS) and for
vitreous degeneration (VD) in the Italian Greyhound (IT). A genomewide association study (GWAS) was performed using canine specific
SNP array to map the associated loci in both disorders.
Results: The GWAS data identified a genome-widely significant
association for MRD in ACS on canine chromosome 22 (CFA22)
with praw=1.9*10-5, pgenome=0.01 and a tentative association, with
a 2 Mb homozygous haplotype, for VD in IT on CFA15 with
praw=5.1x10-5, pgenome=0.27. Further replication studies are being
performed to confirm and to define the critical associated regions.
Conclusions: We have mapped two novel loci for two separate
canine vision disorders. Ongoing candidate gene sequencing is likely
to open new insights to the molecular background of the studied
conditions. The identification of genes behind these disorders will
establish a large animal model for the corresponding human disorders
and the associated genes can be studied in human patients with
similar phenotypes. While the associated genes will become
candidates for human studies, a genetic test can be offered for the
studied breeds.
Commercial Relationships: Saija Ahonen, None; Hannes Lohi,
None
Program Number: 1309 Poster Board Number: A0003
Presentation Time: 8:30 AM - 10:15 AM
Identification of Novel Homozygous Deletions in Consanguineous
Pedigrees as a Shortcut to Candidate Gene Discovery in Retinal
Dystrophies
Kristof Van Schil1, Françoise Meire2, Thomy de Ravel3, Bart P.
Leroy4, Hannah Verdin1, Frauke Coppieters1, Elfride De Baere1.
1
Center for Medical Genetics, Ghent University Hospital, Ghent,
Belgium; 2Department of Ophthalmology, Huderf, Brussels,
Belgium; 3Center for Human Genetics, Leuven University Hospitals,
Leuven, Belgium; 4Center for Medical Genetics, Department of
Ophthalmology, Ghent University Hospital, Ghent, Belgium.
Purpose: To identify the underlying genetic cause in 25 pre-screened
consanguineous families diagnosed with autosomal recessive retinitis
pigmentosa (ARRP) or Leber congenital amaurosis (LCA) using
identity-by-descent (IBD) mapping. To demonstrate the power of
mapping of homozygous deletions as a shortcut to candidate gene
identification in retinal dystrophies (RDs).
Methods: IBD mapping was performed by genome-wide SNP chip
analysis (HumanCytoSNP-12, Illumina). For IBD data analysis we
integrated PLINK with arrayCGHbase, a platform for data analysis of
microarray based comparative genome hybridization. Deletions were
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
confirmed and fine-mapped by conventional PCR. Segregation
analysis was performed by qPCR (LightCycler, Roche; qBase Plus,
Biogazelle).
Results: Homozygous deletions were identified in 3 out of 25
families. The first deletion (133 kb) was found in an ARRP patient
and removes the first non-coding exon of the known gene EYS. The
second deletion (112 kb) was found in a patient with early-onset RD.
It disrupts the last 3 exons of RERG and the lncRNA RERG-AS1.
The third one (416 kb) is a partial deletion of GRID2, found in a
patient with LCA. It leads to an in-frame deletion
(p.Gly30_Glu81del). All of them were located in the one but largest
IBD region. The deletions could be confirmed and segregation could
be demonstrated. They were absent in our local database containing
more than 3000 subjects.
Non-coding deletions of EYS have not yet been described. As to the
RERG and GRID2 genes, both are regulated by the transcription
factor CRX (Corbo et al., 2010). Although the function of RERG
remains unclear, it was found in several retinal transcriptome datasets
(Gamsiz et al., 2012 and Booij et al., 2009). GRID2 encodes a
neurotransmitter receptor that plays an important role in the brain, a
homozygous mutation in mouse was found to be lethal (Zuo et al.,
1997). The in-frame GRID2 deletion found here might represent a
hypomorphic allele leading to a milder phenotype. Further
characterization and functional validation of these deletions is
underway.
Conclusions: This study revealed involvement of a homozygous
5’UTR deletion of EYS in ARRP, and uncovered RERG and GRID2
as two potential novel candidate genes for RD. We demonstrated that
homozygous deletion detection in consanguineous families might be
a powerful approach for gene discovery of RDs.
Commercial Relationships: Kristof Van Schil, None; Françoise
Meire, None; Thomy de Ravel, None; Bart P. Leroy, None;
Hannah Verdin, None; Frauke Coppieters, None; Elfride De
Baere, None
Support: FWO, Funds for Research in Ophthalmology (FRO), IWT
Program Number: 1310 Poster Board Number: A0004
Presentation Time: 8:30 AM - 10:15 AM
Novel de novo mutations in CRX gene associated with Leber
congenital amaurosis in Chinese patients
Ruifang Sui, Xuan Zou, Fangtian Dong. Ophthalmology, Peking
Union Med College Hosp, Beijing, China.
Purpose: To analyze the cone-rod homeobox gene (CRX) mutations
in a cohort of Chinese patients with Leber congenital amaurosis
(LCA) and to describe clinical features of patients with CRX
mutations.
Methods: Genomic DNA was isolated with standard methods for
genetic diagnosis. All three exons of CRX were amplified with PCR
and screened for mutation with direct DNA sequencing. 100
unrelated healthy Chinese subjects were screened to exclude
nonpathogenic polymorphisms. Retinal phenotypes were
characterized by ophthalmic examination, including optical
coherence tomography (OCT) and standardized electrophysiological
tests.
Results: 110 unrelated LCA patients were selected for mutation
screening in the CRX gene. Two novel de novo CRX mutations,
c.421delT (p.Ser141Pro fsX46) and c.571delT (p.Tyr191Met fsX3),
were found related to 2 index patients. Affected daughter of patient
two also carriers the mutation. Their visual acuity were ranged from
hand motion to light perception. Pigmentary retinopathy in the
peripheral retina and macular atrophy were observed. OCT tests
showed macular atrophy without normal lamination structure.
Conclusions: Two novel de novo mutations in CRX were found in
Chinese patients with LCA. The CRX mutation might create a
dominantly inherited trait.
Commercial Relationships: Ruifang Sui, None; Xuan Zou, None;
Fangtian Dong, None
Support: FFB grant: CD-CL-0808-0470-PUMCH; China grant:
2010DFB33430
Program Number: 1311 Poster Board Number: A0005
Presentation Time: 8:30 AM - 10:15 AM
RDH12 mutations associated with Leber congenital amaurosis
and early-onset severe retinal dystrophy in Chinese patients
Xuan Zou, Fangtian Dong, Ruifang Sui. Ophthalmology, Peking
Union Medical College Hospital, Beijing, China.
Purpose: To investigate the Leber congenital amaurosis (LCA) and
early-onset severe retinal dystrophy (ESRD) phenotypes associated
with mutations in RDH12 in Chinese population.
Methods: Genomic DNA was isolated with standard methods for
genetic diagnosis. All 7 exons of RDH12 were amplified with PCR
and screened for mutation with direct DNA sequencing. One hundred
unrelated healthy Chinese subjects were screened to exclude
nonpathogenic polymorphisms. Retinal phenotypes were
characterized by ophthalmic examination, including optical
coherence tomography (OCT) and standardized electrophysiological
tests.
Results: 128 unrelated LCA/ESRD patients were screened for
mutations in the RDH12 gene. Homozygous or compound
heterozygous RDH12 mutations were found in 12/128 (9.4%)
patients, including ten novel mutations and three reported mutations.
The petal-like macular coloboma is a characteristic retinal feature
related to RDH12 mutation. Marked pigmentary retinopathy,
including bone spicules and yellow pin-point pigments in the midperipheral retina, was very common in these patients. The OCT
showed macular depression and choroiretinal atrophy without normal
lamination structure.
Conclusions: RDH12 is a common cause for LCA/ESRD in Chinese
population, with a frequency of 9.4%. The petal-like macular
coloboma with dense pigmentation in the peripheral retina may be a
characteristic feature related to RDH12 mutation.
Commercial Relationships: Xuan Zou, None; Fangtian Dong,
None; Ruifang Sui, None
Program Number: 1312 Poster Board Number: A0006
Presentation Time: 8:30 AM - 10:15 AM
NMNAT1 p.Arg237Cys mutation in Japanese patients with Leber
congenital amaurosis
Tomoka Kambe1, Takuro Fujimaki2, Shuri Kawamorita3, Eisuke
Arai2, 4, Ai Miyazaki2, Keiko Fujiki2, Fumino Iwata5, Chieko
Tamura6, Akira Murakami2. 1Ophthalmology, Saitama Children's
Medical Center, Saitama, Japan; 2Ophthalmology, Juntendo
University School of Medicine, Tokyo, Japan; 3Japanese Red Cross
Medical Center, Tokyo, Japan; 4The Tokyo Metropolitan Children’s
Medical Center, Tokyo, Japan; 5Hatanodai Iwata Eye Clinic, Tokyo,
Japan; 6Kiba Park Clinic, Tokyo, Japan.
Purpose: Leber congenital amaurosis (LCA) is a group of earlyonset childhood retinal dystrophies characterized by loss of vision,
nystagmus, and severe retinal dysfunction. Two-thirds of autosomal
recessive LCA cases are caused by mutations in 17 known diseaseassociated genes. Here we report on NMNAT1 gene analysis in two
cases of early-onset macular dystrophy and suspected LCA.
Methods: The first subject was a four-month-old proband with
nystagmus. Several sites of retinal pigment disruption and atrophic
changes of the macula were found. The second subject was also a
four-month-old proband with nystagmus and similar fundus findings.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
Both marriages were not consanguineous. Genomic DNA was
extracted from peripheral blood leukocytes of the two subjects
according to standard procedures, after obtaining informed consent.
We amplified the coding regions of 17 AR-LCA genes from the
genomic DNA of the patients by PCR, while two micro-arrays and
the dye terminator method were used for sequencing.
Results: A homozygous missense mutation (c.709C>T,
p.Arg237Cys) of NMNAT1 was detected in both probands. This
mutation has been reported in Mongoloid patients by Chiang et al.
(Nat Genet., 2012).
Conclusions: We detected the homozygous mutation p.Arg237Cys in
the NMNAT1 gene of two unrelated probands by screening of 17
genes associated with LCA in Japanese patients. This may be a
characteristic mutation of Mongoloid AR-LCA.
Commercial Relationships: Tomoka Kambe, None; Takuro
Fujimaki, None; Shuri Kawamorita, None; Eisuke Arai, None; Ai
Miyazaki, None; Keiko Fujiki, None; Fumino Iwata, None;
Chieko Tamura, None; Akira Murakami, SEED(Japan) JP4855782
(P), SEED(Japan) JP5132958 (P)
Support: Grants-in-Aid for Scientific Research, 24592654
Program Number: 1313 Poster Board Number: A0007
Presentation Time: 8:30 AM - 10:15 AM
Regulatory mutations in the 5’UTR of NMNAT1, encoding the
nuclear isoform of nicotinamide nucleotide adenylyltransferase 1,
cause Leber Congenital Amaurosis
Frauke Coppieters1, Annelot Baert1, Caroline Van Cauwenbergh1,
Miriam Bauwens1, Sarah De Jaegere1, Thomy de Ravel2, Françoise
Meire3, Bart P. Leroy4, 1, Elfride De Baere1. 1Center for Medical
Genetics Ghent, Ghent University, Ghent, Belgium; 2Center for
Human Genetics, Leuven University Hospitals, Leuven, Belgium;
3
Hôpital Des Enfants Reine Fabiola, Brussels, Belgium; 4Department
of Ophthalmology, Ghent University Hospital, Ghent, Belgium.
Purpose: Leber Congenital Amaurosis (LCA) is the earliest inherited
retinal dystrophy (RD). Recently, coding mutations in NMNAT1
uncovered this gene as the causal disease gene for the LCA9 locus
(Falk et al. 2012). In this study, we aimed to identify the genetic
defect in an LCA9-linked consanguineous Sub-Saharan African
family (F1) and determine the contribution of NMNAT1 mutations in
pre-screened LCA patients.
Methods: The proband of F1 underwent massive parallel sequencing
(MPS) of all coding and promotor regions located in the 4 largest
IBD regions (NimbleGen Sequence Capture 385K array, Roche GS
FLX Titanium). NMNAT1 expression analysis was performed on
leukocyte cDNA with qPCR. NMNAT1 Sanger sequencing was
performed on gDNA (exons) and cDNA. F1 and F2 underwent
thorough phenotyping.
Results: MPS identified a novel homozygous 5’UTR variant in F1,
c.-70A>T, which segregated with disease. This variant was predicted
to alter 5’UTR secondary structure. Sequencing of c.-70A>T on
cDNA revealed loss of heterozygosity in three heterozygous carriers
of F1, suggesting NMNAT1 mRNA degradation.
Subsequent sequencing of NMNAT1 in 76 unrelated probands with
LCA or early-onset RD revealed mutations in 6 additional probands.
Of note, a second 5’UTR variant, c.-69C>T, was found in
homozygous state in a Moroccan LCA patient. Interestingly, it is
located 1 nucleotide downstream of c.-70A>T and predicted to cause
similar aberrant folding. In both F1 and F2, significantly lower
mRNA expression was shown for the homozygous carriers in
comparison with healthy controls. Moreover, phenotypic evaluation
of both families revealed LCA with evolutive macular involvement,
typical for NMNAT1-related disease. Luciferase assays are currently
ongoing for both 5’UTR variants.
Lastly, three probands were compound heterozygous for known
mutations, whereas two other probands were found to carry a single
heterozygous missense variant. Resequencing of the entire genomic
region and copy number screening is currently ongoing.
Conclusions: In conclusion, this study sustained the role of coding
NMNAT1 mutations in LCA. Moreover, the identification of two
neighboring 5’UTR variants in NMNAT1 makes this the first study to
link 5’UTR regulatory variants to LCA with macular involvement.
Overall, this study may impact upon the role of 5’UTR variations in
other RDs in general.
Commercial Relationships: Frauke Coppieters, None; Annelot
Baert, None; Caroline Van Cauwenbergh, None; Miriam
Bauwens, None; Sarah De Jaegere, None; Thomy de Ravel, None;
Françoise Meire, None; Bart P. Leroy, None; Elfride De Baere,
None
Support: FWO (12D8712N), Funds for Research in Ophthalmology
(FRO), BOF Ghent University
Program Number: 1314 Poster Board Number: A0008
Presentation Time: 8:30 AM - 10:15 AM
Wasf3 is required for photoreceptor sensory cilia (PSC)
formation
Jingfa Zhang, Qi Zhang, Conghui Zhang, Chan Wu, Eric A. Pierce,
Qin Liu. Ocular Genomics Institute, Department of Ophthalmology,
Massachusetts Eye and Ear Infirmary, Harvard Medical School,
Boston, MA.
Purpose: Inherited retinal degenerations (IRDs) are important causes
of blindness. These disorders are now recognized to be a sub-class of
the larger group of ciliopathies caused by dysfunction of cilia. Over
190 IRD disease genes have been identified to date, yet mutations in
these genes account for only 50-60% of IRD patients. To help
identify additional IRD disease genes, we are evaluating the locations
and functions of novel proteins in the photoreceptor sensory cilium
(PSC) proteome. In these studies, we evaluated the role of Wasf3, a
member of the WAS family of proteins associated with cytoskeleton
and actin complexes, in PSC biology.
Methods: The human WASF3 ORF was cloned into expression
vectors with V5 or Flag tag and expressed in rat retinas via subretinal injection and in vivo electroporation. The location of tagged
WASF3 protein was evaluated 21 days after transfection. The
location of mouse endogenous Wasf3 protein was verified using antiWasf3 antibodies. The tissue distribution of Wasf3 was studied using
immunoblot analyses. Morpholino oligonucleotide (MO) - mediated
knockdown of wasf3 was investigated in zebrafish and the phenotype
was evaluated by histology analysis.
Results: In vivo expression of V5 or Flag tagged WASF3 revealed its
location at the junction between the base of axoneme and the distal
tip of the transition zone, with a clear gap to the distal end of inner
segment marked by EGFP. Immunostaining of mouse endogenous
Wasf3 confirmed its location at the proximal end of axoneme that costained with Rp1 antibody. Immunoblot demonstrated that Wasf3
protein was expressed mainly in the retina and brain. wasf3
morphants displayed smaller eyes than those of wild type zebrafish,
and a curved body appearance. Histologic analysis showed that wasf3
morphants exhibited compromised photoreceptor ciliogenesis, with
only a few shortened outer segments present.
Conclusions: Our results indicate that Wasf3 is a novel cilia protein
in vertebrate retina. Loss of wasf3 function prevents normal
morphogenesis or maintenance of PSC outer segments. This suggests
that WASF3 plays an important role in the biologic function of
photoreceptor cells and screening WASF3 for mutations in IRD
patients is warranted.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
Commercial Relationships: Carlo Rivolta, None; Giulia
Venturini, None; Shyana Harper, None; Hanna Koskiniemi,
None; Eliot L. Berson, None
Support: Swiss National Science Foundation (grants 320030-121929
and 310030_138346), the Gebert Rüf Foundation, Switzerland (Rare
Diseases - New Technologies grant), a Center Grant to the BermanGund Laboratory from the Foundation Fighting Blindness, Columbia,
Maryland
Wasf3 (Red) and Rp1 (Green) co-staining in mouse retina
Immunostaining with anti-V5 antibody (Red) in rat retina 3 weeks
after subretinal injection of V5-WASF3 plasmid and then followed
electroporation.
Commercial Relationships: Jingfa Zhang, None; Qi Zhang, None;
Conghui Zhang, None; Chan Wu, None; Eric A. Pierce, None;
Qin Liu, None
Support: NEI (EY12910), the Foundation Fighting Blindness USA
and P30EY014104 (MEEI core support)
Program Number: 1315 Poster Board Number: A0009
Presentation Time: 8:30 AM - 10:15 AM
A 353-bp Alu insertion in MAK is a prevalent cause of recessive
retinitis pigmentosa in North American Jewish patients
Carlo Rivolta1, Giulia Venturini1, Shyana Harper2, Hanna
Koskiniemi1, Eliot L. Berson2. 1Department of Medical Genetics,
University of Lausanne, Lausanne, Switzerland; 2Berman-Gund
Laboratory for the Study of Retinal Degenerations, Harvard Medical
School, Massachusetts Eye and Ear Infirmary, Boston, MA.
Purpose: A 353-bp Alu insertion in exon nine of male germ cellassociated kinase (MAK) gene was previously shown to be a cause of
autosomal recessive retinitis pigmentosa (arRP), with a prevalence of
1.2% within a large cohort of unrelated patients. All of the carriers of
the homozygous insertion were of Jewish ancestry. Our aim was to
ascertain the prevalence of this mutation in a group of North
American patients with arRP and of Jewish ancestry, compared with
a cohort of patients of mixed ethnicity.
Methods: Patient sets included 82 unrelated individuals with arRP
and of Jewish ancestry, assessed from a questionnaire, and a group of
190 arRP patients of mixed ethnicity (mostly Caucasians). DNA from
peripheral leukocytes was extracted and used as template for PCR
amplifications. PCR produces were then analyzed by agarose gel
electrophoresis and Sanger sequencing.
Results: The homozygous Alu-element insertion in exon nine of
MAK was identified in 9 out of 82 patients (~11%) of Jewish ancestry
and in 4 out of 190 patients (~2%) of mixed ethnicity. These 4 latter
positive individuals reported generic East European origin.
Conclusions: Our data indicate that a single mutation in the MAK
gene is responsible for arRP in a large fraction of North American
Jewish patients, while being a relatively rare occurrence in other
Caucasians individuals with the same disease.
Program Number: 1316 Poster Board Number: A0010
Presentation Time: 8:30 AM - 10:15 AM
Clinical and Molecular Findings in Japanese Cases with KCNV2retinopathy: Report of Novel Variants
Yu Kato1, Kaoru Fujinami1, Natsuko Nakamura1, Masakazu
Akahori2, Takeshi Iwata2, Kazushige Tsunoda1. 1Lab. of Visual
Physiology, National Institute of Sensory Organs, Tokyo, Japan;
2
Molecular & Cellular Biology Division, National Institute of
Sensory Organs, Tokyo, Japan.
Purpose: To describe clinical and molecular characteristics of four
Japanese patients with "Cone Dystrophy with Supernormal Rod
Electroretinogram (ERG)."
Methods: Four individuals form three families with clinical
diagnosis of cone dystrophy with supernormal rod ERG were
recruited; two siblings from one family {Patient 1 (24 yrs), Patient 2
(17 yrs)} and two simplex cases {Patient 3 (17 yrs), Patient 4 (21
yrs)}. Full clinical examinations were undertaken, including fundus
photography, fundus autofluorescence (AF) imaging, spectral-domain
optical coherence tomography (OCT) imaging and full-field ERG
incorporating to international standard. Mutational screening of
KCNV2 was performed by direct sequencing.
Results: All patients had central visual disturbances and night
blindness. Two patients (Patients 2, 3) complained of photophobia
and one (Patient 3) had nystagmus. The best corrected visual acuity at
the latest examination was variable ranging from 0.08 to 0.7. Fundus
imaging identified RPE mottling at the macula, which was welldefined in AF imaging. ERGs showed identical features with a
decreased rod response, a square-shaped a-wave and a highamplitude b-wave in bright flash response, and decreased conederived responses. OCT imaging detected absence of cone outer
segment tip (COST) line in the macula in all patients, with
discontinuous inner and outer segment (IS/OS) junction line in 2
subjects (Patient 3, 4). Molecular analysis revealed compound
heterozygosity for two alleles (p.C177R and p.G461R) in three
individuals (Patient 1, 2, 4) and homozygosity for complex alleles
(p.R27H and p.R206P) in Patient 3; three were putative novel
(p.R27H, p.C177R, p.R206P).
Conclusions: The clinical features of Japanese patients with KCNV2retionpathy, including ERG, OCT and AF findings, were similar to
those reported in previous studies. Three putative novel variants were
identified in four subjects and there may be a distinct spectrum of
KCNV2 alleles in Japanese population.
Commercial Relationships: Yu Kato, None; Kaoru Fujinami,
None; Natsuko Nakamura, None; Masakazu Akahori, None;
Takeshi Iwata, None; Kazushige Tsunoda, None
Program Number: 1317 Poster Board Number: A0011
Presentation Time: 8:30 AM - 10:15 AM
Bioinformatic identification of altered splicing motifs resulting
from the Alu insertion in exon 9 of the RP gene MAK
S Scott Whitmore1, Shemin Zeng1, Heather T. Daggett1, Adam P.
DeLuca2, Budd A. Tucker1, Terry A. Braun2, 1, Robert F. Mullins1,
Edwin M. Stone1, Todd E. Scheetz1, 2. 1Ophthalmology & Visual
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
Sciences, University of Iowa, Iowa City, IA; 2Biomedical
Engineering, University of Iowa, Iowa City, IA.
Purpose: An Alu insertion into exon 9 of male germ cell-associated
kinase (MAK) causes the exclusion of that exon and the retinaspecific exon 12, resulting in retinitis pigmentosa. We hypothesized
that insertion of the Alu element triggers exclusion of exon 9 by
altering an existing exonic splicing enhancer (ESE) or by introducing
an exonic splicing silencer (ESS).
Methods: Putative ESE and ESS motifs were identified using Human
Splicing Finder (v2.4.1) in MAK exon 9 with and without the Alu
insertion. Scores for 5’ and 3’ splice sites of MAK exons were
calculated using Analyzer Splice Tool. The ExonScan tool was used
to computationally predict exons from the human reference sequence
(GRCh37/hg19) between the initial and terminal exons of MAK.
Results: Insertion of the Alu in exon 9 introduces a complex set of
splicing motifs; however, the run of adenosines near the 3’ end of the
Alu generates a strong signal for exonic splice silencing. Of note, the
retina-specific exon 12 is computationally predicted by ExonScan,
with splice site scores that are similar to the constitutively expressed
exons.
Conclusions: Exclusion of exon 9 may result from the introduction
of exonic splicing silencers within the Alu. Exon 12 can be predicted
by an existing algorithm indicating that the exon shares features with
other known exons. We are analyzing RNA-Seq data from retina and
other tissues to characterize retina-specific splicing regulatory motifs
shared by MAK exon 12 and other retina-specific exons.
Commercial Relationships: S Scott Whitmore, None; Shemin
Zeng, None; Heather T. Daggett, None; Adam P. DeLuca, None;
Budd A. Tucker, None; Terry A. Braun, Alcon Research, LTD (F);
Robert F. Mullins, Alcon Research Ltd (F); Edwin M. Stone, None;
Todd E. Scheetz, None
Support: Howard Hughes Medical Institute, NIH Grant EY017451,
NIH Grant DP20D-007483, NIH T32 Training Grant
5T32GM082729-03
Program Number: 1318 Poster Board Number: A0012
Presentation Time: 8:30 AM - 10:15 AM
Extended Phenotypic Characteristics of NR2E3-related
Enhanced S-cone Syndrome
Ajoy Vincent1, 2, Cynthia VandenHoven1, Carol A. Westall1, 2, Elise
Heon1, 2. 1Ophthalmology and Vision Sciences, Hospital for Sick
Children, Toronto, ON, Canada; 2Ophthalmology and Vision
Sciences, University of Toronto, Toronto, ON, Canada.
Purpose: To evaluate a cohort of patients to enhance the phenotypic
description of NR2E3-related enhanced S-cone syndrome (ESCS).
Methods: Eight patients (seven families; Age range: 11 - 56 years)
with characteristic full-field electroretinographic (ERG) features of
ESCS were screened for mutations in the NR2E3 gene. All underwent
detailed ophthalmological evaluation, which included distance and
color vision testing, contrast sensitivity measurement, fundus
photography and Goldmann visual field (GVF) perimetry. Fundus
autofluorescence (AF) imaging and optical coherence tomography
(OCT) were performed in 7 cases.
Results: Two pathogenic mutations in NR2E3 were identified in 7
cases; one pathogenic mutation was identified in case 8. The dimlight scotopic ERGs (DA0.002) were non-recordable in all cases. The
mixed rod-cone ERGs (DA2.29) were simplifed and delayed in all
cases; a- and b-wave amplitudes were subnormal in all except case 8
(aged 11 years). Single flash photopic ERGs (LA2.29) were delayed
and had similar waveform morphology as DA2.29 ERGs in all cases;
LA2.29 ERG b-wave amplitudes were sub/low-normal in all except
case 8. Photopic flicker amplitude was smaller than LA2.29 ERG awave in all cases. Best corrected vision (BCVA) ranged from 20/20
to 20/200 (median: 20/50). BCVA was 20/30 or better in three cases
(Ages: 11, 21 and 57 years); two of whom had normal contrast
sensitivity. Color vision was normal in the tritan axis in all cases; redgreen deficits were noted in 6 cases. The classical ESCS fundus
description of mid-peripheral nummular pigment clumping or
yellowish retinal deposition was noted only in 6 cases. GVF showed
mid-peripheral field deficits to I4e target in 15/16 eyes. The OCT
showed schitic changes at the fovea in three cases; 2 improved on
carbonic anhydrase inhibitors. Retinal structural disorganization
within and/or beyond the vascular arcades were noted in all tested.
Hyper-intense AF was noted within the arcades in all tested, but the
pattern was variable.
Conclusions: The strict genotype-ERG phenotype correlation in
ESCS is reconfirmed. The classical fundus changes are found in only
75% of cases. The OCT abnormalities observed in this series enhance
the description of NR2E3-related ESCS, and can be a useful adjunct
to direct ERG testing and genetic screening in atypical cases. This
study also demonstrates early field constriction in ESCS.
Commercial Relationships: Ajoy Vincent, None; Cynthia
VandenHoven, None; Carol A. Westall, Lunbeck Pharmaceuticals
(F); Elise Heon, None
Program Number: 1319 Poster Board Number: A0013
Presentation Time: 8:30 AM - 10:15 AM
Phenotypic variability in paediatric cases of enhanced S-cone
syndrome (ESCS)
Gavin Arno1, Panagiotis I. Sergouniotis1, Arundhati Dev Borman1, 2,
Aman Chandra1, 2, Graham E. Holder3, Anthony G. Robson3, Andrew
R. Webster1, 2, Anthony T. Moore1, 2. 1Inherited Eye Diseases, UCL
Institute of Ophthalmology, London, United Kingdom; 2Moorfields
Eye Hospital, London, United Kingdom; 3Electrophysiology,
Moorfields Eye Hospital, London, United Kingdom.
Purpose: ESCS is consequent upon mutation in NR2E3 and is
usually characterised by absence of rod photoreceptors and a retina in
which the majority of cones are short-wavelength sensitive. It is one
of the few disorders in which the electroretinogram (ERG) can be
pathognomonic. The aim of this study was to examine the phenotypic
features of ESCS in children.
Methods: Twelve children from 8 families with ECSC were
ascertained. Most underwent fundus examination, ERG, fundus
autofluorescence imaging (FAF) and OCT. Mutations in NR2E3
were investigated in 7 patients from 6 families where DNA was
available by bidirectional sequencing of all exons and intron/exon
boundaries.
Results: All had nyctalopia from early childhood. Hypermetropia
was documented in 6 cases, and 5 children from 3 families had a
convergent strabismus. Median visual acuity was 0.17 logMAR.
Funduscopic abnormalities along the vascular arcades were observed
in 11 of 12 cases, the remaining case had a normal fundus. ERG
revealed typical features associated with ECSC in 11 of 12 cases
including the patient with a normal fundus. One patient had typical
cone-mediated ERG abnormalities but with only mildly abnormal rod
responses. Fundus autofluorescence imaging revealed high density
foci along the arcades in 7 of 8 cases. Spectralis OCT assessment
confirmed macular schisis-like changes in 3 of 7 cases.
Mutations in NR2E3 were identified in 6 individuals from 5 families
available for DNA analysis. These comprised compound
heterozygous changes: c.119-2A>C and c.1194delT identified in two
affected family members; c.305C>A and c.767C>A; c.119-2A>C and
c.1025T>C and homozygous changes: c.1101-1G>A and c.310C>T.
The novel c.1194delT (p.P399Qfs78*) mutation is predicted to cause
a frameshift abolishing the correct termination codon.
Conclusions: This study further characterises the clinical phenotype
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
of ESCS in children. The recognition of this phenotype allows
targeted molecular screening and molecular diagnosis.
Commercial Relationships: Gavin Arno, None; Panagiotis I.
Sergouniotis, None; Arundhati Dev Borman, None; Aman
Chandra, None; Graham E. Holder, Servier (C); Anthony G.
Robson, None; Andrew R. Webster, None; Anthony T. Moore,
None
Support: Fight for Sight UK
Program Number: 1320 Poster Board Number: A0014
Presentation Time: 8:30 AM - 10:15 AM
Exon 3 genotypes of OPN1LW/OPN1MW associated with Xlinked congenital cone dysfunction
Susanne Kohl1, Britta Baumann1, Christian P. Hamel2, Peter
Gustavsson3, Thomas Rosenberg4, Astrid S. Plomp5, Bart P. Leroy6,
Joke BGM Verheij7, Bernd Wissinger1. 1Institute for Ophthalmic
Reseach, Centre for Ophthalmology, Tuebingen, Germany; 2Genetic
Sensory Diseases - Hopital Gui de Chauliac, Centre Hospitalier
Universitaire, Montpellier, France; 3Department of Molecular
Medicine and Surgery, Karolinska Instituet, Stockholm, Sweden;
4
National Eye Clinic for the Visually Impaired, Kennedy Center,
Glostrup, Denmark; 5Department of Clinical and Molecular
Ophthalmogenetics, NetherlandsInstitute for Neuroscience,
Amsterdam, Netherlands; 6Department of Ophthalmology & Center
for Medical Genetics, Ghent University Hospital, Ghent, Belgium;
7
Universitair Medisch Centrum Groningen, Groningen University
Hospital, Groningen, Netherlands.
Purpose: To assess exon 3 genotypes of the X-linked
OPN1MW/OPN1LW gene cluster encoding the cone photoreceptor
red and green opsins, in patients with X-linked blue cone
monochromatism (BCM) and congenital cone dysfunction (XLCD).
Large deletions affecting the locus control region (LCR), sometimes
also extending into the opsin genes or complete gene cluster
deletions, genomic rearrangements leading to hybrid gene formation
and simultaneous inactivating missense or nonsense mutations are the
cause of BCM. Yet in a small percentage of cases, the causative
mutation could not be identified. Recent publications have shown that
certain combinations of rare sequence variants in exon 3 of the
OPN1MW/OPN1LW genes can result in missplicing and exon
skipping, leading to XLCD.
Methods: DNA sequencing and genotype evaluation of PCR
amplified exon 3 of OPN1MW/OPN1LW in clinically diagnosed
BCM or XLCD patients in whom the structure of the
OPN1MW/OPN1LW gene cluster had previously been determined
by means of PCR and PCR/RFLP analysis.
Results: In our cohort of 187 clinically diagnosed BCM patients,
~80% carry either large genomic deletions (28.8%), the common
missense mutation p.C203R (46%), or other inactivating missense or
nonsense mutations (5.9%). We here describe the genetic basis of
BCM/XLCD of at least 11 patients from 7 independent families
carrying genotypes that have recently been acknowledged by the
encoded amino acid determinants p.L/M153 - p.V/I171 - p.V/A174 p.V/I178 - p.A/S180.
In 3 families the structure of the OPN1MW/OPN1LW gene cluster
was essentially intact, but the proximal gene was shown to carry the
deleterious LIAVA (n=2) or LVAVA (n=1) genotype, while the
distal gene(s) were either LVAIA, MVVVA or MIAVA. In the
remaining 4 families the gene cluster was replaced by a single red- or
red-green hybrid gene that carried the deleterious genotypes LIAVA
(n=2 families, 4 patients) or LVAVA (n=2 families and patients).
Conclusions: Certain rare sequence variants in exon 3 of the
OPN1MW/OPN1LW gene cluster give rise to missplicing and exon
skipping. We conclude that these kinds of previously unrecognized
splice mutations may account for many of the remaining genetically
uncharacterized BCM/XLCD cases, as our first analysis has shown
that 5.9% of patients of our total BCM cohort carry these deleterious
genotypes.
Commercial Relationships: Susanne Kohl, None; Britta
Baumann, None; Christian P. Hamel, None; Peter Gustavsson,
None; Thomas Rosenberg, None; Astrid S. Plomp, None; Bart P.
Leroy, None; Joke BGM Verheij, None; Bernd Wissinger, None
Support: BPL is Senior Clinical Investigator of Fund for Research,
Flanders
Program Number: 1321 Poster Board Number: A0015
Presentation Time: 8:30 AM - 10:15 AM
Fine analysis of the deletions in red/green opsin genes and the
upstream locus control region (LCR) found in two Japanese
families with blue cone monochromacy (BCM)
Chun-xia Wang1, 3, Katsuhiro Hosono1, 2, Shu Kachi4, Hiroko
Terasaki4, Yoshihiro Hotta1, Shinsei Minoshima2. 1Ophthalmology,
Hamamatsu University School of Medicine, Hamamatsu, Japan;
2
Medical Photobiology, Photon Medical Research Center,
Hamamatsu University School of Medicine, Hamamatsu, Japan;
3
Ophthalmology, The Fourth Affiliated Hospital of China Medical
University, Shenyang, China; 4Ophthalmology, Nagoya University
School of Medicine, Nagoya, Japan.
Purpose: BCM is a rare X-linked disorder of color vision
characterized by the absence of both red and green cone sensitivities.
BCM is caused by discrete mutations in each of red (R) and green
(G) opsin genes or a single mutation within the LCR in the upstream
of R. We performed a fine analysis of the mutations in two Japanese
families (W and N) with BCM.
Methods: Affected males were examined clinically, and their DNA
was obtained with informed consents. Various parts of R and G genes
and the LCR were amplified by PCR, and direct sequencing was
performed to find mutations. A PCR-based genomic walking was
done to determine the unknown DNA sequence when necessary.
Results: Affected males were examined clinically, and their DNA
was obtained with informed consents. Various parts of R and G genes
and the LCR were amplified by PCR, and direct sequencing was
performed to find mutations. A PCR-based genomic walking was
done to determine the unknown DNA sequence when necessary.
In family W, a 16,803-bp deletion was found. The proximal boundary
of the deletion was 8,845-bp upstream from the translation initiation
codon of R gene, and the distal boundary was within intron 2 of R
gene. While, family N revealed a complicated 87,355-bp deletion
with 328-bp inversion and 3-bp insertion. The proximal boundary of
the deletion was 28,144-bp upstream from the initiation codon of R
gene, and the distal boundary was within the downstream region of
the first G gene. In both cases, the deletion was associated with a
highly repetitive sequence AluSz.
Conclusions: In two Japanese families with BCM, the diseasecausative mutations were the deletion of long region including the
LCR. This is the first report of the single base-level analysis of BCM
cases caused by the deletion including the LCR.
Commercial Relationships: Chun-xia Wang, None; Katsuhiro
Hosono, None; Shu Kachi, None; Hiroko Terasaki, None;
Yoshihiro Hotta, None; Shinsei Minoshima, None
Program Number: 1322 Poster Board Number: A0016
Presentation Time: 8:30 AM - 10:15 AM
Allelic variation of visual pigments in capuchin monkeys, Sapajus
spp
Daniela M. Bonci1, 2, Maureen Neitz3, Paulo Roney K. Goulart4,
Juliana G. Soares5, Mario Fiorani5, Olavo F. Galvão4, Ricardo
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
Gattass5, Luiz Carlos L. Silveira6, 7, Dora F. Ventura1, 2. 1Psicologia
Experimental, Universidade de Sao Paulo, Sao Paulo, Brazil; 2Nucleo
de Neurociencias e Comportamento, Universidade de Sao Paulo, Sao
Paulo, Brazil; 3Departmento of Ophtalmology, University of
Washington, Seattle, WA; 4Nucleo de Teroria de Pesquisa do
Comportamento, Universidade Federal do Para, Belem, Brazil;
5
Instituto de Biofisica, Universidade Federal do Rio de Janeiro, Rio
de janeiro, Brazil; 6Nucleo de Medicina Tropical, Universidade
Federal do Pará, Belém, Brazil; 7Instituto de Ciências Biológicas,
Universidade Federal do Pará, Belém, Brazil.
Purpose: To study the variation and frequency of M/L visual
pigment genes capuchin monkeys, Sapajus spp from Brazil.
Methods: blood samples were analyzed from capuchin monkeys
maintained in different Brazilian cities: Belém (N=18) and Rio de
Janeiro (N=13). The M/L opsin genes of all monkeys were sequenced
after DNA extraction and PCR amplification procedures. The 180,
277 and 285 amino acids were identified and the spectral absorption
curve was predicted for each animal. The type and frequency of
visual pigment alleles were determined.
Results: 31 capuchin monkeys were evaluated for M/L genes. All
males (N=21) and some females (N=5) had only one M/L allele and
were identified as protanope or deuteranope dichromats. Five females
showed 2 different alleles which characterized them as trichromats.
For the 18 deuteranope monkeys, the SYT (λmax 560-563) or AYT
(λmax 550-556) alleles were present in 15 and 3 animals,
respectively. Eight monkeys had AFA (λmax 532) (N=4) or AFT
(λmax 542-547) (N=4) alleles characterizing them as protanope. The
trichromat females showed two alleles combinations, AFA+AFT
(N=2) and SFT (λ max 546-553) + SYT (N=3). A total of 5 different
alleles were described for Sapajus spp. The frequency of SYT allele
(58%) was higher than AYT (12%), AFA (15%) and AFT (15%)
alleles in dichromats animals. In trichromatic females, the
combination SFT+SYT was more frequent (60%) than AFA+AFT
combination (40%).
Conclusions: The present work is the first to describe 5 different
M/L opsin alleles for Sapajus spp genus. The SYT allele was the
most frequent, which can be explained by the evolution of mammal
opsin genes.
Commercial Relationships: Daniela M. Bonci, None; Maureen
Neitz, Genzyme (F), Alcon (F), Alcon (P); Paulo Roney K. Goulart,
None; Juliana G. Soares, None; Mario Fiorani, None; Olavo F.
Galvão, None; Ricardo Gattass, None; Luiz Carlos L. Silveira,
None; Dora F. Ventura, None
Support: FAPESP
Program Number: 1323 Poster Board Number: A0017
Presentation Time: 8:30 AM - 10:15 AM
Retinal gene expression in mice lacking cones and/or rods
identifies genes potentially involved in human eye function and
disease
Richard J. Holt1, Laurence Brown1, Rachel Butler1, Susan M.
Downes1, 2, Stuart N. Peirson1, Stephanie Halford1. 1Nuffield
Department of Clinical Neurosciences, University of Oxford, Oxford,
United Kingdom; 2Oxford Eye Hospital, National Health Service,
Oxford, United Kingdom.
Purpose: Advances in sequencing have increased the potential to
identify genetic variants in individuals with retinal disease; however
determining which are causal remains a challenge. In order to
generate a resource to help identify genes involved in retinal
function, and so provide candidates for disease studies, we examined
gene expression in retinae from 3 transgenic mouse strains; rd/rd
(rodless), Cl (coneless) and rd/rdCl (rodless and coneless) and
compared them to wild type.
Methods: Retinae were excised from rd/rd, Cl, rd/rdCl and wild type
mice, and RNA extracted. cDNA for probes was generated using the
Ambion® WT Expression Kit. Gene expression was examined using
the GeneChip® mouse exon 1.0 ST arrays. Data was processed using
AltAnalyze v2.0.7 and a Benjamini-Hochberg correction for multiple
testing applied. qPCR validation was performed using the
QuantiFast® SYBR® Green PCR kit. Clustering and pathway
analysis was performed in Cytoscape v2.8. Gene set enrichment
analysis was performed using GSEA v2.07.
Results: The expression of 27,966 genes was determined in retinae
from each of rd/rd, Cl, rd/rdCl and wild type mice and compared
between each transgenic strain and wild type. Clustering analysis was
performed for all genes with a P <0.001 in at least one of the
transgenic strains. Clustering was similar for rd/rd and rd/rdCl, but
with a striking difference to Cl mice. Expected functions such as
phototransduction were highlighted, but also revealed other functions
such as cell projection and transferase activity. Human orthologues of
many genes with the largest decreases in expression are involved in
retinal disease, confirmed by gene set enrichment analysis (P <0.0001
for rd/rd, Cl and rd/rdCl). Comparison to human disease loci with
unknown causal genes identified candidates with significant
decreased expression demonstrating the utility of the data to aid
identification of novel candidates.
Conclusions: We have generated catalogues of genes with decreased
expression in mice lacking cones, rods or both. This provides
information on new genes for investigation into the normal function
of the eye, and will help determine genes involved in human retinal
disease by identifying candidate genes in disease loci, or acting as a
filter for high-throughput sequencing data.
Commercial Relationships: Richard J. Holt, None; Laurence
Brown, None; Rachel Butler, None; Susan M. Downes, Novartis
(F); Stuart N. Peirson, None; Stephanie Halford, None
Support: RP Fighting Blindness Grant GR571
Program Number: 1324 Poster Board Number: A0018
Presentation Time: 8:30 AM - 10:15 AM
Progressive Fundus Autofluorescence Patterns in Achromatopsia
Abigail T. Fahim1, Naheed W. Khan1, Sarwar Zahid1, Ira H.
Schachar1, Kari E. Branham1, Susanne Kohl2, Bernd Wissinger2,
Victor M. Elner1, John R. Heckenlively1, Kanishka T. Jayasundera1.
1
Ophthalmology, Kellogg Eye Ctr, Univ of Michigan, Ann Arbor,
MI; 2Ophthalmology, University of Tuebingen, Tuebingen, Germany.
Purpose: To describe the progression of fundus autofluorescence
(FAF) patterns in patients with achromatopsia and the associated
findings on optical coherence tomography (OCT).
Methods: A restrospective review was conducted of eight patients
with achromatopsia caused by mutations in CNGA3 or CNGB3.
Patients were evaluated with best-corrected visual acuity (BCVA),
ophthalmoscopy, Goldmann visual field (GVF), OCT, full-field
electroretinography (ffERG), and FAF photography. FAF patterns
were compared and correlated with patient age and foveal changes on
OCT.
Results: Patients fell into two dichotomous age groups at the time of
evaluation, with 5 patients ranging from 11 to 23 years old and 3
patients ranging from 52 to 63 years old. All patients had severely
reduced photopic ffERG parameters. The younger patients had mild
to no foveal atrophy on OCT, and four out of five demonstrated focal
foveal or parafoveal hyperfluorescence on FAF. In addition, a 7
month-old child with compound heterozygous mutations in CNGA3
demonstrated similar parafoveal hyperfluorescence. The older
patients had advanced foveal atrophy with cavitation on OCT, and all
three demonstrated punched-out foveal hypofluorescence with
discreet borders on FAF imaging.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
Conclusions: In our cohort, achromatopsia patients demonstrate a
unique pattern of foveal hyperfluoresence with age-dependent
progression to foveal hypofluoresence that correlates with outer
retinal atrophy and cavitation on OCT. Foveal hyperfluorescence
may be an early sign of pathologic change that should prompt further
diagnostic testing for achromatopsia. Furthermore, FAF patterns in
achromatopsia may be useful in charting the natural course of disease
and in defining a therapeutic window for treatment in future
investigations.
Commercial Relationships: Abigail T. Fahim, None; Naheed W.
Khan, None; Sarwar Zahid, None; Ira H. Schachar, None; Kari E.
Branham, Arctic DX (P); Susanne Kohl, None; Bernd Wissinger,
None; Victor M. Elner, OcuSciences, Inc. (I), OcuSciences, Inc. (P);
John R. Heckenlively, None; Kanishka T. Jayasundera, None
Support: FFB C-CL-0710-497-UMICH01
Program Number: 1325 Poster Board Number: A0019
Presentation Time: 8:30 AM - 10:15 AM
Molecular modeling of functional domain of ABCA4: towards
understanding the genotype-to-phenotype relationships in
Stargardt’s disease
Yuri V. Sergeev1, Katherine L. Pogrebniak1, 3, Benedetto Falsini1, 2,
Wadih M. Zein1, Kerry Goetz4, Jillian Huang5, Crandall E. Peeler5,
Kanishka T. Jayasundera5, Brian P. Brooks1, Paul A. Sieving1.
1
OGVFB, National Eye Institute, Bethesda, MD; 2Ophthalmology,
Catholic University, Rome, Italy; 3Princeton University, Princeton,
NJ; 4EyeGene, OGVFB, National Eye Institute, Bethesda, MD;
5
Kellogg Eye Center, University of Michigan, Ann Arbor, MI.
Purpose: ABCA4 is an ATP binding cassette protein transporter.
Mutations affecting the ABCA4 transporter protein are known to
cause Stargardt’s macular degeneration, but very little is known about
the structure of ABCA4, making it difficult to understand how these
mutations affect the function of ABCA4 as a protein transporter.
Here we performed molecular modeling of the functional domain
which includes trans-membrane and nucleotide-binding domains, a
part of ABCA4 polypeptide responsible for the flipping of Nretinylidene PE over the disc membrane.
Methods: The functional domain was modeled by homology, refined
and equilibrated using molecular dynamics. This model was used to
predict structural perturbations caused by mutations found in affected
human subjects. Overall genotype scoring function was derived from
free energy changes for the ABCA4 open/close conformers was
calculated for each mutation (Sergeev et al, Hum Mol Genetics,
2010). We then evaluated possible correlations of this modeling with
phenotypes of patients from the NEI, EyeGENE database, Italian and
Kellogg Eye Center Stargardt’s patients (42 genotypes total).
Results: In the selected group of 15 patients we found an association
between the overall genotype score and the age of disease onset
known to be related to the severity of retinal disease (Pearson’s r = 0.71, Adj. R-square = 0.46). Similar association between these
parameters was observed for 7 NEI patients enrolled in EyeGENE
(Pearson’s r = - 0.76, Adj. R-square = 0.53). Finally, these data were
combined with data from Italian Stargardt’s patients. The total dataset
of 28 patients showed a linear association between predicted
severities and the age of disease onset (Pearson’s r = - 0.67, Adj. Rsquare = 0.42). Similar result was obtained for 12 patients from the
Kellogg Eye Center (Pearson’s r = - 0.76, Adj. R-square=0.54).
Conclusions: The overall impact score for each individual analyzed
was associated with the age of retinal disease onset. The functional
activity suggested by the model is in agreement with recent
experimental data on ABCA4 as a ‘flippase’ protein. Thus, molecular
modeling of the ABCA4 functional domains and the scoring system
derived in our work has a potential to be used for a study of
genotype-to-phenotype relationships in Stargardt’s disease.
Commercial Relationships: Yuri V. Sergeev, None; Katherine L.
Pogrebniak, None; Benedetto Falsini, None; Wadih M. Zein,
None; Kerry Goetz, National Eye Institute/NIH (E); Jillian Huang,
None; Crandall E. Peeler, None; Kanishka T. Jayasundera, None;
Brian P. Brooks, None; Paul A. Sieving, None
Support: EY000476-02 OGVF
Program Number: 1326 Poster Board Number: A0020
Presentation Time: 8:30 AM - 10:15 AM
The frequency of 5 mutations in the ABCA4 gene in Russian
patients with a clinical diagnosis of Stargardt disease / Fundus
Flavimaculatus
Maria Shurygina1, Sergey Borzenok1, Olga Khlebnikova2, Oksana
Nekrasova3, Olga Kravchuk1. 1Center for Fundamental Medicine,
S.Fyodorov Eye Microsurgery Complex, Moscow, Russian
Federation; 2Department of Genetic Epidemiology, Research Centre
for Medical Genetics, Russian Academy of Medical Sciences,
Moscow, Russian Federation; 3M.M. Shemyakin and Yu.A.
Ovchinnikov Institute of bioorganic chemistry of RAS, Moscow,
Russian Federation.
Purpose: To study the five mutations frequency occurrence of
(Leu541Pro, Gly863Ala, Ala1038Val, Leu1940Pro, Gly1961Glu) in
ABCA4 gene in patients with clinical diagnosis of Stargardt disease /
Fundus Flavimaculatus in the Russian Federation.
Methods: DNA samples (n = 24) of unrelated patients with Stargardt
disease / Fundus Flavimaculatus. In 5-exons ABCA4 gene five
mutations were searched (12, 17, 21, 41, 42, Leu541Pro, Gly863Ala,
Ala1038Val, Leu1940Pro, Gly1961Glu respectively). DNA was
extracted from white blood cells using a set of reagents DIA / om ™
DNA RgerIII according to the manufacturer's protocol. To obtain
fragments of exons ABCA4 gene polymerase chain reaction was
performed. The amplification and analysis of restriction fragments
length polymorphism were assessed by electrophoresis in a 7%
polyacrylamide gel followed by staining the gel with a solution of
ethidium bromide, and UV radiation registration .
Results: In 25% cases (n = 6) compound heterozygotes mutation
Gly1961Glu was found,
in 8,3% cases (n=2) complex mutation Leu541Pro - Ala1038Val,
in 8,3% cases (n=2) homozygous mutation Ala1038Val,
in 4,2% cases (n=1) complex mutation Ala1038Val - Gly1961Glu,
compound heterozygotes mutation Gly863Ala in 4,2% cases (n=1)
and
compound heterozygotes mutation Ala1038Val in 4,2% cases (n=1).
In 45,8% cases (n = 11) five mutations were not detected.
Thus, the allelic frequency of mutations in our sample of patients
with a clinical diagnosis of Stargardt disease / Fundus Flavimaculatus
was Ala1038Val 44,4%, Gly1961Glu 38,9 %, Leu541Pro 11,1%,
Gly863Ala 5,6%.
Conclusions: The most frequent mutation in ABCA4 gene at Russian
patients with Stargardt disease / Fundus Flavimaculatus was
Ala1038Val, the second place in the frequency of the mutation was
Gly1961Glu.
Commercial Relationships: Maria Shurygina, None; Sergey
Borzenok, None; Olga Khlebnikova, None; Oksana Nekrasova,
None; Olga Kravchuk, None
Program Number: 1327 Poster Board Number: A0021
Presentation Time: 8:30 AM - 10:15 AM
Visual Prognosis and Association Between Geno - and Phenotype
in Families with ABCA4 Mutations
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
Ulrika Kjellstrom1, 2, Sten Andreasson1, 2. 1Department of
Ophthalmology, University of Lund, Lund, Sweden; 2Department of
Ophthalmology, Skane University Hospital, Lund, Sweden.
Purpose: To evaluate visual outcome and phenotypes in families
with ABCA4 mutations.
Methods: Three families with one or more members with known
homozygous or compound heterozygote ABCA4 mutations were
examined with visual acuity (VA), funduscopy and photography, fullfield electroretinography (ff-ERG), multifocal ERG (mERG) and
optical coherence tomography (OCT). Microarray was used for
ABCA4 genotyping (Asper).
Results: In family 1, the proband was homozygote for c768 G>T.
Her aunt was compound heterozygote for c768 G>T and c2894 A>G.
The proband had low VA, large central scotomas, reduced ff-ERG
cone - and mERG responses. She was diagnosed with cone rod
dystrophy (CRD). Her aunt was diagnosed with pigmentosa (RP)
with low VA, small crescent-shaped visual fields (VF), reduced ffERG and mERG. In family 2, the proband was homozygote for
c5917del. She was diagnosed with CRD with reduced VA, ff-ERG
cone responses and mERG. Her VFs were constricted with large
scotomas. Her father and two sisters were compound heterozygote for
c5917del and c5882 G>A. The eldest sister was diagnosed with
Stargardt disease (STGD) with low VA, central scotomas, normal ffERG and reduced mERG. The youngest sister and the father had no
symptoms at the age of 12 and 48 respectively. They demonstrated
normal ff-ERGs but reduced mERGs. The sister had normal VA and
VFs, while the father had low VA and central scotomas. In family 3,
the proband was compound heterozygote for c768 G>T and c3113
C>T. She was diagnosed with STGD with VA 0.1, small central
scotomas, normal ff-ERG and reduced mERG. Her son had the same
findings except for wider scotomas. Three of the patients with
progressive disorders (CRD or arRP) were examined repeatedly
during 5-15 years and were found to have prolonged implicit times
(ITs) of the 30 Hz flicker and mERG. Patients with two mutations in
the ABCA4 gene demonstrated attenuated retina on the OCT macular
map.
Conclusions: ABCA4 mutations can lead to STGD, CRD or arRP. At
the time of diagnose it is hard to predict the severity of the condition
only from genotyping. In this study, prolongation of ITs in the ff
and/or mfERG seems to be associated with progressive conditions.
ABCA4 mutations are common in the population, thus family
members can harbor various combinations of mutations resulting in
diverse phenotype and prognosis, further emphasizing the importance
of both genetic and electrophysiological tests.
Commercial Relationships: Ulrika Kjellstrom, None; Sten
Andreasson, None
Program Number: 1328 Poster Board Number: A0022
Presentation Time: 8:30 AM - 10:15 AM
Complement Factor Y402H polymorphism is not associated with
Stargardt’s Disease in Italian Patients
Andrea Sodi1, Ilaria Passerini2, Vittoria Murro1, Luca Boni3,
Giacomo Abbruzzese1, Alba Miele1, Matteo Giuntoli1, Giulio
Mecocci4, Francesca Torricelli2, Ugo Menchini1. 1Department Of
Specialistic Surgical Sciences, Eye Clinic AOU Careggi, University
Of Florence, Firenze, Italy; 2Department of Genetic Diagnosis,
Azienda Ospedaliero Universitaria Careggi, Firenze, Italy;
3
Department of Oncology, Azienda Ospedaliero Universitaria
Careggi, Firenze, Italy; 4Imperial College, London, United Kingdom.
Purpose: As CFH Y402H polymorphisms has been associated with a
higher risk of AMD, clinical, physiopathological and genetic
affinities between atrophic AMD and STGD may suggest a possible
role of CFH abnormalities also in STGD. The possible association of
Y402H and STGD has been investigated in Italian patients.
Methods: One-hundred unrelated STGD patients and 116 patients
with advanced atrophic AMD were recruited through the Hereditary
Retinal Degenerations Referring Center and the Medical Retina
Service of the Eye Clinic, University of Florence . The control group
consisted of 100 healthy volunteers. Each patient was genotyped for
the single nucleotide polymorphism rs1061170 (Y402H) in the CFH
gene using Sequencer 3730 DNA Analyzer. Hardy Weinberg
equilibrium was tested using the two sided Pearson's chi-squared test
with Yates' continuity correction. Genotype frequency distributions
between cases and controls were compared by means of chi squared
test for heterogeneity. Univariate unconditional logistic regression
was used to calculate odds ratios (OR) and 95% confidence intervals
(CIs) for the association between genotypes and diseases under
investigation. In addition, the multiplicative and additive genetic
models were used for the analysis. All statistical analyses were
performed using SAS System 9.2.
Results: Among all the three groups of controls, patients with AMD
and patients with STGD, the genotype distribution of Y402H1 was in
Hardy Weinberg equilibrium (P=0.327, P=0.320 and P=0.450,
respectively).
The genotype frequency of Y402H1 were different between patients
with AMD and controls (P=0.0003). In the additive model CT
genotype was associated with approximately 2.5 times higher odds of
disease compared with TT genotype, while CC genotype
demonstrated a risk of disease about 5 times higher than the reference
group (P for trend<0.0001).
Similar genotype frequencies of Y402H1 were observed between
patients with STGD and controls (P=0.4882). In this case, the
estimates of risk were compatible with the multiplicative genetic
model, however results did not achieve the statistical significance
(P=0.2639).
Conclusions: In Italian patients CFH Y402H genotype is associated
with atrrophic AMD but not with Stargardt’s Disease. This result
does not support the hypothesis of a complement system
dysregulation in the pathogenesis of Stargardt’s Disease.
Commercial Relationships: Andrea Sodi, None; Ilaria Passerini,
None; Vittoria Murro, None; Luca Boni, None; Giacomo
Abbruzzese, None; Alba Miele, None; Matteo Giuntoli, None;
Giulio Mecocci, None; Francesca Torricelli, None; Ugo Menchini,
None
Program Number: 1329 Poster Board Number: A0023
Presentation Time: 8:30 AM - 10:15 AM
SNRNP200 Mutations Account for 2% of Autosomal Dominant
Retinitis Pigmentosa
Lori S. Sullivan1, Sara J. Bowne1, Cheryl E. Avery1, Dianna K.
Wheaton2, David G. Birch2, Kari E. Branham3, John R.
Heckenlively3, Stephen P. Daiger1. 1Human Genetics Center SPH,
Univ Texas Hlth Sci Ctr Houston, Houston, TX; 2Retina Foundation
of the Southwest, Dallas, TX; 3Kellogg Eye Center, University of
Michigan, Ann Arbor, MI.
Purpose: Mutations in the pre-mRNA splicing gene SNRNP200
have been identified as a cause of autosomal dominant retinitis
pigmentosa (adRP). In order to determine the prevalence of
SNRNP200 mutations, a cohort of well characterized adRP patients
was screened.
Methods: A cohort of 258 probands has been systematically
screened for mutations in the genes currently known to cause adRP.
Of these 258 individuals, mutations have been identified in 185,
leaving a set of 73 with no known disease-causing mutation. All 45
exons of SNRNP200 were screened by fluorescent di-deoxy
sequencing in this subset of the adRP cohort. Identified variants were
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
tested for segregation in additional family members when available.
Results: In the set of 73 adRP patients screened, six were found to
have missense mutations in SNRNP200. Three families have a
previously identified change (Arg681His) while the other three were
found to have novel variants (Ala542Val, Pro682Ser, Gly1162Glu).
The Ala542Val mutation was found in two additional affected family
members. All three novel variants are predicted to be pathogenic and
are not found in databases of normal controls.
Conclusions: A comprehensive screen of the SNRNP200 gene has
identified likely disease-causing mutations in 6 of 73 individuals
tested. The overall prevalence of SNRNP200 mutations in our adRP
cohort is 2.3% (6/258). This further confirms the role of proteins in
the U4/U6-U5 tri-snRNP splice complex, including PRPF3, PRPF8,
PRPF31 and SNRNP200, in adRP.
Commercial Relationships: Lori S. Sullivan, None; Sara J.
Bowne, None; Cheryl E. Avery, None; Dianna K. Wheaton, None;
David G. Birch, Acucela (C), QLT (C), Neurotech, USA (C); Kari
E. Branham, Arctic DX (P); John R. Heckenlively, None; Stephen
P. Daiger, None
Support: This work was supported by grants from the Foundation
Fighting Blindness and EY007412 from the National Eye
Institute/NIH
Program Number: 1330 Poster Board Number: A0024
Presentation Time: 8:30 AM - 10:15 AM
Impact of CHM Mutations at the mRNA level
Markus N. Preising, Nayyir Alavi, Birgit Lorenz. Department of
Ophthalmology, Justus-Liebig-University, Giessen, Germany.
Purpose: Purpose: Pathogenic mutations underlying choroideremia
fall into four general groups (1) big deletions covering the whole
CHM gene or major parts of it, and preterm translation stops by (2)
either intragenic deletions or insertions causing frameshifts, (3) splice
site mutations causing exon skipping, or (4) nonsense mutations.
Therefore, a protein truncation test and western blots were proposed
as general diagnostic tools to identify mutations in CHM. Such a
general loss of the functional gene product would not be in line with
the phenotypical heterogeneity seen in patients.
In a previous pilot study on lyonization in female carriers we
recognized an escape of frameshifting mutations of CHM from
nonsense-mediated decay (NMD). Therefore, we sought to identify
the effect of CHM mutations at the mRNA level in males to explain
the phenotypical heterogeneity.
Methods: Material and Methods: We generated EBV-immortalized
lymphocyte cultures from 11 male index cases with frameshifting
mutations (intragenic small deletions (n=3), small insertions (n=4),
splice site mutations (n=1)), and nonsense mutations (n=3) in the
CHM-gene. Whole RNA was extracted using the Qiagen Whole
Blood Mini DNA/RNA Kit. The RNA was subjected to RT-PCR
(Qiagen one-step RT-PCR Kit) using mutation-specific primers
spanning 3 or more flanking exons. The PCR-products were analyzed
on capillary-gel electrophoresis.
Results: Results: The presence of CHM< transcripts was confirmed
by successful RT-PCR of all mutations tested excluding deleted
regions.
Conclusions: Discussion: Here we confirmed the escape from NMD
by preterm translation stops created by frameshifting mutations in
males in CHM. Therefore, at least 2 types of mutations in CHM have
to be considered: (1) mutations producing a translatable mRNA and
(2) mutations preventing the production of a translatable mRNA
(extended deletions). The effect of post-translational quality control
mechanisms must be subject of further studies.
Commercial Relationships: Markus N. Preising, None; Nayyir
Alavi, None; Birgit Lorenz, Optos (F)
Program Number: 1331 Poster Board Number: A0025
Presentation Time: 8:30 AM - 10:15 AM
Genetic and clinical features of FEVR and Norrie disease
Eisuke Arai1, 2, Takuro Fujimaki1, Ai Miyazaki1, Keiko Fujiki1,
Fumino Iwata3, Takenori Inomata1, Hiroyuki Kawano1, Toshiyuki
Yokoyama4, Akihisa Okumura5, Akira Murakami1. 1Ophthalmology,
Juntendo University School of Medicine, Tokyo, Japan;
2
Ophthalmology, The Tokyo Metropolitan Children’s Medical
Center, Tokyo, Japan; 3Iwata Ophthalmology Clinic, Tokyo, Japan;
4
Ophthalmology, Juntendo University Nerima Hospital, Tokyo,
Japan; 5Pediatrics and Adolescent Medicine, Juntendo University
School of Medicine, Tokyo, Japan.
Purpose: Analysis of the FZD4, NDP, LRP5, and TSAPN12 genes
was conducted in patients with suspected familial exudative
vitreoretinopathy (FEVR) or Norrie disease based on their clinical
findings.
Methods: Nine families (22 cases) with suspected FEVR and one
family (three cases) with suspected Norrie disease were studied.
Direct sequencing was performed by extracting genomic DNA from
the leukocytes of patients and amplifying each exons of four genes by
the PCR method. Multiple ligation-dependent probe amplification
(MLPA) was also performed in the patient with suspected FEVR and
the Norrie disease families for whom mutations were not found by
PCR analysis. To perform MLPA, two probes corresponding to each
exon of a target gene were hybridized and ligated together, followed
by amplification using the PCR method. Then the intensity of the
band for each probe was measured by electrophoresis and the copy
number was determined.
Results: Mutation of FZD4 was observed in four families (six
patients) with suspected FEVR and deletion of exon 2 of NDP was
observed in one family (three siblings) with suspected Norrie disease.
Conclusions: The present gene analysis identified pathogenic gene
mutations, in families with suspected FEVR and Norrie disease.
Although the direct sequencing method was useful for genetic
diagnosis, there were cases in which the detection of deletion was
difficult. In such cases, MLPA proved to be effective.
Commercial Relationships: Eisuke Arai, None; Takuro Fujimaki,
None; Ai Miyazaki, None; Keiko Fujiki, None; Fumino Iwata,
None; Takenori Inomata, None; Hiroyuki Kawano, None;
Toshiyuki Yokoyama, None; Akihisa Okumura, None; Akira
Murakami, SEED(Japan) JP4855782 (P), SEED(Japan) JP5132958
(P)
Program Number: 1332 Poster Board Number: A0026
Presentation Time: 8:30 AM - 10:15 AM
Presentation and Progression of the Ocular Manifestations of
Methylmalonic Acidemia in Children
Jacqueline K. Ng, Daniel J. Karr, Leah Reznick, Mark E. Pennesi.
Ophthalmology, Oregon Health & Science Univ, Portland, OR.
Purpose: To describe the spectrum of ocular findings of
methylmalonic acidemia (MMA) in children.
Methods: A review of patients was performed for a diagnosis of
cobalamin-A (cba1) and cobalamin-C (cbc1) type disease. We
identified seven cases of MMA (1 case of cba1 and 6 cases of cbc1
disease) and compiled clinical course, spectral-domain optical
coherence tomography (SD-OCT), and electroretinogram (ERG)
findings.
Results: Three patients with confirmed MMACHC gene mutations
had initial normal fundus exams. One case with heterozygous
c.271dupA and novel invariant splicing donor site mutation presented
at 8 months and developed optic atrophy and Bull’s eye maculopathy
consistent with SD-OCT by 4 years old. ERG showed mildly
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
decreased rod and cone amplitudes. One case with homozygous
615C>G (p.Y205X) mutation presented at 3 months and progressed
to severe macular atrophy at 1 year confirmed on SD-OCT. The third
patient with homozygous c.271dupA mutation remained stable. Two
additional patients were siblings homozygous for MMACHC gene
c.271_272dupA mutation. The older one presented at 3 years with
optic atrophy, attenuated vessels, peripheral pigment dusting that
progressed to bony spicules, and prominent Bull’s eye maculopathy
that progressed to macular atrophy. ERG showed severely subnormal
rod and cone responses. The younger one was diagnosed and started
on treatment prenatally but presented at 7 months with a mild Bull’s
eye maculopathy. The final patient presented with Bull’s eye
maculopathy at 6 months that progressed to macular atrophy with
peripheral pigment changes by 2 years. Initial ERG showed
prolongation of the cone-rod implicit times but normal amplitudes
that was stable at 2 years. The cba1 case showed initial mild
peripapillary pigment epithelial mottling that progressed over 21
years to mild optic atrophy in both eyes with associated mild
decreased ERG responses but intact vision.
Conclusions: This series suggests a natural history of initial normal
fundus exams progressing to early optic atrophy, Bull’s eye
maculopathy, and late findings of peripheral pigmentary changes in
MMA. SD-OCT and ERG may be useful before onset of clinical
findings. Systemic treatment did not halt progression, but early
prenatal treatment may limit the severity of disease.
Commercial Relationships: Jacqueline K. Ng, None; Daniel J.
Karr, None; Leah Reznick, None; Mark E. Pennesi, Pfizer (F)
Support: Foundation Fighting Blindness
Program Number: 1333 Poster Board Number: A0027
Presentation Time: 8:30 AM - 10:15 AM
Genotype-Phenotype correlations in patients with Basal Laminar
Drusen and Systemic Associations
Suman Pilli1, Martin McKibbin2, Clare Bailey3, Sheila Pinto4, Jesus
Garcia-Fernandez5, Helen L. Griffiths6, Santiago Rodriguez de
Cordoba7, Andrew J. Lotery8. 1Southampton Eye Unit, Southampton,
United Kingdom; 2St James's University Hospital, Leeds, United
Kingdom; 3Bristol Eye Hospital, Bristol, United Kingdom; 4Centro
de Investigaciones Biologicas, Madrid, Spain; 5Centro de
Investigaciones Biologicas, Madrid, Spain; 6University of
Southampton, Southampton, United Kingdom; 7Centro de
Investigaciones Biologicas, Madrid, Spain; 8University of
Southampton, Southampton, United Kingdom.
Purpose: To evaluate the genotypic features of patients with basal
laminar drusen (BLD) in association with mesangiocapillary
glomerulonephritis (MGN type II) or systemic lupus erythematosus
(SLE).
Methods: Five probands with BLD were studied; of which 4 patients
had MGN type II, while 1 patient had SLE. Three forms of genetic
analysis were performed. These were DNA sequencing of CFH,
analysis of age-related macular degeneration (AMD) associated
single nucleotide polymorphisms (SNPs) in CFH, CFB ARMS2 and
CFHR1 genes, and high-resolution comparative genomic
hybridization (CGH) arrays for the CFH-CFHR1 region for three of
the samples.
Results: A variety of SNPs were identified in CFH. Val62Ile,
Gln672Gln, Glu936Asp and Intron 12 (rs6677604) were observed in
all patients; while, Asn1050Tyr was noted in two patients (MGN and
SLE patient each). Three 402His alleles in a total of 8 chromosomes
(37.5% frequency) were observed in the MGN patients. These
variants were predicted to provide a range of risk for AMD, ranging
from very low (for the SLE patient) to high in the MGN patients. One
patient with MGN type II also had a homozygous deletion at the
CFHR3-CFHR1 locus.
Conclusions: BLD is a distinct drusen phenotype seen in young
patients with MGN type II or SLE. It is associated with genetic
variation within CFH and at the CFH locus. MGN, but not SLE, has a
significant risk of progressing to AMD in later life.
Commercial Relationships: Suman Pilli, None; Martin McKibbin,
None; Clare Bailey, None; Sheila Pinto, None; Jesus GarciaFernandez, None; Helen L. Griffiths, None; Santiago Rodriguez
de Cordoba, Alexion (C); Andrew J. Lotery, Novartis (F), Bayer
(R)
Support: Spanish Ministerio de Economia y Competitividad
(SAF2011-26583) and Comunidad de Madrid (S2010/BMD-2316)
Program Number: 1334 Poster Board Number: A0028
Presentation Time: 8:30 AM - 10:15 AM
Novel mutations of the RS1 gene in a cohort of patients with
retinoschisis
Yang Li, Jieqiong Chen, Yanfan Ren, Xiaohui Zhang, Zhe Pan.
Beijing Inst of Ophthalmology, Beijing Tongren Hospital, Beijing,
China.
Purpose: X-linked retinoschisis (XLRS) is a retinal dystrophy caused
by mutations in the RS1gene in Xp22.1, which leads to schisis
(splitting) of the neural retina leading to reduced visual acuity in
affected men (OMIM #312700). The aim of this study was to identify
the RS1 gene mutations in a small cohort of Chinese patients with Xlinked retinoschisis, and to describe the associated phenotypes.
Methods: Detailed ophthalmic examinations were given to the
patients and unaffected individuals from the nine unrelated families.
After informed consent was obtained, genomic DNA was extracted
from the venous blood of all participants. All exons including the
splicing region of the RS1 gene was amplified by polymerase chain
reaction (PCR). The PCR products were analyzed using direct
sequencing. Long-range PCR followed by DNA sequencing was used
to define the breakpoints of the large deletion.
Results: By clinical examination, ten male individuals from nine
families were diagnosed of retinoschisis. The median age at review
was 12.4 years (range from 5-34 years); the median best-corrected
visual acuity upon review was 0.22 (range 0.02-0.5). Typical foveal
schisis was found in 83.3% eyes examined (15/18) while
peripheralschisis was present in 33.3% of eyes (6/18). Six novel
mutations including: two nonsense c.370C>T(p.Q124X) and
c.98G>A(p.W33X), one missense c.581T>A(p.I194N), one small
deletionc.330delT (p.C110fs) , and one splicing mutation c.113G>T,
and one large deletion, and three recurrent missens mutations were
identified.
Conclusions: The mutations found in our study broaden the spectrum
of RS1 mutations. The identification of each pedigree’s specific
mutation allows future determination of female carrier status for
genetic consulting purposes.
Commercial Relationships: Yang Li, None; Jieqiong Chen, None;
Yanfan Ren, None; Xiaohui Zhang, None; Zhe Pan, None
Program Number: 1335 Poster Board Number: A0029
Presentation Time: 8:30 AM - 10:15 AM
A knock- in mouse model for recessive RP-foveoschisis-optic disc
drusen and nanophthamos syndrome due to a mutation in the
Mfrp gene
Bhubanananda Sahu1, Venkata R. Chavali1, John Suk1, Rachel
Poleman1, Akhila Alapati1, Bruno Maranhao1, 2, Monica M.
Jablonski3, Dirk-Uwe G. Bartsch1, Radha Ayyagari1. 1Shiley Eye
Center, University of California, San Diego, San Diego, CA;
2
Bioengineering, University of California, San Diego, San Diego,
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
CA; 3Hamilton Eye Institute, University of Tennessee Health Science
Center, Memphis, TN.
Purpose: To characterize the phenotype of a mouse model for
retinitis pigmentosa(RP)-foveoschisis-optic disc drusen and
nanonophthalmos syndrome due to a single base pair insertion
(c498_499insC) in exon 5 of Membrane-type frizzled related protein
(MFRP) gene.
Methods: The Mfrp homozygous c498_499insC mutation knock-in
(KI) mice were generated on a C57BL/6 background. The ocular
phenotype was characterized by fundus imaging, optical coherence
tomography (OCT), fundus autofluorescence and measuring axial
length. Retinal morphology was evaluated by microscopy.
Expression of selected marker genes was measured by RT-PCR and
marker proteins were studied by immunohistochemistry and western
blot analysis.
Results: The Mfrp c498_499InsC mutation KI mice developed RPE
atrophy with, progressive loss of photoreceptors beginning before
post-natal day 21. Both retinal histology and OCT revealed a
significant decrease in the thickness of the retina by 1 month. The
outer nuclear layer (ONL) has about 8 rows of nuclei at 21 days, 2 to
3 rows at two months and just a single row of nuclei by 12 months.
Presence of hyperautofluorescent spots throughout the retina was
observed starting at age one month. The focal length to the retinal
nerve fiber layer (RNFL), a surrogate measure for the axial length of
the eye, showed the eyes of Mfrp homozygous KI mice to be
significantly smaller compared to age matched wild-type mice
(P<0.0001). Expression of Mfrp was noted to be significantly
depleted.
Conclusions: Mice carrying the Mfrp c498_499InsC mutation in the
homozygous state developed retinal degeneration and
microphthalmos mimicking the human phenotype in patients with the
same MFRP mutation.
Commercial Relationships: Bhubanananda Sahu, None; Venkata
R. Chavali, None; John Suk, None; Rachel Poleman, None; Akhila
Alapati, None; Bruno Maranhao, None; Monica M. Jablonski,
8,092,825 (P); Dirk-Uwe G. Bartsch, None; Radha Ayyagari, None
Support: Foundation Fighting Blindness, Research to Prevent
Blindness, NIH-EY13198, NIH-EY21237, P30-EY22589
Program Number: 1336 Poster Board Number: A0030
Presentation Time: 8:30 AM - 10:15 AM
Screening for Usher Syndrome in an established cochlear implant
program: the merits of a collaborative paradigm
Elise Heon1, Ajoy Vincent1, Joanne E. Sutherland1, Megan Day1,
Blake C. Papsin2, Sharon L. Cushing2. 1Ophthalmology & Vision
Sciences, The Hospital for Sick Children, Toronto, ON, Canada;
2
Otolaryngology Head and Neck Surgery, Archie’s Cochlear Implant
Laboratory, The Hospital for Sick Children, Toronto, ON, Canada.
Purpose: To evaluate the role of systematic ophthalmic assessment
in children with sensori-neural hearing loss (SNHL) and peripheral
vestibular impairment.
Methods: Twenty-three children with profound SNHL and bilateral
vestibular dysfunction of unknown etiology were referred for an
ophthalmic assessment to evaluate their risk for Usher syndrome. All
children had received cochlear implants.
Bilateral vestibular impairment was confirmed by clinical exam,
abnormal vestibular end-organ testing (caloric, rotational chair and
vestibular evoked myogenic potential testing) and poor static and
dynamic balance was identified using the balance subset of the
Bruininks-Oseretsky test of motor proficiency. Ophthalmic
assessment included a comprehensive eye exam and
electroretinography (ERG). Genetic testing and counseling were
provided when appropriate.
Results: The average age at ophthalmic examination was 12.8 years
(7-19 years). Over 75% had a refractive error warranting correction;
only one had hypermetropia. Anisometropic amblyopia was seen in 2
cases. Half of the patients had an abnormal ERG suggestive of a rod
cone dystrophy/retinitis pigmentosa (RP). Only a third of these cases
had symptoms of decreased night vision while 80% showed some
visual field constriction. Mutations of the MYO7A and CDH23 genes
supported a diagnosis of Usher Syndrome type 1 in these patients.
Conclusions: The high prevalence of refractive errors (80%) and that
of retinal dystrophies (50%) was higher than previously published in
congenital SNHL. Children with SNHL with a vestibular deficit of
unknown etiology should receive an ocular exam including ERG.
This can contribute to the identification of the molecular cause and
allows optimal genetic counseling.
Commercial Relationships: Elise Heon, None; Ajoy Vincent,
None; Joanne E. Sutherland, None; Megan Day, None; Blake C.
Papsin, None; Sharon L. Cushing, None
Support: The Mira Godard Research Fund
Program Number: 1337 Poster Board Number: A0031
Presentation Time: 8:30 AM - 10:15 AM
Tietz syndrome (albinism and congenital deafness): Description
of a familiar case carrying a novel MITF gene mutation and
associated with nanophthalmos
Luz V. Cortés1, Martin Guzman-Sanchez1, Dalia C. Guadarrama2,
Juan C. Zenteno2, Cristina Villanueva-Mendoza1. 1Department of
Genetics, Asociación para Evitar la Ceguera en México I.A.P.
Hospital "Dr. Luis Sánchez Bulnes", Mexico, Mexico; 2Research
Unit and Department of Genetics, Institute of Ophthalmology "Conde
de Valenciana", Mexico, Mexico.
Purpose: To report a familial case of Tietz syndrome (TS) caused by
a novel MITF mutation and nanophthalmos.
Methods: Affected patients underwent a complete ocular and
systemic examination. In addition, B scan echography was
performed. Molecular studies included PCR amplification and
automated DNA sequencing of the complete MITF coding sequence.
Results: The propositus is an 11 years old boy with severe congenital
deafness and cutaneous, hair and ocular hypopigmentation. No
nystagmus, iris transillumination, or foveal hypoplasia were seen.
Refraction was +11= -7.00 x 175° (OD) and +12=-6.50x0° (OS). His
mother has a similar phenotype with deafness, hypopigmentation and
high hyperopia and his brother has hypopigmentation but not
deafness. A B scan echography showed an anterior posterior axial
length of 19.47mm for OD and 19.70mm for OS. MITF gene analysis
disclosed a novel c.216A>C heterozygous mutation in exon 8.
Conclusions: TS is an autosomal dominant condition characterized
by albinoid-like hypopigmentation of the skin and hair and severe
hearing loss. The disorder is caused by mutations in MITF, a gene
encoding a basic helix-loop-helix leucine zipper transcription factor,
which regulates melanocyte development and the biosynthetic
melanin pathway. TS is a rare entity that should be differentiated
from oculocutaneous albinism. In addition to the typical
abnormalities of the syndrome, nanophthalmos was observed in the
case described here. Interestingly, Mitf mutation in mice causes
microphthalmia. Our results expand both the clinical and molecular
spectrum of TS.
Commercial Relationships: Luz V. Cortés, None; Martin
Guzman-Sanchez, None; Dalia C. Guadarrama, None; Juan C.
Zenteno, None; Cristina Villanueva-Mendoza, None
Program Number: 1338 Poster Board Number: A0032
Presentation Time: 8:30 AM - 10:15 AM
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
A novel COL2A1 nonsense mutation causes Stickler syndrome
type I
Mohamed M. Khafagy1, 2, Daniel F. Schorderet3, 4, Hana Abouzeid5.
1
Ophthalmology Department, Kasr-Alainy Faculty of medicine Cairo University, Cairo, Egypt; 2Pediatric Ophthalmology Unit,
Abou-Elreish Pediatric Hospital - Cairo University Hospitals, Cairo,
Egypt; 3IRO - Institut de Recherche en ophtalmologie, University of
Lausanne, Sion, Switzerland; 4EPFL - Ecole polytechnique fédérale
de Lausanne, Lausanne, Switzerland; 5Jules-Gonin Eye Hospital,
University of Lausanne, Lausanne, Switzerland.
Purpose: To investigate the clinical features and perform a genetic
analysis of an Egyptian family with dominant rhegmatogenous retinal
detachment and high myopia.
Methods: Five members of an Egyptian family with autosomal
dominant rhegmatogenous retinal detachment and high myopia were
examined clinically. Peripheral blood samples were collected and
genomic DNA was extracted from blood leukocytes using standard
techniques.All 54 exons, promoter and intron-exon junctions of
COL2A1 gene were PCR amplified, and both forward and reverse
strands were sequenced with the Sanger sequencing technique.
Results: Features of Stickler syndrome type I (arthroophthalmopathy) were recognized in three members of the family
with a dominant inheritance pattern. All the affected patients (the 38year-old father, 7-year-old son, and 5-year-old daughter) had bilateral
high myopia with membranous vitreous liquefaction, micrognathia
and flattened facial appearance. Two of them, the father and 7-yearold son suffered from bilateral rhegmatogenous retinal detachment.
The father had retinal detachment surgery done in the left eye by the
second decade of life. His right eye passed into atrophia due to
neglected long-standing retinal detachment. The 7-year-old son had
corrective surgery for a cleft soft palate and claw feet done during the
first year of his life. By the age of 6, he needed surgery for bilateral
retinal detachment. The unaffected mother and the unaffected 1.5year-old son had a normal general and ophthalmic examination. We
identified a novel heterozygous C to T substitution in exon 24 at
position 1558 of the cDNA sequence of COL2A1 (Ensembl:
ENST00000380518). This [c. 1558C>T] causes a premature stop
codon to replace Glutamine amino acid (Q) at position 520 [p.
Q520X], potentially truncating 967 amino acids from the protein.
This mutation was found in all three affected members of the family
and was not in the two unaffected members nor in 46 Egyptian and
96 Caucasian controls.
Conclusions: We identified a novel heterozygous nonsense mutation
[p.Q520X] of the COL2A1 gene segregating in an Egyptian family
with Stickler syndrome. To our knowledge, this mutation has not
been previously reported.
Commercial Relationships: Mohamed M. Khafagy, None; Daniel
F. Schorderet, None; Hana Abouzeid, None
Program Number: 1339 Poster Board Number: A0033
Presentation Time: 8:30 AM - 10:15 AM
Ocular Manifestations and Genotype-Phenotype Correlations in
a group of 12 patients with the Marfan Syndrome
Marta Latasiewicz1, Elena Milla1, Christian Fontecilla1, Aurora
Sanchez2. 1Ophthalmology, Hospital Clinic de Barcelona, Barcelona,
Spain; 2Biochemistry and Molecular Genetics, Hospital Clinic de
Barcelona, Barcelona, Spain.
Purpose: The aim of this study was to analyze ocular involvement in
patients diagnosed with the Marfan syndrome, study their clinical
course and prognosis based on the type of FBN1 mutation, and
possibly indicate a genotype-phenotype correlation.
Methods: Observational study of 12 patients, (8 male and 4 female,
median age 35, range 6 to 65) diagnosed with the Marfan syndrome.
The patients included in the study all met the Ghent criteria and in 9
cases the diagnosis was confirmed with genetic testing. In all patients
a complete ophthalmologic examination was performed. We
evaluated the clinical data, the incidence of ectopia lentis and other
eye disorders. We analyzed the association of ocular signs with the
type of mutation.
Results: Of the 12 patients 11 (91.67%) had myopia, four (33.3%)
had ectopia lentis, of which three developed secondary glaucoma
(25%). Other ocular abnormalities found were strabismus (16.67%),
retinal tears (8.33%), retinal detachment (8.33%), congenital cataract
(8.33%), and amblyopia (8.33%).
We encountered three classes of mutations in our patients: the most
frequent comprised mutations resulting in a premature termination
codon (PTC, nonsense mutations) found in 7 patients (c.7180C>T in
3 patients, c.3795T>A in 2 patients, c.5493C>G in 1 patient, and c.
5863delC in 1 patient), one patient with a missense mutation
(c.504C> G), and one patient with aberration of splicing (c.1468 +5
G>A). Of patients with ectopia lentis one (1 eye) had a nonsense and
one (2 eyes) a missense mutation.
Conclusions: Myopia was the most frequent ocular involvement,
affecting almost all patients.
In our group PTC mutations of the FBN1 gene revealed to have a
smaller risk of ectopia lentis, as observed in previous reports. Within
the missense mutations ones affecting calcium binding sites and
cysteine substitutions are probably associated with a more severe
ocular involvement. The prevalence and severity of ocular
involvement in relation with the type of FBN1 mutation, and
especially the broad spectrum of ocular manifestations in patients
with the same mutation is still poorly understood. Therefore more
studies are required to explain the genotype-phenotype correlation,
which could serve in the direction of appropriate medical treatment
and monitoring.
Commercial Relationships: Marta Latasiewicz, None; Elena
Milla, None; Christian Fontecilla, None; Aurora Sanchez, None
Program Number: 1340 Poster Board Number: A0034
Presentation Time: 8:30 AM - 10:15 AM
Ocular development and axial length in the Bestrophinopathies
Julie De Zaeytijd1, Tine Sabbe1, Elfride De Baere2, Bart P. Leroy1, 2.
1
Dept of Ophthalmology, Ghent University Hospital, Ghent,
Belgium; 2Ctr for Medical Genetics, Ghent University Hospital,
Ghent, Belgium.
Purpose: To describe the relationship between the retinal pigment
epithelium (RPE) and ocular development in the bestrophinopathies.
The bestrophinopathies are a group of three diseases in which the
pathogenic defect is located at the level of the RPE: Best vitelliform
macular dystrophy (BVMD), autosomal dominant
vitreoretinochoroidopathy (ADVIRC) and autosomal recessive
bestrophinopathy (ARB).
Methods: Thirty patients with BVMD, 15 patients with ADVIRC
and 8 patients with ARB, with proven BEST1 mutations, underwent a
detailed ophthalmological examination, including measurement of
axial length and corneal diameter. In addition, psychophysical and
electrophysiological testing (ISCEV-standard ERG, PERG and EOG)
was performed in the majority of cases.
Results: With a mean value of 21.8mm, the axial length is
significantly shorter in both BVMD and ARB, than the normal mean
value of 23.6mm. In ADVIRC patients, ocular length showed a mean
value of 23.4mm, a difference that was not statistically significant. In
ADVIRC, the corneal diameter is reduced with a mean value of
9.7mm. In BVMD and ARB with a mean value of respectively
10.7mm and 11.2mm, microcornea is less pronounced. Visual field
defects were limited to (peri)central defects in addition to enlarged
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
blind spots in all ADVIRC patients, and (peri)central sensitivity loss
in ARB patients. Full-field flash ERGs were normal in 10/10 eyes
with BVMD, abnormal in 24/30 eyes in ADVIRC and 12/16 eyes in
ARB. A Lp/Dt-ratio < 150% on EOG was seen in 48/60 eyes with
BVMD, and all patients with ADVIRC and ARB.
Conclusions: These results show that ocular development is
abnormal in the bestrophinopathies and thus suggest that either the
presence of an abnormal bestrophin protein, or the complete absence
of it in the RPE, influences ocular development. The normal axial
length in ADVIRC is rather unexpected since the disease has been
associated with ocular developmental problems. In BVMD and ARB,
the axial length is significantly shortened. Microcornea is seen in all
three conditions, but is more pronounced in ADVIRC.
Commercial Relationships: Julie De Zaeytijd, None; Tine Sabbe,
None; Elfride De Baere, None; Bart P. Leroy, None
Program Number: 1341 Poster Board Number: A0035
Presentation Time: 8:30 AM - 10:15 AM
Comparison of human and murine ocular findings in the
Knobloch syndrome caused by mutations in COL18A1
Behrad Y. Milani1, Stephen H. Tsang2, Irene H. Maumenee1.
1
Ophthalmology, Univ of Illinois at Chicago, Chicago, IL; 2Columbia
Univ-Harkness Eye Inst, Columbia Coll phys Surg, newyork, NY.
Purpose: To compare the ocular findings of men and mice with the
Knobloch syndrome to advance understanding of the pathogenesis of
low vision and retinal detachments.
Methods: A 17 year old patient was diagnosed with the Knobloch
syndrome at age 5 months because of congenital nystagmus, poor fix
and following, high myopia of -24D and aplasia of the macular area
as well as a congenital scalp defect. His VA remained stable at
20/200 OU until he developed cataracts requiring surgery at age 15y.
He developed a peripheral retinal hole for which he was successfully
treated with laser therapy. He underwent a complete eye evaluation
including biometry and OCT. Three animals homozygous for a
col18a1 knock-out model mouse model underwent OCT and
fluorescein angiography.
Results: Corneal biometry gave K readings of 39.9 + 3.3D axis 160
and 32.8+11.2D axis 109; pachymetry of the right eye was 578μm
and of the left eye 529μm; OCT scans of the patient showed
tessellated fundus and marked thinning of the retina of the posterior
pole with absence of the foveal depression, apparent lack of
photoreceptors, and an eccentric staphyloma. DNA analysis showed a
two base pair deletion leading to a homozygous frameshift mutation
in exon 6 of COL18A1. OCT and FA findings in a knock-out mouse
model of col18a1 (Olsen and col.) showed apparent absence of
photoreceptors, attenuated retinal and irregular choroidal vasculature.
Conclusions: The Knobloch syndrome is classified as heritable
disorders of connective tissue and combines ocular, CNS and skeletal
findings. It is a complex autosomal recessive syndrome, involving
intrauterine developmental anomalies of the brain
(meningoencephaloceles) and the macula (ateliotic macula) as well as
acquired anomalies of the eye and the skeleton. Ocular complications
consist of irregular astigmatism, juvenile cataracts and retinal
detachments. The patients have a Stickler like facial appearance.
OCT of a patient with a homozygous frameshift mutation showed a
thinned retina with apparent absence of photoreceptors. OCT in the
knock-out mouse showed comparable findings, making this a valid
animal model.
Commercial Relationships: Behrad Y. Milani, None; Stephen H.
Tsang, None; Irene H. Maumenee, None
Support: Illinois society for the preventing of blindness research
grant
Program Number: 1342 Poster Board Number: A0036
Presentation Time: 8:30 AM - 10:15 AM
Jalili syndrome: retinal dystrophy and amelogenesis imperfecta:
genotype-phenotype analysis in four new cases
Christina Gerth-Kahlert1, Britta Seebauer2, 3, Sandra Dold2,
Johannes Fleischhauer1, Hubertus van Waes4, Wolfgang Berger2, 5.
1
Ophthalmology, University of Zurich, Zurich, Switzerland; 2Institute
of Medical Molecular Genetics, University of Zurich, Zurich,
Switzerland; 3Neuroscience Center Zurich, University of Zurich,
Zurich, Switzerland; 4Center of Dental Medicine, University of
Zurich, Zurich, Switzerland; 5Zurich Center for Integrative Human
Physiology, University of Zurich, Zurich, Switzerland.
Purpose: Jalili syndrome is a rare autosomal-recessive disorder,
which was first described in 1988 and recently linked to mutations in
the CNNM4 gene. Two phenotypes are proposed: associated with
bull’s eye maculopathy and peripheral retinal degeneration (type A)
or with minor retinal dystrophy (type B). (Jalili IK, Smith JD. J Med
Gen 1988 Jalili IK. Eye 2010)
Methods: Patients who meet the criteria of Jalili syndrome received a
comprehensive eye exam and a detailed retinal function (fullfield
electroretinogram- ERG) and morphology (optical coherence
tomography-OCT) assessment. A detailed dental examination was
performed. Genetic testing was performed by Sanger DNA
sequencing of all 7 exons (protein coding parts and flanking splice
sites) of the CNNM4 gene. Sequence data were aligned to the
CNNM4 reference sequence from ENSEMBL.
Results: Two unrelated sibling pairs ages 4.5 to 15 years, all
originating from families in Kosovo, were identified. Visual acuity
deteriorated over age and was severely reduced in all 4 patients to
20/200 or less at the last exam. Nystagmus was present in 3/4
patients. Fundus exam revealed mild to severe bull’s eye
maculopathy in 3/4 and diffuse retinal dystrophy in 1/4 patients. ERG
showed normal scotopic responses but non-recordable cone responses
in all patients. OCT revealed outer retinal abnormalities. All four
patients show severe dental abnormalities as described in
amelogenesis imperfecta. All four patients showed the same
homozygous mutation in the CNNM4 gene.
Conclusions: Patients with Jalili Syndrome show a progressive
retinal dystrophy with predominantly cone dysfunction. Retinal
changes can vary from mild maculopathy to diffuse retinopathy.
Retinal phenotype variability can occur within the same family.
Dental abnormalities seem to be more uniform among the patient
affected. The proposed strict differentiation between type A and B
might not be applicable to all patients affected.
Commercial Relationships: Christina Gerth-Kahlert, None;
Britta Seebauer, None; Sandra Dold, None; Johannes
Fleischhauer, None; Hubertus van Waes, None; Wolfgang Berger,
None
Program Number: 1343 Poster Board Number: A0037
Presentation Time: 8:30 AM - 10:15 AM
The RICO mouse - a novel model of dominant uveal coloboma
Ramakrishna Alur1, Felix I. Onojafe1, Amalia Dutra5, James
Thomas6, Marcus Fruttiger4, William D. Richardson3, Lorenzo
Nichols1, Peter F. Hitchcock2, Brian P. Brooks1, Sandra Pieke-Dahl2.
1
Ocular Genetics, NEI / NIH, Bethesda, MD; 2Ophthalmology and
Visual Sciences, University of Michigan, Ann Arbor, MI; 3Cell and
Developmental Biology, Wolfson Institute for Biomedical
Research,University College London, London, United Kingdom;
4
Cell Biology, UCL Institute of Ophthalmology, London, United
Kingdom; 5GENETIC DISEASE RESEARCH BRANCH,
NHGRI/NIH, Bethesda, MD; 6NIH INTRAMURAL SEQUENCING
CENTER, NHGRI/NIH, Rockville, MD.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
Purpose: Uveal coloboma is a potentially blinding congenital ocular
malformation caused by failure of the optic fissure to close during the
5th week of human gestation and at around 11.5 days in the mouse.
Mutations in the developmentally-regulated genes have been
described in some patients; however, the genetic cause of coloboma
(if any) in many individuals has remained elusive. Here we describe
the initial genetic and phenotypic characterization of the RICO
(Retinal & Iris COloboma) mouse, a novel model of dominant uveal
coloboma.
Methods: RICO was inadvertently created by the transgenic insertion
of a human VEGF (hVEGF) expression vector. The insertion site was
localized with FISH and commercially available BAC clones for
mouse chromosome 13. Embryos and mice were genotyped using a
combination of FISH and PCR and were phenotyped grossly and
histopathologically. The insertion site was evaluated using a
combination of comparative genomic hybridization (CGH), BAC
library construction, shot gun sequencing of two BAC clones and
‘Whole-genome shotgun sequencing’ of RICO genomic DNA.
Results: The uveal coloboma in C57BL/6J-RICO mice was generally
bilateral, affecting the iris, retina/choroid, and optic nerve.
Transmission was autosomal dominant with nearly 100% penetrance.
RICO mice breed well, do not express hVEGF, and display no
apparent systemic abnormalities. CGH array of heterozygote RICO
DNA shows no large duplications or deletions on Chr.13. BAC
library construction resulted in two BAC clones containing the VEGF
transgene (15Kb and 65Kb). Shotgun sequencing of BAC clones
showed several contigs with Chr 13 sequence from two different
regions (4Mb apart) suggesting a genomic rearrangement. ‘Wholegenome shotgun sequencing’ of RICO DNA suggested multiple
copies of transgene insertion but identified the insertion in a wellconserved, noncoding site on chromosome 13.
Conclusions: Insertion of NSE-hVEGF transgene on mouse
chromosome 13C results in a dominant uveal coloboma in C57BL/6J
mice with homozygous lethality, reminiscent of human disease. The
phenotype is not related to the expression of transgene. From the
BAC library, shotgun sequencing of BAC clones and ‘Wholegenome shotgun sequencing’ of RICO genomic DNA, we propose a
model for the genetic insertion/re-arrangement responsible for the
phenotype. Further, analysis of this genomic region will be carried
out to elucidate the key elements causing this disease in RICO mouse
model.
Commercial Relationships: Ramakrishna Alur, None; Felix I.
Onojafe, None; Amalia Dutra, None; James Thomas, None;
Marcus Fruttiger, AstraZeneca (F), Novartis (F), Novartis (C),
Amakem (F); William D. Richardson, None; Lorenzo Nichols,
None; Peter F. Hitchcock, None; Brian P. Brooks, None; Sandra
Pieke-Dahl, None
Support: NIH Intramural research support
Program Number: 1344 Poster Board Number: A0038
Presentation Time: 8:30 AM - 10:15 AM
Variable Phenotype & Retinal Abnormalities in Ectopia Lentis
Et Pupillae
Bart P. Leroy1, Cathérine Boileau2, Nadine Hanna2. 1Dept
Ophthalmology & Ctr Med Genetics, Ghent Univ Hosp & Ghent
Univ, Ghent, Belgium; 2Lab de Biochimie, Toxicologie,
Hormonologie et Génétique Moléculaire, Hôpital Ambroise Paré,
Boulogne, France.
Purpose: To describe the clinical and molecular findings in affected
members of a Flemish Belgian family with ectopia lentis et pupillae
due to biallelic mutations in ADAMTSL4.
Methods: Three patients with ectopia lentis et pupillae and two
obligate heterozygotes (parents of proband) from three generations
from one family underwent aan extensive ophthalmological
examination.
Results: The 25-year old male proband (patient 1) was referred with
unilateral disease but showed extremely asymmetrical signs in both
eyes. BCVA was 20/20 in the RE, and LP with localization in the LE.
Slit-lamp examination showed an abnormal, corrugated inferior lens
border and an abnormally low number and abnormal insertion of the
inferior zonular fibers in RE, and correctopia, extreme superior
displacement of a cataractous lens with very few zonular remnants in
the LE. Gonioscopy revealed bilateral angle recession bridged by iris
processes inferiorly. Fundoscopy revealed outer retinal abnormalities,
with partial hyperautofluorescence in the macula of the RE, and
transretinal macular abnormalities in the LE. OCT confirmed loss of
PRs and RPE in the RE, and abnormalities at all retinal levels in the
left macula. The ERG was normal in BE.
The 75-year old maternal grandmother of the proband (patient 2) and
her 73-year old sister (patient 3) showed bilateral intravitreal lens
luxation. The grandmother did not show pupillary ectopia, whereas
her sister showed mildly upwardly displaced pupils, more
pronounced after dilatation. Fundoscopy of the RE of patient 2 was
normal in the RE, whereas foveal aplasia was evident in the LE.
Macular ectopia was seen in BE of patient 3.
No ocular abnormalities were observed in the two obligate
heterozygotes.
Molecular analysis showed segregation of two ADAMTSL4
mutations in the family, confirming autosomal recessive inheritance:
c.767_786del and c.1656delG.
Conclusions: The phenotype of ectopia lentis et pupillae due to
biallelic ADAMTSL4 mutations is variable. Presumed unilateral
disease should trigger an extensive ocular examination. This is the
first report of retinal abnormalities other than retinal detachments.
Commercial Relationships: Bart P. Leroy, None; Cathérine
Boileau, None; Nadine Hanna, None
Support: Senior Clinical Investigator Grant, Fund for Research
Flanders
Program Number: 1345 Poster Board Number: A0039
Presentation Time: 8:30 AM - 10:15 AM
Inositol 5-Phosphatases in Primary Cilia Formation in Lowe
syndrome
Yang Sun1, Akhilesh Kumar1, Michael Conwell1, Jingyun Wang1,
Robert N. Weinreb2, Na Luo1. 1Department of Ophthalmology, Glick
Eye Institute Indiana University, Indianapolis, IN; 2Ophthalmology,
University of California, San Diego, San Diego, CA.
Purpose: Inositol phosphatases are important regulators of cell
signaling, polarity, and vesicular trafficking. Mutations in OCRL, an
inositol polyphosphate 5-phosphatase, result in Oculocerebrorenal
syndrome of Lowe—an X-linked recessive disorder that presents
with congenital cataracts, glaucoma, renal dysfunction and mental
retardation[1]. The function of OCRL in causing cataracts and
glaucoma is not understood.
Methods: Using human eyes previous enucleated for uveal
melanoma, we examined the distribution of OCRL and INPP5B in
trabecular meshwork, lens epithelium, and ciliary body epithelium.
We also examined the subcellular distribution of INPP5B by
immunofluorescence in HTM and RPE cells. Using zebrafish
embryos, antisense morpholinos against INPP5B and OCRL were
used to examine the function of these 5-phosphatases in cilia
formation.
Results: We have showed that endogenous OCRL is localized in the
trabecular meshwork and Schlemm’s canal endothelial cells in human
and murine eyes. We also showed that INPP5B, a paralog of OCRL,
is expressed in low levels in human trabecular meshwork but is
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
highly expressed in murine trabecular meshwork. We show that the
knockdown of INPP5B resulted in a decrease in primary cilia
formation using INPP5B specific shRNA in hTERT-RPE1 cell line.
In a zebrafish model, INPP5B morpholino knockdown morphants
presented a dose-dependent phenotype of microphthalmia, body axis
asymmetry and kinked tail, which is a phenotype caused by Kupffer’s
vesicle defect and also found in OCRL morphants. The decreased
cilia in Kupffer vesicles of INPP5B morphants are shorter than
control morphants, and cilia-dependent melanosome transport of
INPP5B morphants delayed much longer when embryos exposed to
epinephine. All these cilia related phenotypes could be rescued by coinjected with mouse INPP5B mRNA.
Conclusions: OCRL and INPP5B are differentially expressed in the
human and murine eyes, which may explain the ocular phenotypes of
Lowe syndrome.
1. Luo N, West CC, Murga-Zamalloa CA, Sun L, Anderson RM, et
al. (2012) OCRL localizes to the primary cilium: a new role for cilia
in Lowe syndrome. Human molecular genetics.
Fig 1. Expression of OCRL and INPP5B in trabecular meshwork in
human eyes.
Fig 2. Generation of zebrafish morphants of INPP5B. A) Knockdown
of INPP5B in zebrafish. B) microphthalmia and anophthalmia of
INPP5B zebrafish morphants.
Commercial Relationships: Yang Sun, NIH (F), American
Glaucoma Society (R), Knights Templar Eye Foundation (R), Reeves
Foundation (R); Akhilesh Kumar, None; Michael Conwell, None;
Jingyun Wang, Fight for Sight (F), IUPUI EMPOWER (F); Robert
N. Weinreb, Aerie (F), Alcon (C), Allergan (C), Altheos (C),
Amakem (C), Bausch&Lomb (C), Carl Zeiss-Meditec (C), Genentech
(F), Haag-Streit (F), Heidelberg Engineering (F), Konan (F), Lumenis
(F), National Eye Institute (F), Nidek (F), Optovue (C), Quark (C),
Solx (C), Topcon (C); Na Luo, None
Support: NIH Grant EY-K08022058, American Glaucoma Society,
Knight Templar Eye Foundation
Program Number: 1346 Poster Board Number: A0040
Presentation Time: 8:30 AM - 10:15 AM
Evidence of the role of ADAMTS18 in ocular development
Aman Chandra1, 2, Gavin Arno1, Panagiotis I. Sergouniotis1,
Arundhati Dev Borman1, Andrew R. Webster1, 2, Anthony T. Moore1,
2 1
. Cell Biology, UCL Institute of Ophthalmology, London, United
Kingdom; 2Moorfields Eye Hospital, London, United Kingdom.
Purpose: To investigate the molecular pathology of two subjects
with atypical Knobloch syndrome (KNO) and to describe the detailed
ocular phenotype.
Methods: Detailed phenotype data was collected from affected
members of one consanguineous Pakistani family with a rare
developmental ocular abnormality. Autozygosity mapping and
candidate gene sequencing was undertaken.
Results: Two brothers were identified as having with congenital lens
opacities. One sibling developed axial myopia and had
electrophysiological evidence of a cone rod dystrophy. The other had
ectopia lentis, unilateral posterior Staphyloma with contralateral
hypermetropia. He subsequently developed bilateral rhegmatogenous
retinal detachments. No skull defects were present. Genotyping with
Affymetrix SNP6 array© was performed and analysis highlighted
40mB of autozygous DNA with a 6.9mB region in 16q22.2-23.2, a
region which included ADAMTS18. Bidirectional sequencing of this
gene revealed a novel missense mutation (c.536C>T (p.Ser179Leu)
predicted to be pathogenic (SIFT, PolyPhen, Mutation Taster).
This is the second report of mutations in ADAMTS18 causing a
developmental eye phenotype confirming the role of this gene in
Knoblock syndrome.
Conclusions: We report the second ADAMTS18 mutation causing
human disease, thus confirming the importance of its role and
broadening the understanding of this family of proteins in ocular
development.
Commercial Relationships: Aman Chandra, None; Gavin Arno,
None; Panagiotis I. Sergouniotis, None; Arundhati Dev Borman,
None; Andrew R. Webster, None; Anthony T. Moore, None
Support: Fight for Sight UK
Program Number: 1347 Poster Board Number: A0041
Presentation Time: 8:30 AM - 10:15 AM
Allelic Heterogeneity Contributes to Variability in Ocular
Dysgenesis, Myopathy, and Brain Malformations Caused by
Col4a1 and Col4a2 Mutations
Debbie S. Kuo1, Cassandre Labelle-Dumais1, Mao Mao1, Marion
Jeanne1, William B. Kauffman1, Jennifer Allen1, Jack Favor3,
Douglas B. Gould1, 2. 1Ophthalmology, University of California, San
Francisco, San Francisco, CA; 2Anatomy and Institute for Human
Genetics, University of California, San Francisco, San Francisco,
CA; 3Institute of Human Genetics, Helmholtz Zentrum München,
Neuherberg, Germany.
Purpose: Collagen type IV alpha 1, COL4A1, and its binding partner
COL4A2 are present in nearly all basement membranes. COL4A1 and
COL4A2 mutations are pleiotropic and associated with a broad
spectrum of disorders including ocular dysgenesis and glaucoma.
Recently we reported COL4A1 mutations in two patients with
Muscle-Eye-Brain disease (MEB) and Walker-Warburg Syndrome
(WWS) and demonstrated that Col4a1 mutant mice have ocular
dysgenesis, myopathy and brain malformations modeling
MEB/WWS. We showed that mutations in COL4A1 can affect the
biosynthesis of COL4A1/COL4A2 heterotrimers, typically resulting
in intracellular retention and impaired secretion of mutant protein. In
this study, we investigate the contribution of allelic differences to the
phenotypic variability associated with Col4a1 and Col4a2 mutations
using an allelic series of mice.
Methods: We crossed eight distinct Col4a1 mutations and one
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
Col4a2 mutation to C57BL/6J mice to generate an allelic series on a
uniform genetic background. Clinical examination of the anterior
segment and histological analyses of optic nerve, muscle and brain
sections were performed to characterize ocular dysgenesis, myopathy
and brain malformations. The effects of mutations on
COL4A1/COL4A2 heterotrimer biosynthesis were evaluated using
immunofluorescent labeling and Western blot analyses on primary
mouse embryonic fibroblasts derived from mutant mouse lines.
Results: We identified allele-specific effects on the expression and
severity of ocular dysgenesis, myopathy and brain malformations.
We also found allelic differences in the levels of intracellular and
extracellular COL4A1 and COL4A2. While most mutations
demonstrated increased intracellular and decreased extracellular
levels of COL4A1 and COL4A2, one mutation showed no significant
difference in intracellular or extracellular levels of COL4A1 and
COL4A2 compared to controls and one mutation showed both
decreased intracellular and extracellular levels of COL4A1 and
COL4A2.
Conclusions: Col4a1 and Col4a2 mutations can have distinct
molecular consequences and lead to heterogeneous ocular, cerebral
and myopathic phenotypes of variable severity. Allelic differences
may reflect mechanistic heterogeneity and could provide valuable
insight toward developing prognostic tools and targeted therapeutics
for patients with COL4A1 and COL4A2 mutations.
Commercial Relationships: Debbie S. Kuo, None; Cassandre
Labelle-Dumais, None; Mao Mao, None; Marion Jeanne, None;
William B. Kauffman, None; Jennifer Allen, None; Jack Favor,
None; Douglas B. Gould, None
Support: The following funding sources contributed to this work:
National Institutes of Health (EY019887), Muscular Dystrophy
Association, Research to Prevent Blindness (all to DBG), Knights
Templar Eye Foundation (MM), Howard Hughes Medical Institute
(DSK), National Institutes of Health (core grant EY02162) and
Research to Prevent Blindness (unrestricted grant) (both to UCSF
Department of Ophthalmology).
Program Number: 1348 Poster Board Number: A0042
Presentation Time: 8:30 AM - 10:15 AM
MIR184 c.57C>T mutation is responsible for congenital cataracts
and corneal abnormalities in a five-generation family from
Galicia, Spain
Yelena Bykhovskaya1, 2, Kenneth W. Wright3, Yaron S. Rabinowitz1, 4,
Ana Laura C. Canedo1. 1Cornea Genetic Eye Institute, Beverly Hills,
CA; 2Regenerative Medicine Institute, Cedars-Sinai Medical Center,
Los Angeles, CA; 3Wright Center for Pediatric Ophthalmology and
Strabismus, Los Angeles, CA; 4The Jules Stein Eye Institute,
University of California Los Angeles, Los Angeles, CA.
Purpose: To identify genetic defect (s) in members of the fivegeneration family originated in Galicia, Spain with early onset
cataracts and variable corneal abnormalities which include nonectatic corneal thinning and severe early-onset keratoconus.
Methods: Family history was collected from the parents of a 9 year
old boy with progressive loss of vision in both eyes due to congenital
posterior cataracts and keratoconus. After it has identified additional
affected family members thus suggesting a potential genetic nature of
the boy’s condition, proband’s parents and his maternal and paternal
grandmothers underwent a complete ophthalmological evaluation
which included slit-lamp bioicroscope evaluation, retinoscopy,
videokeratography, and OCT pachymetry measurements. We PCR
amplified stem-loop region of MIR184 gene in the proband, his
parents, and maternal grandmother. Amplified DNA was separated
by agarose gel electrophoresis and correctly sized PCR product was
sequenced using Sanger DNA sequencing. Sequence was confirmed
by both forward and reverse sequencing.
Results: We identified a heterozygous c.57C>T mutation in the stem
loop of micro RNA-184 gene in the proband affected with bilateral
congenital posterior cataract and keratoconus, his mother with
bilateral cataracts and extremely thin corneas, and maternal
grandmother (Figure). This is a same mutation that was earlier
identified in both Northern Irish family with autosomal dominant
keratoconus with early-onset anterior polar cataract and in family
with autosomal dominant EDICT syndrome. Based on close genetic
relationship between current inhabitants of Iberian Peninsula,
especially Galicia, and Ireland, we suggest a single ancestral point of
origin of this highly deleterious mutation in the family described in
this study, Northern Irish family, and possibly EDICT family.
Conclusions: In this study we present only third known family with
the c.57C>T MIRN184 mutation and variable abnormalities of the
cornea and lens. We hypothesize that considerable differences in the
clinical presentation should be attributed to the additional genetic
modifier (s). We are working on establishing of an iPS cell model of
MIRN184 defect in the proband and his mother which may shed
some light on such modifier (s).
Commercial Relationships: Yelena Bykhovskaya, None; Kenneth
W. Wright, None; Yaron S. Rabinowitz, None; Ana Laura C.
Canedo, None
Support: NEI Grant 09052
Program Number: 1349 Poster Board Number: A0043
Presentation Time: 8:30 AM - 10:15 AM
Congenital Cataract Locus in a Seven Generation Family
Sarah J. Garnai1, Jeroen R. Huyghe2, David M. Reed1, Kathleen M.
Scott1, Michael Boehnke2, Julia E. Richards1, 3, Robert Ritch4, 5,
Hemant S. Pawar1. 1Ophthalmology and Visual Sciences, University
of Michigan, Ann Arbor, MI; 2Biostatistics, University of Michigan,
Ann Arbor, MI; 3Epidemiology, University of Michigan, Ann Arbor,
MI; 4Einhorn Clinical Research Center, New York Eye and Ear
Infirmary, New York, NY; 5Department of Ophthalmology, New
York Medical College, Valhalla, NY.
Purpose: Congenital cataract (CC) appears in the first year of life
and can be a simple ocular trait or be part of a multi-system disorder.
Hereditary cataracts account for up to 25% of congenital cataracts,
and multiple different modes of inheritance have been observed. The
purpose of this study was to map a CC locus in a 7 generation
Ashkenazi Jewish family and evaluate candidate genes in the region.
The family exhibits additional phenotypes including microphthalmia,
glaucoma, CC+glaucoma, CC+microphthalmia, and there is evidence
of consanguinity.
Methods: This IRB-approved study obtained informed consent from
16 members in three generations of the CC family. Participants were
examined at the New York Eye and Ear Infirmary. A genome-wide
SNP-based linkage analysis was performed using Illumina's Human
Omni-Quad BeadChip and whole exome sequencing (Illumina)
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
provided information on a subset of the family. Assuming complete
penetrance, the power to detect linkage was determined by simulation
using FastSLINK. Multipoint parametric linkage analysis was
performed using MERLIN on a subset of ~10,000 inheritanceinformative SNPs with intermarker distances of ~250 kb.
Results: Multipoint parametric linkage analysis was carried out
assuming both autosomal dominant and recessive modes of
inheritance. The autosomal recessive model indicated significant
linkage on chromosome 22 from 16.918 to 22.437 Mb (Chr
22q11.21) with a maximum LOD score of 3.61.
Conclusions: This CC locus maps to chromosome 22 with
statistically defined boundaries excluding CRYBB2 and CRYBB3, but
haplotyping will be needed to tell whether hard recombination break
points exclude them. The coding sequence of these known CC genes
showed no missense or nonsense mutations, but one silent mutation
(not near a splice site) raises the possibility of alternative splicing.
The sequence of additional genes inside of the interval will also need
to be examined.
Commercial Relationships: Sarah J. Garnai, None; Jeroen R.
Huyghe, None; David M. Reed, None; Kathleen M. Scott, None;
Michael Boehnke, None; Julia E. Richards, None; Robert Ritch,
None; Hemant S. Pawar, None
Support: NIH-R01-EY011671, NIH-P30-EY007003 (core),
Research to Prevent Blindness
Program Number: 1350 Poster Board Number: A0044
Presentation Time: 8:30 AM - 10:15 AM
Crystal deposits in the anterior lens cortex in Bietti crystalline
corneoretinal dystrophy and first report of chroidal neovascular
membrane in patient affected with Bietti
Veronika Vaclavik1, 2, Francis L. Munier1, 3, Daniel F. Schorderet1, 3,
Viet H. Tran1. 1unité oculogénétique, Hosp Ophtalmique Jules Gonin
Lausanne, Lausanne, Switzerland; 2Ophtalmology department, HUG,
Geneva, Switzerland; 3Institut de recherche en Ophtalmology, IRO,
Sion, Switzerland.
Purpose: To report a previously unknown finding of glistening
crystal-like deposits on the
anterior lens cortex in 4 patients from 3 different kindred suffering
of Bietti crystalline corneoretinal dystrophy (BCD). To describe a
first case of CNV with good response to ranibizumab intravitreal
injections
Methods: Four consecutive patients presenting with limbal corneal
and intraretinal crystals
were enrolled. One of patient developed a CNV, with
metamorphopsias sudden onset of reduced vision
Results: Mean patient age was 60.0 years (range, 40-81 years). Mean
best Snellen visual
acuity was 0.5 (range, light perception-1.0). All patients, except one,
had
homozygous or compound heterozygous mutations in CYP4V2 gene.
Slit lamp
biomicroscopy showed multiple crystal-like deposits in the anterior
lens cortex
and as well on the anterior lens capsule. Those deposits were
highlighted on
Scheimpflug camera. Fundus examination showed different stages of
severe
chorioretinal atrophy.
The patient with CNV responded to intravitreal injections of
Lucentis, improved her visual acuity
Conclusions: Crystal-like deposits might appear in advanced disease,
although their exact
etiology remains yet to be determined.
To the best of our knowledge, this is the first time that crystal-like
deposits are
described on the lens cortex of patients with BCD associated with a
mutation in
the CYP4V2 gene. This is also a first report of a choroidal
neovascular membrane complicating BCD, with favourable response
to intravitreal injections of Lucentis
Commercial Relationships: Veronika Vaclavik, None; Francis L.
Munier, None; Daniel F. Schorderet, None; Viet H. Tran, None
Program Number: 1351 Poster Board Number: A0045
Presentation Time: 8:30 AM - 10:15 AM
Molecular characterization in Mexican patients with anterior
segment dysgenesis including primary congenital glaucoma,
aniridia, Peters anomaly and Axenfeld-Rieger anomaly and
syndrome
Cristina Villanueva-Mendoza1, Nancy Hernández-Martínez2, Miguel
A. Alcántara-Ortigoza2, Omar Honerlage-Ceniceros1, Ariadna
González-Del Angel2, Rehotbevely B. Barrientos-Ríos2, Luz M.
González-Huerta3. 1Genetics, Asociación Para Evitar La Ceguera En
México, México, Mexico; 2Molecular Biology, Instituto Nacional de
Pediatría, Mexico, Mexico; 3Genetics, Hospital General de México,
SS, Mexico, Mexico.
Purpose: The anterior segment dysgenesis (ASD) are a group of
developmental abnormalities that share some common features and
have a high prevalence of associated glaucoma. ASD include
abnormalities of the cornea, angle and iris such as corectopia,
polycoria, iris hypoplasia, corneal leucoma and posterior
embryotoxon. The purpose of this study was to evaluate CYP1B1,
PAX6 and FOXC1 gene mutations in Mexican patients with primary
congenital glaucoma, aniridia, Peters anomaly and Axenfeld-Rieger
anomaly and syndrome.
Methods: A complete systemic and ophthalmologic evaluation for
phenotypic characterization was done. DNA was collected from
patients and parents. Mutations were detected by single-strand
conformation polymorphism (SSCP) and direct sequencing.
Results: There were 9 patients with aniridia, 8 with primary
congenital glaucoma, 3 with Peters anomaly, 6 with Axenfeld-Rieger
anomaly or syndrome. Also we had 8 patients with overlapping
phenotypes most of them with congenital glaucoma. We found three
pathological mutations in PAX6 in patients with aniridia. We didn't
find mutations in CYP1B1 in patients with primary congenital
glaucoma. At this moment we have not found any pathological
mutation in FOXC1.
Conclusions: Heterozygous PAX6 mutations are found in 40-80% in
patients with aniridia. We found pathological mutations in 30% of
aniridia patients. The frequency of CYP1B1 mutations in primary
congenital glaucoma varies among populations, it is probably that in
Mexican patients it is a rare cause of the pathology as we didn't find
any pathological mutation, including two familiar cases. A complete
assessment of FOXC1 gene (and in a near future PITX2 gene) is
needed in patients with Axenfeld-Rieger anomaly and syndrome in
order to detect the causal mutation. This analysis may help to
improve the characterization of this group of abnormalities from a
molecular and clinical standpoint.
Commercial Relationships: Cristina Villanueva-Mendoza, None;
Nancy Hernández-Martínez, None; Miguel A. AlcántaraOrtigoza, None; Omar Honerlage-Ceniceros, None; Ariadna
González-Del Angel, None; Rehotbevely B. Barrientos-Ríos,
None; Luz M. González-Huerta, None
Program Number: 1352 Poster Board Number: A0046
Presentation Time: 8:30 AM - 10:15 AM
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
Functional characterization of a homozygous nonsense FOXE3
mutation that causes Peters anomaly in a consanguineous family
Arif O. Khan1, Shahid Y. Khan2, Zhiwei Ma3, Saleh Al-Mesfer1,
Shahira Al Turkmani1, Sheikh Riazuddin4, Walter Stark2, James F.
Hejtmancik3, John D. Gottsch2, S. Amer Riazuddin2. 1Division of
Pediatric Ophthalmology, King Khaled Eye Specialist Hospital,
Riyadh, Saudi Arabia; 2The Wilmer Eye Institute, Johns Hopkins
University School of Medicine, Baltimore, MD; 3Ophthalmic
Genetics and Visual Function Branch, National Eye Institute,
Bethesda, MD; 4National Centre of Excellence in Molecular Biology,
University of the Punjab, Lahore, Pakistan.
Purpose: Peters anomaly is a congenital anterior segment dysgenesis
characterized by central corneal opacity, posterior corneal defect, and
irido-lenticular adhesions. As part of our ongoing investigation of the
genetics of Peters anomaly, we report an underlying recessive
mutation in an affected consanguineous family and its functional
characterization.
Methods: A consanguineous family with familial Peters anomaly
(four affected individuals) was ascertained. Blood samples were
collected and genomic DNA was extracted from white blood cells
using a non-organic method. A genome-wide linkage scan was
completed to localize the disease phenotype and two-point LOD
scores were calculated. The causative mutation was identified by bidirectional Sanger sequencing of candidate genes present in the
linkage interval. A standard immunofluorescence staining protocol
was used to localize the mutant protein in HeLa cells.
Results: Affected individuals had clinical Peters anomaly (bilateral
corneal opacities, irido-lenticular adhesions) and developmental
glaucoma. Genome-wide linkage analysis identified an informative
homozygous region on chromosome 1p with significant two-point
LOD scores. Bi-directional Sanger sequencing of candidate genes on
chromosome 1p identified a homozygous substitution (c.720C>A;
p.C240X) present in all four affected individuals. The 11 unaffected
individuals were heterozygous carriers (nine) or were homozygous
for the wild type allele (two). This variant is predicted to prematurely
truncate the FOXE3 protein and was absent in 192 ethnically
matched control chromosomes. Immunofluorescence tracking
confirmed nuclear localization of the mutant protein similar to that of
the wild type FOXE3 protein.
Conclusions: Our data strongly suggest that homozygous nonsense
mutation in FOXE3 causes Peters anomaly and subsequent in vivo
analyses confirm that the mutation does not affect the localization of
FOXE3 protein to the nucleus. We speculate that premature
termination of FOXE3 compromises its ability to regulate genes
critical for ocular developmental leading to the disease phenotype.
Commercial Relationships: Arif O. Khan, None; Shahid Y. Khan,
None; Zhiwei Ma, None; Saleh Al-Mesfer, None; Shahira Al
Turkmani, None; Sheikh Riazuddin, None; Walter Stark,
VueCare (C); James F. Hejtmancik, None; John D. Gottsch, None;
S. Amer Riazuddin, None
Support: This study was supported in part by the KKESH-JHU
collaborative agreement (AOK & SAR) and the Kwok Research
Fund (JDG)
Program Number: 1353 Poster Board Number: A0047
Presentation Time: 8:30 AM - 10:15 AM
Mutant LTBP-2 proteins lack secretion ability and fibrillin-1
binding activity
Tomoya O. Akama1, 2, Yusuke Fujikawa1, Tadashi Inoue1, Tomoyuki
Nakamura1. 1Pharmacology, Kansai Medical University, Moriguchi,
Japan; 2Tumor Microenvironment, Sanford-Burnham Med Res Inst,
La Jolla, CA.
Purpose: Latent TGFβ-binding protein 2 (LTBP-2) is an
extracellular matrix protein that belongs to LTBP family proteins.
Homozygous mutations on LTBP2, which encodes LTBP-2, have
been found on several congenital ocular diseases such as primary
congenital glaucoma, Weill-Marchesani syndrome and congenital
megalocornea, indicating an important function of LTBP-2 on eye
development. Previous reports including ours pointed out direct
binding between LTBP-2 and fibrillin-1, an essential extracellular
matrix protein for microfibril formation, and suggested involvement
of LTBP-2 in microfibril organization. Thus we assumed that
mutations found on LTBP2 affect fibrillin-1 binding activity of
LTBP-2. In this work, we studied ability of mutant LTBP-2 to bind to
fibrillin-1 molecule.
Methods: We constructed expression vectors encoding mutant
LTBP-2s and produced the mutants in culture medium of transfected
HEK293T cells. These mutant proteins were individually reacted to
recombinant fibrillin-1 fragment and subjected to
immunoprecipitation to detect specific binding.
Results: By recombinant protein production and
immunoprecipitation, we identified that LTBP-2 binds to fibrillin-1
by the latter half of a large domain consists of tandem Ca2+ binding
EGF-like repeats. By searching literatures, we obtained four
pathogenic mutant LTBP-2s that possess intact fibrillin-1 binding
domain, and constructed expression vectors for the mutant proteins.
We then transfected each of the expression vectors into HEK293T
cells to produce recombinant LTBP-2 in culture medium, however,
all the mutant LTBP-2s were not recovered in the medium although
all the mutant proteins were detected in cell lysates, indicating the
mutant LTBP-2s were produced but not secreted. We further
analyzed binding activity of mutant LTBP-2s to fibrillin-1, by
recovering the mutant proteins from cell lysates, and found three out
of four mutant LTBP-2s did not bind to fibrillin-1.
Conclusions: We found that pathogenic LTBP-2 mutants were
secretion-deficient and inactive to fibrillin-1 binding. Because all the
tested mutants possess minimal mutations (single amino acid
replacement or alteration of C-terminal region), we assume these
mutations lead to conformational alteration and result in secretion
arrest as well as functional deficiency. These findings may link to
explain pathogenensis of ocular diseases caused by LTBP2 mutation.
Commercial Relationships: Tomoya O. Akama, None; Yusuke
Fujikawa, None; Tadashi Inoue, None; Tomoyuki Nakamura,
None
Support: NIH grant EY014620
Program Number: 1354 Poster Board Number: A0048
Presentation Time: 8:30 AM - 10:15 AM
Multiplex Ligation-dependent Probe Amplification (MLPA) for
detection of large deletions in CYP1B1 in congenital glaucoma
patients from the US
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
Keri Allen, Maria Janessian, Kevin Linkroum, Wael Abdrabou, Janey
L. Wiggs. Ophthalmology, Massachusetts Eye and Ear Infirmary,
Boston, MA.
Purpose: Mutations in CYP1B1 were first identified in
consanguineous Saudi Arabian and Turkish primary congenital
glaucoma (PCG) pedigrees. However, this gene accounts for only a
small percentage of PCG cases in ethnically heterogeneous
populations such as the United States. In a previous study conducted
at the Massachusetts Eye and Ear infirmary (MEEI), a collection of
50 U.S. congenital glaucoma families were screened for mutations in
CYP1B1 and only five probands were found to have causative
mutations for CYP1B1. Testing for copy number variation using
fragment analysis can significantly increase the detection rate of
many genetic disorders. The purpose of this study is to test for whole
gene, or whole exon deletions in CYP1B1 that are not detected by
Sanger sequencing methods using Multiplex Ligation-dependent
Probe amplification (MLPA) in MEEI congenital glaucoma families
who previously tested negative for mutations for CYP1B1.
Methods: Genomic DNA samples from 32 of the original MEEI
PCG probands were selected to be screened using MLPA. The MRC
Holland SALSA MLPA P128-B2 Cytochrome P-450 probemix kit
was used to assess copy number variation, specifically whole gene or
whole exon deletion of CYP1B1.
Results: MLPA analysis showed deletion of both exons in the
CYP1B1 gene in 1 out of the 32 (3%) probands in the MEEI
collection of congenital glaucoma families.
Conclusions: MLPA was able to detect a large deletion that includes
the entire coding sequence of CYP1B1 in 1/32 of MEEI PCG
probands who previously tested negative for a CYP1B1 mutation.
MLPA should be used to exclude large deletions of CYP1B1 as the
causative mutation in congenital glaucoma patients. Overall these
results are consistent with the findings that CYP1B1 mutations are
rare causes of congenital glaucoma in affected patients residing in the
U.S. These results also suggest that other genes, not yet discovered,
are responsible for the majority of congenital glaucoma cases in the
U.S.
Commercial Relationships: Keri Allen, None; Maria Janessian,
None; Kevin Linkroum, None; Wael Abdrabou, None; Janey L.
Wiggs, None
Support: Research to Prevent Blindness
Program Number: 1355 Poster Board Number: A0049
Presentation Time: 8:30 AM - 10:15 AM
Clinical features of OPA1-related optic neuropathy: a
retrospective case series
Eric Gaier1, 2, Phillip Skidd3, Maria Janessian4, 5, Simmons Lessell3, 6,
Dean Cestari3, 6, Joseph F. Rizzo3, 6, Janey L. Wiggs4, 5. 1School of
Medicine, University of Connecticut Health Center, Farmington, CT;
2
Neuroscience, University of Connecticut Health Center, Farmington,
CT; 3Neuro-Ophthalmology, Massachusetts Eye and Ear Infirmary,
Boston, MA; 4Glaucoma, Massachusetts Eye and Ear Infirmary,
Boston, MA; 5Howe Laboratory, Massachusetts Eye and Ear
Infirmary, Boston, MA; 6School of Medicine, Harvard Medical
School, Boston, MA.
Purpose: Dominant optic atrophy (DOA) is the most common
hereditary optic neuropathy. The most common form of DOA, DOA
type 1 (Kjer’s disease) accounts for 40-60% of cases and is
associated with mutation of the OPA1 gene. Previously, clinical
studies focusing on OPA1 mutations have been limited to a few
mutations and clinical parameters. In the present study, we evaluated
and analyzed detailed clinical information for patients referred to a
major tertiary care center for OPA1 testing for suspected DOA.
Methods: In this retrospective case series, we review the clinical
records of 62 patients referred for PCR-based sequencing of OPA1.
Missense and small deletions/insertions found by sequencing were
compared with those reported in the literature, and objective analyses
were used to determine the likelihood of pathogenicity for specific
DNA changes. A subset of patients was selected for Multiplex
Ligation-dependent Probe Amplification (MLPA) analysis. The
clinical features examined include, but are not limited to, visual
acuity, Ishihara testing, automated visual field testing, and dilated
fundoscopy.
Results: Clinical data was available for 54 patients (31 male; 23
female). The majority (28/41; 68.3%) were referred by an
optometrist/ophthalmologist for a problem related to optic nerve
appearance. Of the 62 patients that underwent diagnostic OPA1
sequencing, 18 (29.0%) were found to have potentially diseasecausing coding mutations: 3 splice site mutations, 3 missense
mutations, and 12 nonsense/truncating mutations. The common
TTAG deletion comprised 7 of the 12 nonsense mutations in 4
unrelated families, confirming previous reports of this mutation hot
spot. Three sequence mutations are novel. MLPA analysis identified
5 patients with large-scale deletions/duplications unrecognized by
sequencing. Deletion spans ranged from 3 exons to the entire gene.
Automated visual field testing was the most sensitive measure of
disease status as assessed in a subset of individuals followed over 1-5
years.
Conclusions: Our results show that OPA1 mutations are a common
cause of optic neuropathy in the tertiary referral setting and largescale genomic aberrations account for a small subset of OPA1-related
neuropathies. Automated visual field testing may be the best measure
of disease progression in these patients.
Commercial Relationships: Eric Gaier, None; Phillip Skidd,
None; Maria Janessian, None; Simmons Lessell, None; Dean
Cestari, None; Joseph F. Rizzo, Bionic Eye Technology (I), Bionic
Eye Technology (P); Janey L. Wiggs, None
Program Number: 1356 Poster Board Number: A0050
Presentation Time: 8:30 AM - 10:15 AM
New candidate genes for inherited optic atrophy
Cecile Delettre, Maxime Hebrard, Francois Halloy, agathe
roubertie, Christian P. Hamel, Guy Lenaers. INSERM, Montpellier,
France.
Purpose: Primary hereditary optic neuropathies comprise a group of
disorders that are characterized by visual loss due to retinal ganglion
cell death. The most common forms of optic neuropathy are Leber
hereditary optic neuropathy (LHON) with mitochondrial transmission
(OMIM 535000) and autosomal dominant optic atrophy (OMIM
165500). Autosomal recessive optic neuropathies are uncommon and
are mostly observed in association with multisystem diseases.
To identify novel candidate genes for inherited optic neuropathies we
have undertaken whole exome sequencing (WES) in patients with
syndromic or non syndromic optic atrophy without mutation in
OPA1, OPA3, and mitochondrial DNA.
Methods: Families with recessive inheritance are selected and passed
on 250k SNP chips. Homozygosity mapping was done using a
homemade software name TASE. We the performed Whole Exome
Sequencing on families.
Results: To prioritise putative causative variants, we filtred using the
1000 Genomes (http://www.1000genomes.org/) and NHLBI Exome
Sequencing Project (ESP: http//evsgs.washington.edu/EVS/) to
exclude known variants and focused on those predicted to alter
protein sequence. Potential candidates genes were confirmed by
sequencing and co-segregation with available family members
examined.
Using this approach disease causing mutations were found in 3
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
families within 3 different genes not previously implicated in optic
atrophy but within pathways relevant to mitochondrial function.
Conclusions: Combination of WES with traditional approaches,
consistent with linkage analysis, has a greatest impact to discover
new genes.
Commercial Relationships: Cecile Delettre, None; Maxime
Hebrard, None; Francois Halloy, None; agathe roubertie,
Genzyme (F); Christian P. Hamel, None; Guy Lenaers, None
Support: UNADEV, RETINA France
Program Number: 1357 Poster Board Number: A0051
Presentation Time: 8:30 AM - 10:15 AM
Pan-American MtDNA haplogroups in LHON patients
Pablo T. Romero1, 2, Mark A. Slabaugh2, Verónica Fernández1,
Nicolás Seleme1, Patricio Pezo1, Luisa M. Herrera1, Mauricio
Moraga1. 1Universidad de Chile, Santiago de Chile, Chile;
2
University of Washington, Seattle, WA.
Purpose: The aim of this research was to determine the clinical
spectrum and molecular characterization of fifteen South American
families with Leber’s Hereditary Optic Neuropathy (LHON)
Methods: This study was conducted between March 2006 and
August 2012. Fifteen index cases were identified during neuroophthalmic examination at different centers throughout Chile. All
patients were referred to the Clinical Hospital of the University of
Chile, where the clinical study was conducted. Molecular study was
conducted at ICBM (University of Chile). Clinical features of LHON
patients and maternal relatives of fifteen families (75 individuals: 26
affected and 49 healthy carriers) were evaluated. The primary
mtDNA mutations (G3460A/ND1, G11778A/ND4, or
T14484C/ND6) were determined with restriction fragment length
polymorphism analysis in all individuals. Mitochondrial haplotypes
were determined by direct sequencing of the regions HV1 and HV2
and compared with the reference sequence
Results: The G11778A/ND4 mutation was found in 59 subjects
(78.7%), the T14484C/ND6 was found in 12 (16.0%) and the
G3460A/ND1 mutation was found in 4 (5.3%) individuals. The
average age of onset of symptoms in affected subjects was 22.2 years
old (range 3 to 53 years); 21 (80.8%) were male and 5 (19.2%) were
female. Average time from symptom onset to presentation was 7.2
days (range 12 hours to 21 days). Time to involvement of the second
eye was 53.3 days on average. Twelve families had Amerindian
haplogroups: 1 family had A2 haplotype, 4 families had B2i2
haplotype, 6 families had C1b haplotype and 1 family had D1g
haplotype. One family had J2b haplotype, associated with European
populations, and two families had L1b haplotype, associated with
African populations. In five patients the disease onset was after 40
years of age. Subjects with A haplotype on average had a later onset
of disease (p=0.001, ANOVA) than those with other haplotypes (47.3
years on average) and subjects with haplotype C were more likely to
retain 20/200 or better vision in the better eye than those with other
haplotypes (p=0.009, ANOVA)
Conclusions: LHON mutations are necessary but not sufficient for
phenotypic expression of disease in patients with LHON mutations
and Amerindian haplotypes."A" haplotype may be associated with a
delayed onset of disease in this population. Patients with haplotype C
retained better vision than patients with other haplotypes in this
population
Commercial Relationships: Pablo T. Romero, None; Mark A.
Slabaugh, None; Verónica Fernández, None; Nicolás Seleme,
None; Patricio Pezo, None; Luisa M. Herrera, None; Mauricio
Moraga, None
Support: Do not use
Program Number: 1358 Poster Board Number: A0052
Presentation Time: 8:30 AM - 10:15 AM
Prenatal Molecular Diagnosis of Oculocutaneous Albinism
(OCA) in a Large Cohort of Israeli Families
Anat Blumenfeld1, Dalia Eli2, Idit Bejarano-Achache1, Efrat
Shemesh1, Irene I. Anteby1, Claudia Yahalom1, 2, Ada Rosenmann2.
1
Department of Ophthalmology, Hadassah—Hebrew University
Medical Center, Jerusalem, Israel; 2Michaelson Institute for
Rehabilitation of Low Vision, Hadassah—Hebrew University
Medical Center, Jerusalem, Israel.
Purpose: To present the prenatal molecular test results of OCA types
1, 2 and 4 caused by mutations in the tyrosinase (TYR), P and
SLC45A2 genes, respectively, in a large cohort of Israeli albino
families.
Methods: Clinical evaluation of subtypes of OCA included hair skin
and eye examination. Detailed genetic investigation included
pedigree analysis and ethnic origin of all 4 grandparents. Further
genetic counseling was performed after the completion of molecular
tests of the propositus and parents, prior to, and after each prenatal
test. Following prenatal molecular test genetic counseling included
prediction of the spectrum of expected phenotypes based on
diagnosed genotypes. Molecular prenatal tests were performed on
extracted DNA using the combination of PCR followed by direct
mutation screen, direct sequencing, and haplotype analysis.
Results: 77 prenatal tests were performed in 48 families; in 35 - the
propositus was the child and in 13 - a parent or a close relative. In 39
families TYR mutations were diagnosed, in 8 families P mutations,
and in one family a SLC45A2 mutation was identified. 19 albino
fetuses were diagnosed. Following further genetic counseling most
couples elected to terminate the pregnancy. Three couples (4
pregnancies) elected not to terminate the pregnancy of an albino fetus
due to expected variable phenotype of albinism. Several additional
pregnancies were terminated for other reasons. In 4 families the
propositus was a close relative of one parent and the other parent
carried the R402Q change in TYR. In most individuals R402Q is a
non-pathogenic polymorphism and compound heterozygotes (CH)
are normally pigmented, but some CH have various degrees of
albinism. One fetus was CH for R402Q / R402X and a normally
pigmented child was born. One couple in which the father was a
grandchild of an albino and carried a “severe” TYR mutation and the
mother carried R402Q elected not to perform prenatal test and an
albino girl CH for the “severe” TYR mutation and R402Q was born.
Conclusions: Families with increased risk for an albino child with
severe visual handicap seek prenatal genetic counseling and testing
for the prevention of affected offspring. Unless mild phenotype of
albinism is predicted, couples elect to terminate the pregnancy of an
albino fetus. Molecular genetic testing at our center enables a
nationwide approach for the prevention of a severe phenotype of
albinism.
Commercial Relationships: Anat Blumenfeld, None; Dalia Eli,
None; Idit Bejarano-Achache, None; Efrat Shemesh, None; Irene
I. Anteby, None; Claudia Yahalom, None; Ada Rosenmann, None
Program Number: 1359 Poster Board Number: A0053
Presentation Time: 8:30 AM - 10:15 AM
Molecular diagnostic testing by eyeGENE®: Analysis of patients
with hereditary maculopathy and/or Cone Rod Dystrophy
John Suk1, Akhila Alapati1, Kerry Goetz2, Santa J. Tumminia2, Radha
Ayyagari1. 1Shiley Eye Center, University of California, San Diego,
La Jolla, CA; 2National Eye Inst/NIH, Bethesda, MD.
Purpose: Analyze 214 probands with a diagnosis of hereditary
maculopathy and/or cone-Rod dystrophy (CRD) referred to
eyeGENE® for molecular diagnostic testing.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
Methods: Thirty nine patients with a clinical diagnosis of Best’s
macular degeneration (BMD), 26 with Doyne’s Honey Comb
dystrophy (DHRD), 9 with Sorsby’s fundus dystrophy (SFD), and 6
with late-onset retinal degeneration (LORD) were screened for
mutations in BEST1, EFEMP1, TIMP3, and CTRP5 genes
respectively. In addition, 74 patients with a diagnosis of pattern
dystrophy alone and 6 with both pattern dystrophy and BMD were
screened for mutation(s) in one or more of the following genes: RDS,
BEST1, ELOVL4 and ABCA4. Furthermore, 54 patients with a
diagnosis of CRD were screened for mutations in one or more of the
following: CRX, ABCA4, RDS, ELOVL4 genes and GUCY2D
codon 838. Mutation analysis was carried out by PCR and dideoxy
sequencing. Impact of novel variants was evaluated using PolyPhen.
Results: Of the 39 patients with BMD, 24 carry a known mutation
and 1 carries a variant of unknown significance (VUS) in BEST1. Of
the 26 patients with DHRD, 2 have a known mutation and one has a
novel VUS in EFEMP1. Among the 9 patients with SFD, 3 have a
known mutation in TIMP3. None with LORD carry causative
mutations in CTRP5. All 80 patients with pattern dystrophy were
screened for mutations in RDS, 10 were additionally screened for
ABCA4, 5 for ELOVL4, 1 for CTRP5, and 6 for BEST1. Twelve of
these patients carry a known heterozygous mutation, 1 has a
heterozygous VUS, and another has 2 known heterozygous mutations
in the RDS gene while one patient has a VUS in BEST1. Three
patients have known causative mutations in ABCA4. Of the 54 CRD
patients, 42 have recessive mutations and 12 have dominant CRD. Of
the 42 recessive CRD patients, 1 has a known GUCY2D codon 838
mutation in the homozygous state, and 17 have known mutations or
VUS in the homozygous or compound heterozygous state in ABCA4.
Of the 12 patients with dominant CRD, 5 have a known GUCY2D
codon 838 mutation. One of these five patients also has a VUS in
CRX.
Conclusions: Molecular diagnostic testing provided by eyeGENE®
for inherited maculopathies identified the underlying cause of disease
in ~33% of referred cases. These data indicate that the candidate gene
approach only has a limited ability for identifying the genetic basis
for maculopathies and/or CRD.
Commercial Relationships: John Suk, None; Akhila Alapati,
None; Kerry Goetz, National Eye Institute (E); Santa J. Tumminia,
None; Radha Ayyagari, None
Support: Foundation Fighting Blindness, Research to Prevent
Blindness, RO1- EY013198, RO1-EY021237, P30-EY22589.
Program Number: 1360 Poster Board Number: A0054
Presentation Time: 8:30 AM - 10:15 AM
VON HIPPEL LINDAU: 3q134X MUTATION FINDING
Juan Pablo Davila1, alejandra valladares1, Thamar Gomez-Villegas2,
Araceli Rojas-Diaz1. 1Hosp Fdtn Nuestra Senora De La Luz, Mexico,
Mexico; 2Instituto Nacional de Neurologia y Neurocirugia,
MEXICO, Mexico.
Purpose: To present a gene mutation in a family with a history of
Von Hippel Lindau (VHL) disease.
Methods: Herein we present a case of a sixteen year old male
asymptomatic. Family diseases: maternal grandmother deceased from
unspecified cancer, mother deceased from renal carcinoma,
pancreatic cysts and central nervous system hemangioblastomas.
Clinical findings were visual acuity in both eyes 1 logmar, unaltered
anterior segment. Right Eye fundus with a single superotemporary
hemangioblastoma (3 Disc Diameters), with a dilated feeding artery,
and a tortuous draining vein from and up to the optic nerve. Left Eye
fundus was normal.
Fluorescent angiography shows early leakage and marked
hyperfluorescence in the tortuous vessels and the site of the
hemangioblastoma, no macular edema was observed. Brain magnetic
resonance imaging was normal, abdominal contrast material
enhanced computerized tomography shows well defined cysts in the
head of pancreas, kidneys were normal.
Patient treated with 3 sessions of cryotherapy (freezing and thawing
technique) on bony vessels and YAG laser 532nm, five weeks after
cryotherapy in afferent and efferent vessels and the second 4 weeks
later. Results indicative of attenuation of bony vessels post-treatment
which constitutes an indicator of treatment success.
Results: Genetic study was performed on the mother prior to death,
to the patient and siblings (2 brothers and 1 sister), finding a mutation
3q134X for VHL in the mother, patient and one of his brothers. Eye,
brain and abdominal scanning was normal in his brother.
VHL disease results from a germ line mutation in the VHL gene
which is a tumor suppressor gene located on the short arm of
chromosome 3 (3p25-26). Discovered by Latif and colleagues in
1993, helped understanding of the molecular pathology of VHL
disease. This gene encodes the VHL protein, which is a tumor
suppressor protein.
In spite of these important advances in the understanding of VHL
disease, the overall extremely variable intra- and interfamilial
expressivity of the disease remains unclear.
Conclusions: Retinal hemangioblastoma is one of the most frequent
occurring tumors during the course of VHL disease and can be
responsible for significant visual impairment.
Gene mutation 3q134X found in this family members will help to
compare the common clinical presentation in patients with VHL
3p25-26.
Commercial Relationships: Juan Pablo Davila, None; alejandra
valladares, None; Thamar Gomez-Villegas, None; Araceli RojasDiaz, None
341 Exome Sequencing: New Genes, Methods and Databases
Tuesday, May 07, 2013 11:00 AM-12:45 PM
Exhibit Hall Poster Session
Program #/Board # Range: 3345-3383/C0001-C0039
Organizing Section: Genetics
Program Number: 3345 Poster Board Number: C0001
Presentation Time: 11:00 AM - 12:45 PM
Exome sequencing identifies RDH12 gene compound
heterozygous mutations in a Mexican inbred family with severe
autosomal recessive retinitis pigmentosa
Juan C. Zenteno, Oscar F. Chacon-Camacho, Beatriz Buentello,
Serguei Jitskii. Genetics, Institute of Ophthalmology, Mexico City,
Mexico.
Purpose: Retinitis pigmentosa (RP) is an inherited retinal dystrophy
caused by a progressive and irreversible loss of photoreceptors. RP
prevalence is approximately 1/4000 worldwide and it may be
transmitted in all inheritance patterns. To date, 36 genes have been
associated with autosomal recessive retinitis pigmentosa (arRP). Due
to arRP’s phenotypic and genetic heterogeneity, its molecular
diagnosis is highly complex and time-consuming. The aim of this
work was to identify the causative mutations in a Mexican inbred
family with arRP.
Methods: A total of 4 affected siblings were clinically evaluated.
Patients underwent full ophthalmologic examination including fundus
examination, ERG, FAG, and OCT. Molecular analysis was
performed in genomic DNA from affected subjects and a number of
non-affected relatives. A genome wide linkage analysis was
performed on DNA from 3 patients by means of an Affymetrix 250K
single-nucleotide polymorphism microarray. Exome sequencing of
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
one affected family member was done using the TrueSeq exome
enrichment kit, captured using Nimblegen SeqCap EZ V2.0 probes
kit, and paired-end sequenced on an Illumina Hiseq 2000 platform at
Ambry Genetics (Aliso Viejo, CA, USA). Dideoxy sequencing was
used for candidate gene variant confirmation.
Results: All affected patients exhibited dense intraretinal pigment
migration, severe retinal pigment epithelium atrophy, and arteriolar
attenuation, with a severe atrophic pigmentary maculopathy. A large
region of homozygosity was observed at chromosome 18q. The
TUBB6 gene was sequenced as a candidate gene but no mutations
were demonstrated. Exome sequencing analysis disclosed two
pathogenic mutations in the RDH12 gene: c.295C>A (p.L99I) and
c.446T>C (p.L149P). Sanger sequencing demonstrated segregation of
both RDH12 mutations in all affected subjects.
Conclusions: While homozygosity mapping is an effective tool for
identification of the underlying causative gene in inbred families,
compound heterozygosity may occur even within the same
consanguineous family. Whole exome sequencing is a powerful and
cost-effective tool for molecular diagnostics in families with inherited
retinal dystrophies.
Commercial Relationships: Juan C. Zenteno, None; Oscar F.
Chacon-Camacho, None; Beatriz Buentello, None; Serguei Jitskii,
None
Program Number: 3346 Poster Board Number: C0002
Presentation Time: 11:00 AM - 12:45 PM
Exome sequencing identifies mutations of both MYO7A and
PDE6B in three siblings with retinitis pigmentosa
Tamar Ben-Yosef1, Nitza Goldenberg-Cohen2, 3, Eyal Banin4, Ben
Cohen1, Yael Zalzstein5, Leah Rizel1, Lina Basel-Vanagaite5.
1
Genetics Dept - Faculty of Med, Technion, Haifa, Israel; 2Eye
Research Laboratory, FMRC, Tel-Aviv University, Petah Tikva,
Israel; 3Ophthalmology, Rabin Medical Center, Petah Tikva, Israel;
4
Ophthalmology, Hadassah- Hebrew University Medical Center,
Jerusalem, Israel; 5Raphael Recanati Genetic Institute, Rabin Medical
Center, Petah Tikva, Israel.
Purpose: Retinitis pigmentosa (RP), the most genetically
heterogeneous disorder in humans, actually represents a group of
pigmentary retinopathies characterized by night blindness followed
by visual-field loss. RP can appear as either syndromic or
nonsyndromic. One of the most common forms of syndromic RP is
Usher syndrome, characterized by the combination of RP and hearing
loss. The current work aimed to identify the underlying cause for the
appearance of both syndromic and nonsyndromic RP in three siblings
from a consanguineous Israeli Muslim Arab family.
Methods: Patients underwent a detailed ophthalmic examination,
including funduscopy, electroretinography (ERG), optical coherence
tomography (OCT), visual field and color vision testing. Affected
individuals were studied by whole-genome homozygosity mapping
followed by whole exome sequencing.
Results: The family was found to segregate novel mutations of two
different genes: MYO7A, causing type 1 Usher syndrome, and
PDE6B, causing nonsyndromic RP. One affected child was
homozygous for both mutations. Since the retinal phenotype seen in
this patient results from overlapping pathologies, one might expect to
find a very severe retinal degeneration. Indeed, he was diagnosed
with RP based on an abnormal ERG at a very young age (9 months).
However, this early diagnosis may be biased, as two of his older
siblings have already been diagnosed, leading to increased awareness.
At the age of 32 months he had relatively good vision with normal
visual fields.
Conclusions: Here we present a rare case of three siblings with the
same retinal phenotype (RP) and three different genotypes (MYO7A
deficiency, PDE6B deficiency or both). This report further exhibits
the genetic heterogeneity of RP, and demonstrates how consanguinity
could increase intrafamilial clustering of multiple hereditary diseases.
Moreover, it provides a unique opportunity to study the clinical
implications of co-existence of null mutations in two RP-causative
genes in a human patient.
Commercial Relationships: Tamar Ben-Yosef, None; Nitza
Goldenberg-Cohen, None; Eyal Banin, None; Ben Cohen, None;
Yael Zalzstein, None; Leah Rizel, None; Lina Basel-Vanagaite,
None
Support: grant 612/09 from the Legacy Heritage Bio-Medical
program of the Israel Science Foundation to T.B.
Program Number: 3347 Poster Board Number: C0003
Presentation Time: 11:00 AM - 12:45 PM
Exome analysis identified novel mutations in the FAM161A gene
in a family with recessive retinal degeneration
Jacque L. Duncan1, Pooja Biswas2, Igor Kozak2, Mili Navani2, Rafael
C. Caruso3, John R. Heckenlively4, Austin Roorda5, Radha Ayyagari2.
1
Ophthalmology, Univ of California - SF, San Francisco, CA; 2Shiley
Eye Center, University of California, San Diego, La Jolla, CA;
3
National Eye Institute, National Institutes of Health, Bethesda, MD;
4
Ophthalmology, University of Michigan, Ann Arbor, MI; 5School of
Optometry, University of California, Berkeley, Berkeley, CA.
Purpose: To describe the clinical phenotype and identify the
molecular basis of disease in an Indian family with autosomal
recessive retinal degeneration (RD).
Methods: Five family members were characterized with visual
acuity, perimetry, fundus photos, full-field electroretinography
(ERG), and optical coherence tomography. Cone photoreceptors
surrounding the fovea were imaged in one affected member, the
proband, using adaptive optics scanning laser ophthalmoscopy. The
exome was captured using Nimblegen SeqCap EZ V4.0 probes and
sequenced on llumina HiSeq. Read mapping and variant calling were
performed with published protocols. Exome variants were analyzed
using exomeSuite. Confirmation of variants and segregation analysis
were performed using dideoxy sequencing.
Results: Exome analysis detected 30,606 single nucleotide variants
(SNVs) and indels in the proband. Analysis of these variants using
exomeSuite identified 5 candidate SNVs. Further analysis revealed a
novel homozygous nonsense change, c.1105 C>T, p.Arg335Ter, and
a rare homozygous probably damaging (PolyPhen score=0.999)
change, c.1791 G>T, p.E597D (rs201052209) in the FAM161A gene
segregating with RD. These sequence variants were not detected in
100 ethnicity matched controls. Similar to prior reports of RD
associated with FAM161A mutations, affected family members were
myopic with visual acuity ranging from light perception to 20/40,
with progressive visual acuity and field loss beginning at different
ages. All affected members showed severely reduced ERG responses
and grayish spots extending from the arcades, while prominent bone
spicule pigmentary changes were observed only in the proband.
Residual cones with increased cone spacing were observed near the
fovea despite severely reduced visual acuity in the proband.
Conclusions: Exome analysis revealed two homozygous probably
damaging variants in the FAM161A gene segregating with
progressive RD. While the novel nonsense mutation alone could be
sufficient to cause RD, the effect of the second missense change is
not known. The severe progressive vision loss with a range of
symptom onset observed among 3 siblings perhaps is a consequence
of genetic or environmental modifiers. High-resolution retinal
imaging of the proband revealed sparse central cones, suggesting
FAM161A is important for normal photoreceptor structure and
survival.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
Commercial Relationships: Jacque L. Duncan, None; Pooja
Biswas, None; Igor Kozak, None; Mili Navani, None; Rafael C.
Caruso, None; John R. Heckenlively, None; Austin Roorda, US
Patent #6890076 (P), US Patent #7118216 (P), UC Berkeley (P);
Radha Ayyagari, None
Support: Foundation Fighting Blindness, Research to Prevent
Blindness, That Man May See, NIH Grants EY014375, R01EY013198, R01-EY021237, P30-EY22589, NEI Intramural Research
Program, NEI Core grant EY002162.
Program Number: 3348 Poster Board Number: C0004
Presentation Time: 11:00 AM - 12:45 PM
Whole Exome Sequencing as a Tool for Identification of Genes
Causing Autosomal Recessive Retinitis Pigmentosa
Dror Sharon1, Lina Zelinger1, Samer Khateb1, Avigail Beryozkin1,
Liliana Mizrahi-Meissonnier1, Samuel G. Jacobson2, Eyal Banin1.
1
Department of Ophthalmology, Hadassah-Hebrew Univ Medical
Ctr, Jerusalem, Israel; 2Department of Ophthalmology, Scheie Eye
Institute, Philadelphia, PA.
Purpose: Over 200 genes are known or suspected to cause inherited
retinal diseases, and the number is likely to rise within the coming
years. This is mainly due to next generation sequencing (NGS)
techniques and whole exome sequencing (WES) in particular. The
aim of this study is to use WES to identify the genetic cause of
autosomal recessive (AR) retinitis pigmentosa (RP) in 30 Israeli
index cases.
Methods: Patients with ARRP who agreed to participate in the study
were recruited at Hadassah medical center. Clinical data included
family history, ocular examination and imaging. Genomic DNA was
extracted from blood samples and analyzed using Affymetrix whole
genome single nucleotide polymorphism (SNP) microarrays and/or
WES (Otogenetics Corporation).
Results: We selected for this study a set of 30 index cases with
ARRP. The DNA sample of each patient was prescreened for all
mutations known to cause retinal degeneration in the appropriate
ethnic group. We obtained an average of about 50 million sequences
per sample and assembled them to the reference human genome
sequence. Each WES sample was initially analyzed to detect
mutations in known retinal degeneration genes. In seven samples we
have identified mutations, most of which are novel, in known RP
genes (RP1, CNGA1, CNGB1, CYP4V2, C2orf71, RDH12, and
ABCA4). In a nonconsanguineous family with three siblings affected
by nonsyndromic RP, we identified compound novel mutations in the
BBS2 gene known to cause Bardel-Biedl syndrome. In three
additional families we identified likely disease causing mutations that
are currently being evaluated. Interestingly, seven out of the 30 index
cases carried single heterozygous null mutations in known diseasecausing genes, that are unlikely to be the cause of disease but might
affect disease severity. The analysis of the remaining exome samples
is being performed mainly using homozygosity data obtained by
whole genome SNP arrays.
Conclusions: We present here the first comprehensive WES analysis
in Israeli and Palestinian patients with RP. Pre-screening for known
founder mutations and the availability of homozygosity mapping data
improve WES efficiency as a tool for identification of novel diseasecausing genes.
Commercial Relationships: Dror Sharon, None; Lina Zelinger,
None; Samer Khateb, None; Avigail Beryozkin, None; Liliana
Mizrahi-Meissonnier, None; Samuel G. Jacobson, None; Eyal
Banin, None
Support: Foundation Fighting Blindness USA grant (BR-GE-05100490-HUJ), the Yedidut 1 research grant, and the Macula Vision
Research Fund (MVRF).
Program Number: 3349 Poster Board Number: C0005
Presentation Time: 11:00 AM - 12:45 PM
Identification of causative mutations in consanguineous pedigrees
from Pakistan with recessive retinal degeneration by whole
exome analysis
Pooja Biswas1, Bruno Maranhao1, 2, Pauline Lee1, John Suk1, Mili
Navani1, Shahid Y. Khan3, Nadeem H. Butt4, Sheikh Riazuddin4, 5, S.
Amer Riazuddin3, Radha Ayyagari1. 1Shiley Eye Center, University
of California, San Diego, San Diego, CA; 2Bioengineering,
University of California, San Diego, San Diego, CA; 3The Wilmer
Eye Institute, Johns Hopkins University School of Medicine,
Baltimore, MD; 4Allama Iqbal Medical College, University of Health
Sciences, Lahore, Pakistan; 5National Centre of Excellence in
Molecular Biology, University of the Punjab Lahore, Lahore,
Pakistan.
Purpose: To describe the clinical phenotype and identify the
causative mutations in four unrelated consanguineous Pakistani
families with autosomal recessive retinal degeneration.
Methods: Clinical phenotype in each case was characterized by
standard ophthalmic evaluation, fundus photography,
electroretinography and optical coherence tomography. Exomes of
probands in each pedigree were captured using Nimblegen V4 kits
and sequencing was performed on illumina HiSeq. The read
mapping, variant calling were performed using published protocols.
Variants were analyzed using exomeSuite software to identify
candidate genes consistent with the predicted pattern of inheritance.
Segregation of candidate mutations in respective families and their
evaluation in unaffected Pakistani control subjects was investigated
by dideoxy sequencing.
Results: The four large inbred families (5-17 available members in
each pedigree) with multiple consanguineous marriages (1-3 per
family) and several affected members (3-13 per family) were
recruited from Pakistan. Affected members in all four pedigrees were
diagnosed with early onset severe non-syndromic retinal
degeneration. Analysis of sequence variants in probands of all four
pedigrees using exomeSuite and stringent filtering criteria detected 1
or 2 potential candidate genes. A novel variant, c.2384G>A;
p.Arg795Gln in GUCY2D that is predicted to be probably damaging
by PolyPhen was found in pedigree 1. Similar analysis of pedigree 2
revealed the presence of a novel possibly damaging variant
c.2189T>C; p.Phe730Ser in the GUCY2D gene. A previously
reported mutation c.1087C>A; p.P363T in the RPE65 gene was
detected in pedigree 3 while a single base deletion in the LCA5 gene,
c.1151delC (p.Pro384GlufsX18) that was previously reported in a
Pakistani family was detected in pedigree 4. All above-mentioned
variations were homozygous in affected individuals and segregated
with the disease in their respective pedigrees. The novel changes
were not observed in 190 ethnicity matched controls.
Conclusions: The exome analysis of samples from 4 pedigrees
revealed two novel missense mutations in GUCY2D and previously
reported mutations in the RPE65 and LCA5. These results strongly
suggest that novel and/or previously reported mutations in genes
implicated in retinal disease contribute to early onset retinal
degeneration in the Pakistani population.
Commercial Relationships: Pooja Biswas, None; Bruno
Maranhao, None; Pauline Lee, None; John Suk, None; Mili
Navani, None; Shahid Y. Khan, None; Nadeem H. Butt, None;
Sheikh Riazuddin, None; S. Amer Riazuddin, None; Radha
Ayyagari, None
Support: Foundation Fighting Blindness, Research to Prevent
Blindness, RO1- EY013198, RO1-EY021237, P30-EY22589.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
Program Number: 3350 Poster Board Number: C0006
Presentation Time: 11:00 AM - 12:45 PM
Whole exome sequencing identifies mutations in LRIT3 as a cause
for autosomal recessive complete congenital stationary night
blindness
Christina Zeitz1, Samuel G. Jacobson2, Christian P. Hamel3, Kinga
M. Bujakowska1, Marion Neuillé1, Elise Orhan1, Xavier Zanlonghi4,
Jose A. Sahel6, Shomi S. Bhattacharya5, 7, Isabelle S. Audo8, 5.
1
Institut de la Vision, Univ Pierre et Marie Curie Paris 6, INSERM,
UMR_S968; CNRS, UMR_7210, Paris, F-75012, France; 2University
of Pennsylvania, Scheie Eye Institute, Philadelphia 19104, PA;
3
Inserm U. 583, Physiopathologie et thérapie des déficits sensoriels et
moteurs, Institut des Neurosciences de Montpellier, Hôpital SaintEloi, Montpellier, 34295 Cedex 05, France; 4Service Exploration
Fonctionnelle de la Vision et Centre basse vision de la Clinique
Sourdille, Nantes 44000, France; 5UCL-Institute of Ophthalmology,
11-43 Bath Street, London EC1V 9EL, United Kingdom; 6Univ
Pierre et Marie Curie Paris 6, INSERM, UMR_S968; CNRS,
UMR_7210; CHNO, INSERM-DHOS CIC 503; Fondation
Ophtalmologique Adolphe de Rothschild, Paris; Académie des
Sciences-Institut de France, Paris, F-75012, France; 7Department of
Cellular Therapy and Regenerative Medicine, Andalusian Molecular
Biology and Regenerative Medicine Centre (CABIMER), Isla
Cartuja, Seville 41902, Spain; 8Univ Pierre et Marie Curie Paris 6,
INSERM, UMR_S968; CNRS, UMR_7210; CHNO, INSERMDHOS CIC 503, Paris, F-75012, France.
Purpose: Mutations in NYX, GRM6, TRPM1 and GPR179 expressed
in ON-bipolar cells, lead to a disruption of the ON-bipolar response
and reduced night vision. This dysfunction is present in patients with
complete congenital stationary night blindness (cCSNB). Although
many cases of cCSNB have been caused by mutations in these genes,
in some of the patients the gene defect remains unknown. Here we
sought to identify the disease-causing gene in the remaining patients
by whole exome sequencing.
Methods: Whole exome sequencing was applied to one simplex
cCSNB case lacking mutations in the known genes. Tissue specific
and prediction databases were used to define the most promising
candidate gene defect. In addition, Sanger sequencing in other CSNB
patients was performed. RT and immunolocalization with confocal
microscopy studies of the candidate gene were applied.
Results: Whole exome sequencing led to the identification of a
missense mutation (c.983G>A p.Cys328Tyr) and nonsense mutation
(c.1318C>T p.Arg440*) in a gene LRIT3 coding for a Leucine-Rich
Repeat (LRR), immunoglobulin-like and transmembrane domains 3
protein. Subsequent Sanger sequencing of 89 individuals with CSNB
identified another cCSNB case harboring a nonsense mutation
(c.1151C>G p.Ser384*) and a deletion (c.1538_1539del
p.Ser513Cysfs*59) in the same gene. Human LRIT3 antibody
staining revealed a punctate-labeling pattern in the outer plexiform
layer of the human retina, resembling dendritic tips of bipolar cells as
found in other proteins implicated in cCSNB.
Conclusions: Including the current study, mutations in five genes
have been implicated in cCSNB. All localize postsynaptically to the
photoreceptors in the retina in ON-bipolar cells. The mutation
spectrum described here affect different domains of LRIT3, including
the Cys328, which is predicted to form a disulfide bond in the Ig-like
domain. The three other mutations represent two nonsense mutations
and a frameshift mutation, which are located in the last exon. Thus it
is most likely that mutant mRNA products escape nonsense-mediated
decay. Further functional studies will eventually clarify the exact role
and pathogenic mechanism of LRIT3 within the ON-bipolar cell
pathway.
Commercial Relationships: Christina Zeitz, None; Samuel G.
Jacobson, None; Christian P. Hamel, None; Kinga M.
Bujakowska, None; Marion Neuillé, None; Elise Orhan, None;
Xavier Zanlonghi, None; Jose A. Sahel, UPMC/Essilor (P), Second
Sight (F); Shomi S. Bhattacharya, None; Isabelle S. Audo, None
Support: The project was supported by Retina France (part of the
100-exome project), Foundation Voir et Entendre, Foundation
Fighting Blindness, Ville de Paris and Région Ile de France, the
French Association against Myopathy and an INSERM/ICMR accord
No.53/1/Indo-Foreign/Oph/10-NCD-II.
Program Number: 3351 Poster Board Number: C0007
Presentation Time: 11:00 AM - 12:45 PM
A Missesne Mutation in the Acetyl-CoA Carboxylase β Gene
Involved in Lipid Metabolism is Associated with Autosomal
Recessive Retinitis Pigmentosa
Lina Zelinger1, Carmen Ayuso2, Eyal Banin1, Dror Sharon1.
1
Ophthalmology, Hadassah Hebrew University Medical Center,
Jerusalem, Israel; 2Departamento de Genética, Instituto de
Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD), Madrid,
Spain.
Purpose: The genetic cause of disease in our Israeli and Palestinian
cohort is still unknown in most cases with autosomal recessive (AR)
retinitis pigmentosa (RP). Aiming to identify additional genes
associated with the disease, we used a combination of whole genome
single nucleotide polymorphism (SNP) analysis followed by whole
exome sequencing (WES) in a family of Iranian Jewish ancestry with
ARRP.
Methods: Patients were evaluated clinically using visual function
testing and electroretinography. Blood samples were obtained from
patients and unaffected family members who agreed to participate in
the study. Homozygosity mapping was performed using the
Affymetrix 6.0 platform. WES was performed on the index case at
Otogenetics with over 60M reads, 91% of which were mapped to
exonic sequences. The identified mutation in the ACACB gene was
verified by Sanger sequencing and screened in additional samples.
Results: We recruited for the study a consanguineous family of
Iranian Jewish origin (MOL0249) with three individuals affected
with RP. Homozygosity mapping (HM) of the three patients revealed
a single large (5 Mb) shared homozygous region on chromosome 12,
containing 71 protein coding genes. A subsequent WES analysis
revealed 304 homozygous variants within the region. Further analysis
narrowed down the number of potential pathogenic changes to 32.
Only 2 changes were within coding sequences and only one was
predicted by online prediction software (Polyphen2 and
MutationTaster) to be damaging: c.G4469A (p.R1490H) missense
change in the Acetyl-CoA carboxylase β (ACACB) gene. The
mutation cosegregated in the family with LOD score of 3.3. The
Arg1490 amino acid is highly conserved throughout evolution.
Acetyl-CoA carboxylase β is an enzyme in the metabolic pathway of
lipids. It catalyzes the carboxylation of acetyl-CoA to malonyl-CoA,
the rate-limiting step in fatty acid synthesis. It is suspected to be
involved in the regulation of fatty acid oxidation, rather than
biosynthesis.
Conclusions: We report here an association between a missense
mutation in the ACACB gene and retinal degeneration. HM
combined with WES allowed us to identify a possible new pathway
that is involved in the phathogenicity of RP. This result, along with
identification of genes such as DHDDS and ELOVL4, shows the
importance of lipids and fatty acids in the proper function of the
retina.
Commercial Relationships: Lina Zelinger, None; Carmen Ayuso,
None; Eyal Banin, None; Dror Sharon, None
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
Support: Foundation Fighting Blindness USA grant (BR-GE-05100490-HUJ) and the Yedidut 1 research grant.
Program Number: 3352 Poster Board Number: C0008
Presentation Time: 11:00 AM - 12:45 PM
RP1L1 variants are associated with retinitis pigmentosa and
occult macular dystrophy
Panagiotis I. Sergouniotis1, 2, Alice E. Davidson1, Donna S. Mackay1,
Genevieve A. Wright2, Michel Michaelides1, 2, Graham E. Holder1, 2,
Anthony G. Robson1, 2, Anthony T. Moore1, 2, Vincent Plagnol3,
Andrew R. Webster1, 2. 1UCL Institute of Ophthalmology, London,
United Kingdom; 2Moorfields Eye Hospital, London, United
Kingdom; 3UCL Genetics Institute, London, United Kingdom.
Purpose: Autosomal dominant mutations in the RP1L1 gene have
been recently associated with occult macular dystrophy (OCMD), a
condition usually characterized by progressive foveal cone
dysfunction and no apparent fundoscopic or full-field
electoretinogram abnormalities. Intriguingly, the Rp1l1 knockout
mouse has been shown to have a phenotype consistent with retinitis
pigmentosa (RP). The aim of this study is to provide insights into the
clinical and genetic characteristics of OCMD and to probe whether
RP1L1 mutations cause RP in man.
Methods: Twenty-eight individuals with OCMD and 286 individuals
with recessive RP were recruited and screened for RP1L1. Exome
sequencing was performed in a consanguineous family with RP.
Sanger sequencing of the RP1 gene as well as haplotype and in silico
analysis of RP1L1 variants were performed. Clinical investigations
included fundus autofluorescence imaging, spectral domain optical
coherence tomography (OCT) and electrophysiology.
Results: Homozygous, likely disease causing, RP1L1 variants were
identified in two individuals with typical sings of RP; one carried a
frameshifting (p.Lys203Argfs*28) and the other a missense
(p.Ser546Thr) mutation. Ten out of twenty-eight OCMD patients
were found to harbour rare (minor allele frequency ≤0.5% in the
1,000 genomes dataset) heterozygous RP1L1 variants. Irregular
foveal fundus autofluorescence was evident in 6 of 10 cases. Spectral
domain OCT showed focal disruption of the outer retina in 8 of 9
cases. Full-field electroretinograms were normal in 8 and revealed
mild generalised cone system dysfunction in 2 individuals. Analysis
of family members revealed unaffected relatives harbouring the same
variant. Linkage analysis excluded recessive inheritance, and
sequencing of RP1, a gene encoding a photoreceptor protein that
interacts with RP1L1, excluded a potential digenic mechanism.
Conclusions: Biallelic RP1L1 variants can be associated with
recessive RP in man. OCMD is a pathogenetically heterogeneous
macular dystrophy and heterozygous RP1L1 variants cause OCMD
with incomplete penetrance; the disorder is not Mendelian and is
likely to have a complex pathology with contribution from other
genetic and environmental factors.
Commercial Relationships: Panagiotis I. Sergouniotis, None;
Alice E. Davidson, None; Donna S. Mackay, None; Genevieve A.
Wright, None; Michel Michaelides, Novartis (R); Graham E.
Holder, Servier (C); Anthony G. Robson, None; Anthony T.
Moore, None; Vincent Plagnol, None; Andrew R. Webster, None
Support: British Retinitis Pigmentosa Society, Fight for Sight,
Moorfields Eye Hospital Special Trustees, the NIHR Biomedical
Research Centre at Moorfields Eye Hospital NHS Foundation Trust
and UCL Institute of Ophthalmology, the Foundation Fighting
Blindness (USA)
Program Number: 3353 Poster Board Number: C0009
Presentation Time: 11:00 AM - 12:45 PM
RNA-Seq Approach for the Refinement of a Modifier Locus in a
Canine Model of Cone-Rod Dystrophy
Keiko Miyadera1, Matthew Brooks2, Gautami Das1, Anand Swaroop2,
Gustavo D. Aguirre1. 1School of Vet Medicine, Univ of
Pennsylvania, Philadelphia, PA; 2National Eye Institute, NIH,
Bethesda, MD.
Purpose: We have previously established a canine model of
oligogenic cone-rod dystrophy (cord1). The primary locus
represented by a homozygous insertion in RPGRIP1
(RPGRIP1ins/ins) was mapped in an early-onset cord1 research
colony. A proportion of RPGRIP1ins/ins pet dogs, however, showed
a slower disease progression with much later or no clinical onset.
Subsequent GWAS in these RPGRIP1ins/ins dogs mapped a 1.5Mb
homozygous modifier interval associated with early onset. As Sanger
sequencing of positional candidate genes failed to identify a
mutation, we have used now a transcriptomics approach to further
refine the modifier locus.
Methods: RNA-seq was performed using retinal tissues from seven
dogs in a newly established colony where the primary and the
modifier loci segregated independently. High-throughput sequencing
reads of retinal cDNA librarys were assembled to the canine
reference. Differential expression of annotated genes/transcripts and
unresolved transcribed sequences was analyzed. Based on sequence
variants, haplotypes were constructed across the modifier interval.
Results: Of 12 annotated genes in the modifier interval, GUCY1A3
and GUCY1B3 were highly expressed in the retina; less abundantly
expressed were MAP9 and CTSO. Other known genes showed trace
or no retinal expression except for LRAT affected by RPE
contamination. Homozygosity for the early-onset haplotype
(modE/E) was confirmed in one RPGRIP1+/ins dog. One wildtype
dog was homozygous only for a telomeric portion of the E haplotype
(modE/TE). There were no double-homozygotes
(RPGRIP1ins/insmodE/E) in the study samples, and early-onset
cord1 was not observed clinically. There was no significant
difference in the expression of annotated genes/transcripts along the
modifier interval associated with the E haplotype. Among the
unresolved regions, differential splicing pattern was found flanking
the 3’-UTR of MAP9.
Conclusions: RNA-seq using canine retinal samples could be applied
to assess differential expression of annotated and unannotated
transcripts across the target interval. Sequence variants enabled
constructing of haplotypes to determine identity by descent and
chromosomal break points. There were no critical changes in
sequences or expression levels corresponding to the E haplotype. The
role of the MAP9 transcript variant in the interaction with the
primary cord1 locus is being explored.
Commercial Relationships: Keiko Miyadera, None; Matthew
Brooks, None; Gautami Das, None; Anand Swaroop, None;
Gustavo D. Aguirre, None
Support: FFB Career Development Award for Veterinarian
Residents, Van Sloun Fund, NIH-EY 06855, 17549
Program Number: 3354 Poster Board Number: C0010
Presentation Time: 11:00 AM - 12:45 PM
Exome sequencing in dogs with progressive retinal atrophy to
facilitate the development of therapeutic intervention studies
Rob W. Collin1, Arjen Henkes1, Galuh D. Astuti1, Christian Gilissen1,
Birgit Lorenz2, Alexander Hoischen1, Frans P. Cremers1, Knut
Stieger2. 1Human Genetics, Radboud University Medical Ctr,
Nijmegen, Netherlands; 2Ophthalmology, Justus-Liebig-University
Giessen, Giessen, Germany.
Purpose: The dog is an attractive model organism to study retinal
degeneration, because the eye is anatomically similar to the human
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
one, and the phenotypic characteristics of progressive retinal atrophy
(PRA) highly resemble that of human rod-cone and cone-rod
dystrophies in terms of onset and progression. Also, canine PRA
models have shown to be instrumental in the development of gene
therapy, for instance in translating RPE65 gene augmentation therapy
to the clinic. In this study, we aim to identify the genetic defect
underlying PRA in several dog breeds with unknown causative
mutation employing next generation sequencing technology.
Methods: In five dog breeds segregating PRA (Airdale Terrier,
Berger des Pyrénées, Chihuahua, Löwchen and Saarloos Wolfshond),
whole exome sequencing was performed in one affected and one
unaffected dog. A completely new pipeline was built to map all reads
to the canine genome and annotate all variants to the corresponding
canine genes. Subsequent filtering of the variants included removing
variants that were present in unaffected dogs, and prioritization of
variants that were highly conserved or present in known human
retinal disease genes. Candidate pathogenic variants were validated
with Sanger sequencing.
Results: Despite the poor annotation of the canine genome, by using
gene prediction software, we have been able to correctly annotate
~20,000 canine genes. In each sample, exome sequencing yielded
approximately 60,000 genomic variants. After filtering and
prioritizing the variants, in each affected dog, on average 37
candidate pathogenic variants were remaining. For the variants that
have been validated so far, the majority (>95%) was confirmed with
Sanger sequencing analysis. Currently, we are assessing the potential
pathogenicity of these variants in larger cohorts and/or by performing
additional experiments.
Conclusions: Our data show that exome sequencing is a powerful
tool to assess genetic variation in protein-coding parts of the genome,
not only in humans but also in dogs. Further characterization of
candidate variants will likely result in the identification of the
underlying genetic defect in canine models of PRA, and may
facilitate the development of novel therapeutic interventions in these
models.
Commercial Relationships: Rob W. Collin, Radboud University
Medical Centre (P); Arjen Henkes, None; Galuh D. Astuti, None;
Christian Gilissen, None; Birgit Lorenz, Optos (F); Alexander
Hoischen, None; Frans P. Cremers, None; Knut Stieger, None
Support: Stichting A.F.Deutman Researchfonds Oogheelkunde
Nijmegen; Landelijke Stichting voor Blinden en Slechtzienden
Program Number: 3355 Poster Board Number: C0011
Presentation Time: 11:00 AM - 12:45 PM
Exome Sequencing Identifies a Novel RP1 Mutation in a Belgian
Family with Autosomal Dominant Retinitis Pigmentosa
Caroline Van Cauwenbergh1, Frauke Coppieters1, Sarah De
Jaegere1, Julie De Zaeytijd2, Bart P. Leroy1, 2, Elfride De Baere1.
1
Centre for Medical Genetics Ghent, Ghent University, Ghent,
Belgium; 2Department of Ophthalmology, Ghent University Hospital,
Ghent, Belgium.
Purpose: To identify the underlying genetic cause in a
multigenerational Belgian family with autosomal dominant retinitis
pigmentosa (adRP) using genome-wide linkage analysis and exome
sequencing.
Methods: Five affected and eight unaffected individuals from a fourgeneration Belgian adRP family were clinically evaluated. They
underwent genome-wide linkage analysis using BeadChips (Illumina)
and multipoint linkage analysis (Merlin, dominant model, 95%
penetrance, disease allele frequency of 0.0001). Exome sequencing
was performed in two affected individuals (TruSeq Exome
Enrichment Kit, HiSeq, Illumina). The CLC Genomics Workbench
(CLC bio) was employed for read mapping and variant calling.
Linkage regions and RetNet genes were used for filtering of exome
variants.
Results: GW linkage analysis revealed two regions with a maximum
LOD score of 1.7. As to the exome data, 11.3/12.7 and 12.9/14.5
million reads could be mapped against the human genome reference
sequence, resulting in an average coverage of 87.8 and 99.3
respectively. Integrated linkage- and RetNet gene-based filtering of
exome data revealed a heterozygous RP1 change in both patients:
p.Leu749fs and p.Leu750fs respectively. Of note, variant calling
failed to correctly call this change. Sanger sequencing confirmed
following mutation: c.2245_2248delinsTGAG (p.Leu749X). This
mutation was found to co-segregate with disease, one unaffected
individual excepted, suggesting incomplete penetrance. This indel
mutation, replacing nucleotides CTCA by their reverse complement
TGAG, creates a premature stop codon that may lead to a truncated
protein. The mutation, present in a hotspot region in exon 4, belongs
to the ‘Class II’ mutations, which are frequently reported nonsense
mediated decay-insensitive truncations, with a net dominant negative
effect.
Conclusions: Combined linkage-based filtering of exome data
revealed a novel RP1 mutation in a Belgian pedigree with adRP. Our
study expands the spectrum of RP1 Class II mutations leading to
adRP with incomplete penetrance. Interestingly, the findings of this
study can be used as a starting point for further research on modifiers
influencing RP1 expression.
Commercial Relationships: Caroline Van Cauwenbergh, None;
Frauke Coppieters, None; Sarah De Jaegere, None; Julie De
Zaeytijd, None; Bart P. Leroy, None; Elfride De Baere, None
Support: FWO, Funds for Scientific Research (FRO), BOF Ghent
University
Program Number: 3356 Poster Board Number: C0012
Presentation Time: 11:00 AM - 12:45 PM
The familial dementia gene revisited: ITM2B missense mutation
causes a new dominant retinal dystrophy
Isabelle S. Audo1, 2, Kinga M. Bujakowska5, Elise Orhan5, Florian
Sennlaub5, Xavier P. Guillonneau5, Thierry D. Leveillard5, Saddek
Mohand-Said1, Shomi S. Bhattacharya2, 3, Jose A. Sahel4, Christina
Zeitz5. 1Univ Pierre et Marie Curie Paris 6, INSERM, UMR_S968;
CNRS, UMR_7210; CHNO, INSERM-DHOS CIC 503, Paris, F75012, France., Paris, France; 2UCL-Institute of Ophthalmology, 1143 Bath Street, London EC1V 9EL, United Kingdom., London,
United Kingdom; 3Department of Cellular Therapy and Regenerative
Medicine,, Andalusian Molecular Biology and Regenerative
Medicine Centre (CABIMER), Isla Cartuja, Seville 41902, Spain.,
Seville, Spain; 4Univ Pierre et Marie Curie Paris 6, INSERM,
UMR_S968; CNRS, UMR_7210; CHNO, INSERM-DHOS CIC 503;
Fondation Ophtalmologique Adolphe de Rothschild, Paris; Académie
des Sciences-Institut de France, Paris, Paris, France; 5Univ Pierre et
Marie Curie Paris 6, INSERM, UMR_S968; CNRS, UMR_7210;
CHNO, Paris, F-75012, France., Paris, France.
Purpose: To identify and characterize the underlying genetic defect
of an unusual autosomal dominant inherited retinal dystrophy
comprising inner retinal dysfunction, ganglion cell abnormalities and
progressive cone degeneration.
Methods: Phenotypic characterization and exclusion of known gene
defect using Sanger and next generation sequencing had been
previously presented (ARVO 2012, abstract # 1226). Whole exome
sequencing was applied to two affected and one unaffected family
members. Stringent filtering with the use of available SNP and retinal
transcriptomic databases, co-segregation analysis and haplotyping
using microsatellite markers were carried out to select the most likely
pathogenic variant. Expression and immuno-localization studies were
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
performed by RT-PCR, in situ hybridization and immunostainning on
mouse and human retinal samples. In addition, subcellular
localization of the wildtype and mutated protein was analyzed after
COS-1 cell transient transfection.
Results: Whole exome sequencing identified a new missense variant
[c.782A>C, p.Glu261Ala], in ITM2B (Integral Membrane Protein
2B) which co-segregates with the disease in this large family.
Subsequent haplotype analysis on chromosome 13q identified a
crossover between markers D13S1272 and D13S275 leading to an
interval of 24.6Mb including ITM2B, with the same haplotype
present in all affected members available. ITM2B mRNA is highly
expressed in the retina and localized in the inner nuclear and ganglion
cell layer. Similarly, immuno-localization shows staining of the same
layers. Functional assay does not show mutant protein membrane
miss-localization.
Conclusions: This is the first report of a missense change in ITM2B
associated with a peculiar autosomal dominant retinal restricted
disease. Mutations in this gene have previously been reported in two
distinct autosomal dominant Alzheimer-like dementia families.
Pathogenic mechanism(s) that lead either to a restricted peculiar
retinal phenotype or dementia in association with ITM2B mutations
need to be further elucidated.
Commercial Relationships: Isabelle S. Audo, None; Kinga M.
Bujakowska, None; Elise Orhan, None; Florian Sennlaub, None;
Xavier P. Guillonneau, None; Thierry D. Leveillard, None;
Saddek Mohand-Said, None; Shomi S. Bhattacharya, None; Jose
A. Sahel, UPMC/Essilor (P), Second Sight (F); Christina Zeitz,
None
Support: GIS-maladies rares (C.Z.), Retina France ([part of the 100Exome Project] (I.A., J.-A.S and C.Z.), Foundation Voir et Entendre
(C.Z.), Agence National de la Recherche (S.S.B), Foundation
Fighting Blindness (FFB) grant CD-CL-0808-0466-CHNO (I.A. and
the CIC503, recognized as an FFB center), FFB grant C-CMM-09070428-INSERM04, Ville de Paris and Region Ile de France
Program Number: 3357 Poster Board Number: C0013
Presentation Time: 11:00 AM - 12:45 PM
Variant prioritization and linkage mapping using whole-exome
sequencing data for families with autosomal dominant retinitis
pigmentosa (adRP)
Daniel C. Koboldt1, David E. Larson1, Lori S. Sullivan2, Sara J.
Bowne2, Robert S. Fulton1, Erica Sodergren1, Susan H. Blanton3,
Stephen P. Daiger2, Richard K. Wilson1, George M. Weinstock1. 1The
Genome Institute, Washington University, St. Louis, MO; 2Human
Genetics Center, Univ. of Texas Health Science Ctr, Houston, TX;
3
Hussman Institute of Human Genomics, Univ. of Miami, Miami, FL.
Purpose: To facilitate the identification of novel retinitis pigmentosa
(RP) genes by family-based exome sequencing of adRP families.
Novel approaches to linkage analysis, coupled with a comprehensive
variant scoring strategy, narrow the search space and prioritize the
most likely disease-causing variants.
Methods: A scoring algorithm was implemented to prioritize
potential causal variants within a family according to segregation
with the phenotype, population frequency, predicted effect, and
retinal gene expression data. The search can be further refined in
families with multiple affected individuals using two complementary
approaches to exome-based linkage analysis: (1) Shared IBD analysis
of common variants identifies segments of maximum identity-bydescent among affected individuals, and (2) Rare heterozygote rule
out nominates regions based on shared rare variants and the absence
of homozygous differences between affected individuals.
Results: Twenty-four families with probable adRP but lacking
common disease-causing mutations were included in the study. We
performed exome capture and sequencing on 75 total samples (2-7
affecteds, 0-2 unaffected controls per family), followed by variant
prioritization and linkage analysis. A subset of these families also had
regions from traditional linkage mapping of extended pedigrees, the
results of which were highly concordant with our linkage analyses.
Seven of 24 families (29%) were revealed to have unrecognized
mutations in known RP genes that were both high-scoring by our
algorithm and deemed likely pathogenic by clinical assessment.
Analysis of the remaining 17 families has identified candidate
variants in a number of interesting genes, some of which have already
undergone further segregation testing in extended pedigrees.
Conclusions: Family-based exome sequencing is a powerful strategy
for the identification of novel RP genes, yet these studies often
identify thousands of potential causative variants. Here, we
demonstrate that comprehensive scoring of individual variants
coupled with two genetic linkage approaches can substantially refine
the search for disease-causing mutations.
Commercial Relationships: Daniel C. Koboldt, None; David E.
Larson, None; Lori S. Sullivan, None; Sara J. Bowne, None;
Robert S. Fulton, Orion Genomics (C); Erica Sodergren, None;
Susan H. Blanton, None; Stephen P. Daiger, None; Richard K.
Wilson, None; George M. Weinstock, None
Support: NIH EY007142, NIH U54HG3079-10, and Foundation
Fighting Blindness
Program Number: 3358 Poster Board Number: C0014
Presentation Time: 11:00 AM - 12:45 PM
Whole-exome sequencing in age-related macular degeneration
Margaret A. Pericak-Vance1, Gaofeng Wang1, William Cade1,
Monique D. Courtenay1, Patrice Gay1, Stephen G. Schwartz3, Jaclyn
L. Kovach3, Anita Agarwal4, Jonathan L. Haines2, William K. Scott1.
1
Human Genomics, Univ of Miami Miller Sch of Med, Miami, FL;
2
Center for Human Genetics Research, Vanderbilt University School
of Medicine, Nashville, TN; 3Bascom Palmer Eye Institute,
University of Miami, Miami, FL; 4Ophthalmology, Vanderbilt
University School of Medicine, Nashville, TN.
Purpose: Genome-wide association meta analysis (GWAMA) has
implicated common variations in 19 genes as AMD risk factors. Rare
variants (RV) are not well captured by genome wide association
studies and may represent some of the unexplained heritability in
AMD risk. We used whole exome sequencing (WES) in
phenotypically extreme individuals to identify RVs implicating novel
AMD genes.
Methods: The GWAS 19 loci were used to calculate a genetic risk
score (summed number of risk alleles weighted by effect sizes) for
each individual. We created an ‘extreme’ case/control sample with
the following characteristics: 20 individuals with bilateral
neovascular AMD and the lowest calculated genetic risk score, and
youngest ages at examination; 20 unaffected controls with no drusen,
the highest calculated genetic risk score, and oldest ages at
examination. Sequencing Capture was by SureSelect. Alignment and
base calling used the Illumina CASAVA 1.6 pipeline, aligned to hg19
using BWA. Single nucleotide variants (SNV) and insertion-deletion
variants (indels) were called by GATK Unified Genotyper with
VQSR recalibration. Variants with VQSLOD<-3 and variant
genotype likelihoods < 99 were excluded. All variants were annotated
using SeattleSeq Annotation. Association of individual SNV with
AMD was assessed by Fisher’s exact test. RVASSOC was used for
the gene-based tests.
Results: No gene-based tests met Bonferroni-corrected significance
(2.2 x 10-6). The most significant genes identified in the overall
analysis are all novel. LRP1 is implicated both in the overall analysis
and when considering only rare and damaging SNVs. The other most
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
significant genes in the two analyses are different. Six of the 19
known loci have nominally significant gene-based tests. Associated
SNVs in each gene are previously known variants.
Conclusions: Initial results of WES suggest additional genes with
multiple rare SNVs may influence AMD. LRP1 is an APOE receptor
and also involved in cholesterol transport. Restricting analysis to rare
damaging variants reveals different results than considering the
overall burden of SNVs, suggesting both analyses are needed to
extract maximum information from sequence data. Novel coding
variants in known AMD genes do not appear to explain the extreme
phenotype studied here. WES of a replication dataset with an
additional 20 cases/20 controls with extreme phenotype is in
progress.
Commercial Relationships: Margaret A. Pericak-Vance, None;
Gaofeng Wang, None; William Cade, None; Monique D.
Courtenay, None; Patrice Gay, None; Stephen G. Schwartz,
Alimera (C), Bausch + Lomb (C), Eyetech (C), IC Labs (P),
ThromboGenics (C); Jaclyn L. Kovach, None; Anita Agarwal,
Vanderbilt University (P); Jonathan L. Haines, Arctic Dx (I), AMD
genes (P); William K. Scott, Duke University/ArcticDx (P)
Support: NIH R01EY012118-11
Program Number: 3359 Poster Board Number: C0015
Presentation Time: 11:00 AM - 12:45 PM
Mutation analysis of B3GALTL in Peters Plus Syndrome and
alike phenotypes
Eric Weh1, 2, Linda M. Reis2, Rebecca C. Tyler2, Elena V. Semina1, 2.
1
Cell Biology, Neurobiology and Anatomy, Medical College of
Wisconsin, Wauwatosa, WI; 2Pediatrics and Children's Research
Institute, Medical College of Wisconsin, Wauwatosa, WI.
Purpose: Peters Plus Syndrome (PPS) is a rare autosomal recessive
disorder characterized by Peters’ Anomaly (or other anterior segment
dysgenesis), brachydactyly, short stature and variable other features.
A number of different causal variants in B3GALTL have been
reported but genotype/phenotype correlations have not yet been
established due to rarity of this condition and insufficient mutation
data. To obtain additional information about B3GALTL mutations in
PPS and related phenotypes, we evaluated 53 human patients for
variation in this gene.
Methods: The sequence of B3GALTL was obtained for each patient
using PCR primers specific to the coding exons and immediate
surrounding intronic regions of B3GALTL. Sequence files were
analyzed using Mutation Surveyor to identify potentially causative
variations. TaqMan Copy Number probes were used to test for copy
number mutations involving B3GALTL.
Results: B3GALTL sequences were obtained from 53 probands
affected by classic PPS or overlapping phenotypes; the screen was
completed for all exons in 50 of the patients. We identified recessive
B3GALTL mutations in all but one classic PPS case in our study: 3
patients were homozygous for c.660+1G>GA, and 4 patients were
compound heterozygotes for the following alleles,
c.660+1G>GA/c.1065-1 G>GA, c.459+1G>GA/c.660+1G>GA,
660+1G>GA/1234C>CT (p.R412RX), 660+1G>GA/c.165insA
(pG56Rfs*10). A c.1045G>GA (p.D349DN) allele was identified in
an additional patient that is still under study for other B3GALTL
exons. Three of the identified alleles, c.165insA (pG56Rfs*10),
c.1234C>CT (p.R412RX), c.1045G>GA (p.D349DN) are novel,
predicted to be deleterious and are not reported in the EVS database.
The remaining patients were negative for any causative mutations.
Conclusions: We identified three novel B3GALTL alleles associated
with Peters’ Plus syndrome: two nonsense/frameshift and one
missense mutation. The missense change p.D349DN alters the first
amino acid in the catalytic core of B3GALTL which is predicted to be
deleterious by PolyPhen-2 and SIFT and is the second missense
mutation reported in B3GALTL. B3GALTL mutations appear to be
strongly associated with classic cases of PPS involving all three
features: anterior segment anomaly, short stature and brachydactyly
with variable other defects. Other PPS-like conditions, despite a
significant phenotypic overlap with classic PPS, are likely to be
associated with mutations in other genetic factors.
Commercial Relationships: Eric Weh, None; Linda M. Reis,
None; Rebecca C. Tyler, None; Elena V. Semina, None
Support: Institutional Research Training Program in Vision Science
T32-EY014537 and R03HD058894 NIH grant from Eunice Kennedy
Shriver National Institute of Child Health & Human Development
and Children’s Research Institute, CHW, WI
Program Number: 3360 Poster Board Number: C0016
Presentation Time: 11:00 AM - 12:45 PM
Next Generation Sequencing Reveals A Novel Gene Mutation in
Primary Congenital Glaucoma Patients
Terri L. Young1, 2, Sing H. Lim2, 3, Tammy Yanovitch1, 3, Thomas P.
Klemm2, Elizabeth St.Germain3, Sebastian Maurer-Stroh5,
Vachiranee Limviphuvadh5, Nicholas Katsanis4, Steve Rozen2,
Khanh-Nhat Tran-Viet3. 1Ophthalmology, Duke University Eye
Center, Durham, NC; 2Computational Biology, Duke-National
University of Singapore, Singapore, Singapore; 3Medicine, Duke
Center for Human Genetics, Durham, NC; 4Medicine, Duke Center
for Human Disease Modeling, Durham, NC; 5Bio-informatics
Institute, Agency for Science, Technology and Research, Singapore,
Singapore.
Purpose: Primary congenital glaucoma (PCG) is a potentiallyblinding disease due to a mal-developed anterior segment structure.
Three major genes have been implicated -CYP1B1, LTBP2, and
MYOC- but do not account for all cases. The study aim was to
determine novel gene sequence variants in PCG families using whole
exome sequencing (WES).
Methods: DNA samples from 13 families negative for known PCG
gene mutations underwent WES. WES was performed using the
NimbleGen v2 exome capture kit and Illumina HiSeq2000 platform.
SNP and Variation Suite 7.5 software was used to filter single
nucleotide variants (SNVs) and insertions/deletions (indels). Public
databases and internal whole exome controls were used to filter out
known variants. Filtered novel SNVs were validated by Sanger
sequencing to confirm familial segregation, and was performed in 32
additional PCG cases. In silico prediction tools (Polyphen/SIFT and
FoldX) were used to determine mutational impact on protein function
and structure.
Results: An average 50X coverage depth for all coding regions was
achieved. Two affected individuals in 1 family inherited a novel,
heterozygous stop codon amino acid change, p.Tyr307*, in the
tyrosine kinase, endothelial, (TEK) gene. Sequencing revealed TEK
mutations in 2 additional families, one as a heterozygous mutation
(p.Lys294Asn), and the other as a compound heterozygous
(p.Pro346Gln and p.Tyr611Cys) mutation. The p.Tyr307* causes
TEK protein truncation, resulting in unstable protein formation, as
several inter-domain interactive hydrophobic residues become
accessible to water. For p.Lys294Asn and p.Pro346Gln, replacement
of the positively charged Lys with a polar Asn molecule perturbs
hydrophilic interactions, while the substitution of non-polar Pro with
a polar Gln affects hydrophobic interactions causing flexible loop
region instability. FoldX predictive effects on protein structure note
an insignificant average free energy change for p.Lys294Asn, while
that of p.Tyr611Cys and p.Pro346Gln had significant destabilization.
cDNA expression panels validated TEK presence in relevant human
ocular tissues.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
Conclusions: In all, TEK SNVs were present in 3/35 PCG families
(6.67%). The absence of the kinase domain due to the stop codon
clearly indicates that p.Tyr307* has deleterious effects on TEK
function. WES is useful in causal gene discovery for rare ocular
disorders.
Commercial Relationships: Terri L. Young, National Institutes of
Health (F); Sing H. Lim, None; Tammy Yanovitch, None; Thomas
P. Klemm, None; Elizabeth St.Germain, None; Sebastian MaurerStroh, None; Vachiranee Limviphuvadh, None; Nicholas
Katsanis, None; Steve Rozen, None; Khanh-Nhat Tran-Viet,
Golden Helix (R)
Support: NIH Grant R01 EY014685
Program Number: 3361 Poster Board Number: C0017
Presentation Time: 11:00 AM - 12:45 PM
CCDC111 mutation was identified in high myopia through
exome sequencing
Fuxin Zhao, Xiangtian Zhou, Anquan Xue, Yanfeng Su, Jia Qu.
School of Ophthal & Optometry, Wenzhou Medical College,
Wenzhou, China.
Purpose: To identify a novel genetic cause of high myopia in
Chinese family
Methods: Exome sequencing was performed on one affected subject
of high myopia family. 170 sporadic patients with high myopia and
170 matched normal controls for genotyping using MassArray. The
100 sporadic patients and the 100 controls for other variant of gene
using Sanger sequencing. Expression was analyzed in human cell
line, especially in eye tissue.
Results: One variant c.9812T>G in coiled-coil domain containing
111 gene (CCDC111) was identified associated with phenotype of
this family. Genotyping was carried on using MassARRAY platform
in additional 170 patients and 170 controls, and the variant
(c.9812T>G) was found in two patients, but did not find in the 170
controls. To identify other variant in CCDC111 gene, Sanger
sequencing was used. In 100 sporadic patients, two variants were
found, one variant, c.9812T>G found in two patients, did not find in
100 controls. Another variant in Exon 6 (m.503G>A), which found in
patients and controls, of PolyPhen-2 was used to predict, the
influence for protein was light, it considered as a single nucleotide
polymorphism. The CCDC111 gene was expressed in the human
Epic-Human gastric epithelial cells (HGM), Human embryonic
kidney epithelial cells (293T), Human corneal epithelial cells
(HCEC), Choroidal melanoma cells (UM-U-96), Human scleral
fibroblasts (Pre-S110), Human embryonic lung fibroblasts (LX-2),
Human retinal epithelial cells (R92), Human retinal müller cells
(MIOMI), Human lens capsule epithelial cells (L1).
Conclusions: Using exome sequencing technique, we have identified
CCDC111 as a causative susceptibility gene of high myopia in a
Chinese family.
Commercial Relationships: Fuxin Zhao, None; Xiangtian Zhou,
None; Anquan Xue, None; Yanfeng Su, None; Jia Qu, None
Support: National Basic Research Program of China (973 project,
NO: 2011CB504604), the National Natural Science Foundation of
China (81170880)
Program Number: 3362 Poster Board Number: C0018
Presentation Time: 11:00 AM - 12:45 PM
The superior ocular fissure: a novel finding in early eye
development
Tara Stach1, 2, Jakub Famulski2, Andrew Waskiewicz2, Ordan J.
Lehmann1, 3. 1Medical Genetics, University of Alberta, Edmonton,
AB, Canada; 2Biological Sciences, University of Alberta, Edmonton,
AB, Canada; 3Ophthalmology, University of Alberta, Edmonton, AB,
Canada.
Purpose: To determine the cause of a novel ocular developmental
anomaly through integrated human and zebrafish model organism
approaches.
Methods: Human genetic analyses encompassing Sanger and exomic
next generation sequencing were combined with zebrafish
morpholino oligonucleotide (MO) inhibition, in situ hybridization,
confocal microscopy and immunofluorescence.
Results: Five patients with superiorly positioned
iris/lens/retinochoroidal colobomata were identified over a 6 year
period, and in one the molecular basis was defined [biallelic CYP1B1
mutations: R368H & A287X]. Zebrafish have a transient superior
fissure, readily visible in the dorsal retina using light microscopy as
well as with anti-Laminin immunofluorescence. Loss of Bone
Morphogenetic Protein (BMP) signaling, as seen in zebrafish gdf6a-/mutants, causes a persistent superior colobomata phenotype. Since
cyp1b1’s expression is restricted to the vertical axis of the developing
retina, and it synthesizes retinoic acid (RA), the contribution of RA to
superior fissure closure was explored. MO inhibition of cyp1b1
results in an increased duration of an open superior fissure [3.5 fold
increased incidence in cyp1b1 MO embryos], and loss of the RARE
transgenic reporter of RA-dependent ocular signalling. Cyp1b1’s
retinal expression is dependent on gdf6a, as reduced expression is
observed in zebrafish gdf6a-/- mutants and gdf6a morphants have a 40
fold increased incidence of delayed fissure closure. Notably, RA
supplementation rescues these phenotypes [2.7 fold decrease in
incidence in RA treated gdf6a morphants]. The superior fissure lies at
the expression interface of two forkhead box genes that specify nasal
(foxg1) and temporal (foxd1) retinal identity. MO inhibition of Foxd1
results in superior fissure twinning whilst inhibition of Foxg1 shifts
the superior fissure nasally.
Conclusions: These studies identify a novel superior fissure in the
developing retina conserved between species separated by 450
million years of evolutionary time. Its location is specified by the
forkhead transcription factors foxd1 and foxg1, whilst its closure is
dependent on CYP1B1, BMP and RA signaling. Although these
findings run contrary to current knowledge of early eye development,
they provide opportunities to enhance understanding of initial steps in
vertebrate retinal development.
Commercial Relationships: Tara Stach, None; Jakub Famulski,
None; Andrew Waskiewicz, None; Ordan J. Lehmann, None
Program Number: 3363 Poster Board Number: C0019
Presentation Time: 11:00 AM - 12:45 PM
ALDH1A3 mutations cause recessive anophthalmia and
microphthalmia
Lucas Fares Taie1, Sylvie Gerber1, Nicolas Chassaing2, Jill ClaytonSmith3, Eduardo D. Silva4, Arnold Munnich1, Patrick Calvas2,
Josseline Kaplan1, Nicola Ragge5, Jean-Michel Rozet1. 1GENETICS,
INSERM U781, Paris, France; 2GENETICS, CHU Toulouse,
TOULOUSE, France; 3Genetic Medicine, St Mary's Hospital,
Manchester, United Kingdom; 4Ophthalmology, Coimbra University
Hospital, Coimbra, Portugal; 5Clinical Genetics, University Hospital
Southampton, Southampton, United Kingdom.
Purpose: Anophthalmia and microphthalmia (A/M) are early eye
development anomalies resulting in absent or small ocular globes,
respectively. A/Ms occur as syndromic or nonsyndromic forms. They
are genetically heterogenous with some mutations in some genes
responsible for both anophthalmia and microphthalmia. The purpose
of this study was to identify the disease gene involved in A/M in a
large inbreed Pakistani Family and other unrelated A/M families.
Methods: A combination of homozygosity mapping, exome
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
sequencing and Sanger sequencing, was used to identify the disease
mutation in the Pakistani family and to screen the ALDH1A3 gene
for mutations in additional unrelated A/M patients.
Results: We identified homozygosity for a missense mutation in the
gene encoding the A3 isoform of the aldehyde dehydrogenase 1
(ALDH1A3) in the Pakistani family. The screening of the gene is a
cohort of A/M patients excluding known A/M genes allowed
identifying two additional homozygote ALDH1A3 mutations
including another missense change and a splice-site mutation in two
consanguineous families. The review of the clinical files of patients
showed that patients with ALDH1A3 mutations had A/M with
occasional orbital cystic, neurological and cardiac anomalies.
Conclusions: ALDH1A3 is a key enzyme in the formation of a
retinoic acid gradient along the dorso-ventral axis during the early
eye development. Transitory expression of mutant ALDH1A3
cDNAs showed that both missense mutations reduce the
accumulation of the enzyme, potentially leading to altered retinoic
acid synthesis. While the role of retinoic acid signaling in eye
development is well established, our findings provide genetic
evidence of a direct link between retinoic acid synthesis dysfunction
and early eye development in human.
Commercial Relationships: Lucas Fares Taie, None; Sylvie
Gerber, None; Nicolas Chassaing, None; Jill Clayton-Smith,
None; Eduardo D. Silva, None; Arnold Munnich, None; Patrick
Calvas, None; Josseline Kaplan, None; Nicola Ragge, None; JeanMichel Rozet, None
Support: Retina France, Clinical Research Hospital Program from
the French Ministry of Health (PHRC 09 109 01), Academy of
Medical Sciences/Health Foundation (NR), VICTA (Visually
Impaired Children Taking Action), MACS (Microphthalmia and
Anophthalmia Children’s Society), and Hampshire and Isle of Wight
CLRN.
Program Number: 3364 Poster Board Number: C0020
Presentation Time: 11:00 AM - 12:45 PM
Whole exome sequencing identifies novel mutations in OTX2 ,
CRYBA4, and PAX6 in microphthalmia patients
Brett Deml1, 2, Linda M. Reis1, Elena V. Semina1, 2. 1Pediatrics,
Medical College of Wisconsin, Milwaukee, WI; 2Cell Biology,
Neurobiology & Anatomy, Medical College of Wisconsin,
Milwaukee, WI.
Purpose: Anophthalmia and microphthalmia (A/M) are defined as
the complete absence or reduction in size of the eye and has a
combined incidence between 1-3.2 cases per 10,000 live births. A/M
is a genetically heterogeneous disorder that has been associated with
mutations in at least 35 genes with SOX2 representing the major
causative factor. Approximately 60% of A/M patients lack a
molecular diagnosis. We submitted nine SOX2-negative A/M patients
for whole exome sequencing to screen for mutations in known genes
as well as identify novel genes involved in A/M.
Methods: DNA samples from nine A/M patients were analyzed by
whole exome sequencing using Agilent Sure Select v4+UTR
platform for exome capture and PerkinElmer sequencing service.
Base/variant calls were made using the Geospiza software package
and patients were examined for mutations in 100 known ocular
genes.
Results: Heterozygous mutations, confirmed with Sanger
sequencing, were identified in three of the nine patients. A novel
deletion, c.651delC, predicted to lead to a frameshift,
p.(Thr218Hisfs*76), was identified in OTX2 in a patient with
bilateral anophthlamia. A novel substitution, c.158+1 G>A, predicted
to lead to aberrant splicing was identified in intron 3 of CRYBA4 in a
patient with microphthalmia, congenital cataracts, and coloboma of
the iris, cornea, and retina. Another novel mutation was identified in
the homeodomain of PAX6, c.767 T>C (p.Val256Ala), in a patient,
and his similarly affected brother, with microphthalmia, iris
hypoplasia, sclerocornea, coloboma, congenital glaucoma, and
aphakia.
Conclusions: The deletion in OTX2 is the most C-terminal deletion
reported to date. The c.158+1 G>A mutation is the first splice site
mutation identified in CRYBA4. To the best of our knowledge, this is
the first report of coloboma of the iris, cornea and retina in
association with CRYBA4 mutation. The p.Val256Ala PAX6 allele is
associated with a more severe phenotype than typically reported for
missense mutations and represents only the sixth missense variant to
be reported in the PAX6 homeodomain. Due to the severity of the
phenotype the absence of a second mutation was confirmed by
sequencing the complete PAX6 gene. Analysis of the remaining six
unexplained patients is ongoing. These results demonstrate the
efficiency of whole exome sequencing to identify causative mutations
for a genetically heterogeneous disorder, such as A/M.
Commercial Relationships: Brett Deml, None; Linda M. Reis,
None; Elena V. Semina, None
Support: NIH Grant EY015518 and Children’s Research Institute,
CHW, WI
Program Number: 3365 Poster Board Number: C0021
Presentation Time: 11:00 AM - 12:45 PM
Exome Sequencing Identification of a Novel Causal Mutation for
Eyelid Dysplasia
Elizabeth St.Germain1, Khanh-Nhat Tran-Viet1, Thomas P. Klemm5,
Vachiranee Limviphuvadh2, Sebastian Maurer-Stroh2, 3, Nicholas
Katsanis6, Yasmin Shayesteh4, 7, James Katowitz4, 7, Steve Rozen5,
Terri L. Young1, 5. 1Center for Human Genetics, Duke University,
Durham, NC; 2Agency for Science Technology and Research,
Bioinformatics Institute, Singapore, Singapore; 3School of Biological
Sciences, Nanyang Technological University, Singapore, Singapore;
4
Division of Pediatric Ophthalmology, The Children's Hospital of
Phiadelphia, The Scheie Eye Institute, Philadelphia, PA; 5DukeNational University of Singapore Graduate Medical School,
Singapore, Singapore; 6Center for Human Disease Modeling, Duke
University, Durham, NC; 7Raymond and Ruth Perelman School of
Medicine, University of Pennsylvania, Philadelphia, PA.
Purpose: Blepharophimosis, ptosis, and epicanthus inversus
syndrome (BPES) is a complex eyelid malformation. A novel
dominantly inherited BPES-like eyelid/conjunctival phenotype was
seen in an Ashkenazi Jewish family. The causal gene for BPES FOXL2- was sequenced, with no mutations detected. Deep
sequencing was performed to identify a causal variant for this novel
phenotype.
Methods: Six members were recruited for study (4 affected, 2
unaffected). The affected phenotype consisted of ptosis, lateral
canthal dystopia, telecanthus, absent lower eyelid lashes, epicanthus
tarsalis rather than inversus, normal horizontal palpebral fissure
lengths, and atypical lateral conjunctival ciliated epibulbar
keratinization. Pathology uniformly revealed inflamed conjunctiva.
Whole exome sequencing was performed with 2 affected DNA
samples. SNP and Variation Suite 7.5 software was used to filter
single nucleotide variants (SNVs) and micro-insertions/deletions
(indels). Public databases and internal exome controls were used to
filter out known variants. PCR and sequencing primers were designed
to confirm co-segregating variants with additional family members.
Variant allelic frequencies were determined by genotyping 672
ethnically matched controls.
Results: An average 50X coverage depth for all coding regions was
achieved. Eleven SNVs/indels passed filtering criteria. A segregating,
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
novel heterozygous change (c.868A>G) was detected in GALNT2
(chromosome 1q42.13), causing an arginine to glycine (R290G)
change in an evolutionarily conserved region. The variant was
negative in 672 controls. cDNA expression panels validated
GALNT2 presence in all human ocular and relevant systemic tissues.
In silico prediction tools (XFold and SIFT/ANNOTATOR) of the
p.R290G suggests both a moderate destabilizing effect of the crystal
structure (2.47 kcal/mol), and affects protein function (score
0.01/0.00), respectively.
Conclusions: We identified a novel causal gene for a BPES-like
eyelid/conjunctival dysplasia. GALNT2 functions in the mucin-type
O-glycan biosynthesis pathway affecting mucin expression in
conjunctival epithelial cells. GALNT2 is also involved in eyelid
development through the epidermal growth factor receptor (EGFR).
EGFR/MAPK/AP-1 signaling at the edge of eyelid keratinocytes is
required for normal eyelid development. Whole exome sequencing is
useful in causal gene discovery for rare ocular disorders.
Commercial Relationships: Elizabeth St.Germain, None; KhanhNhat Tran-Viet, Golden Helix (R); Thomas P. Klemm, None;
Vachiranee Limviphuvadh, None; Sebastian Maurer-Stroh,
None; Nicholas Katsanis, None; Yasmin Shayesteh, None; James
Katowitz, None; Steve Rozen, None; Terri L. Young, National
Institutes of Health (F)
Support: This research was funded by the-National Institutes of
Health Grant R01 EY014685, the Lew Wasserman Award from
Research to Prevent Blindness Inc., Chicago, Illinois, and a DukeNational University of Singapore core grant to Terri Young
Program Number: 3366 Poster Board Number: C0022
Presentation Time: 11:00 AM - 12:45 PM
Identification of Mutations in Candidate Genes in Patients with
Globe Anomalies: A Targeted Next-Generation Sequencing
Approach
Sushil K. Dubey1, Perumalsamy Vijayalakshmi2, Sharwan K. Kedia3,
Periasamy Sundaresan1. 1Genetics, Aravind Medical Research
Foundation, Madurai, India; 2Department of Pediatric
Ophthalmology, Aravind Eye Hospital, Madurai, India; 3Laxmi
Netralaya, Arrah, India.
Purpose: To find out the spectrum of genetic variations in candidate
genes in patients with microphthalmia, anophthalmia and coloboma
(MAC) in South Indian cohort.
Methods: We evaluated 85 MAC patients’ DNA samples for
mutations and sequence variants in the candidate genes by two
different approaches. We performed targeted re-sequencing to
determine the efficacy of next-generation sequencing for the rapid
screening of multiple genes in MAC patients. Targeted re-sequencing
of 67 genes was done in 20 pooled samples without bar-coding.
Targeted regions were captured on a 244K microarray platform and
sequencing was performed on Illumina/ Solexa platform. Data
analysis was done using SeqQC tool and reads were aligned against
Homo sapiens genome using BWA tool. Variation detection was
done using Sam tools. In addition, exons along with flanking exonintrons boundaries of RAX, OTX2, and SOX2 genes were screened
in 65 MAC samples by direct sequencing. Hundred ethnicallymatched control samples were sequenced to confirm the identified
variants as patient specific.
Results: The re-sequencing of 67 genes detected 80 insertions, 108
deletions and 988 SNPs/ substitution mutations and nine of these
variants were confirmed by direct sequencing as population specific
non-pathogenic changes. Screening by direct sequencing identified,
five novel mutations in RAX, OTX2, and SOX2 genes in this cohort.
A homozygous substitution mutation p. Arg179Trp, found in one
bilateral anophthalmia patient, was identified in RAX gene. Three
heterozygous mutations; p. Pro142Fs [c. 426insC (in a proband with
bilateral anophthalmia)], p. Thr186Fs [c. 558delC (in one proband
with microphthalmia in one eye and anophthalmia in other)] and a
compound heterozygote p. Gln104X, p. Gln106 His (in one patient
with microphthalmia) were identified in OTX2 gene. These three
mutations in OTX2 gene cause premature termination of the coding
sequence. Mutation p. Val303Val was identified in SOX2 gene in
only one patient with microphthalmia and iris coloboma.
Conclusions: >96% of the targeted regions were captured and
sequenced by NGS technology. Targeted re-sequencing identifies
1176 variants. Most of the variations would be Indian population
specific changes and may not be playing a role in causing the disease.
Five novel mutations identified in RAX, OTX2, and SOX2 suggest
the role of these genes in ocular development.
Commercial Relationships: Sushil K. Dubey, None; Perumalsamy
Vijayalakshmi, None; Sharwan K. Kedia, None; Periasamy
Sundaresan, None
Support: Indian Council of Medical Research, New Delhi, India
Program Number: 3367 Poster Board Number: C0023
Presentation Time: 11:00 AM - 12:45 PM
Whole Exome Sequencing (WES) indentifies a mutation in
ALPK1 responsible for a novel, autosomal dominant disorder of
vision loss, splenomegaly, and pancytopenia
Lloyd B. Williams1, Chad D. Huff4, Denise Morgan2, Rosann
Robinson2, Margaux A. Morrison2, Krista Kinard2, George Rodgers3,
Kathleen B. Digre2, Margaret M. DeAngelis2. 1Cornea Department,
Wilmer Eye Center, Baltimore, MD; 2Ophthalmology, University of
Utah, Salt Lake City, UT; 3Hematology, University of Utah, Salt
Lake City, UT; 4University of Utah, Salt Lake City, UT.
Purpose: Tantravahi et al. (2012) previously described a novel
inherited syndrome consisting of a cone-rod dystrophy, chronic optic
nerve edema and low-grade ocular inflammation, idiopathic massive
splenomegaly, mild pancytopenia, and anhidrosis with unknown
genetic mechanism. We conducted whole exome sequencing (WES)
on DNA from the members of this pedigree to elucidate the genetic
cause.
Methods: The three affected family members in this pedigree are: the
proband and her mother and half-sister. All family members had
extensive laboratory and clinical evaluations. DNA was collected
from all affected individuals and from the following unaffected: both
maternal grandparents, a maternal aunt, the proband’s father and her
two full sisters. DNA from the proband, her affected mother, her
unaffected father and one unaffected sister underwent WES. Target
enrichment was performed with the Illumina TruSeq Exome
Enrichment Kit followed by quantitative PCR (q-PCR) and
sequencing via Illumina HiSeq2000 101 Cycle Paired End
Sequencing. Analysis was performed using VAAST.
Results: VAAST identified 44 candidate variants. Sanger sequencing
confirmed a missense mutation in ALPK1 segregating with disease.
The mutation, chr4:113348736 C to T, results in a T237M amino acid
change in the ALPK1 protein. Bioinformatic analysis predicts that
this change is damaging to the protein and in a highly conserved
region. ALPK1 mutation is the only candidate SNP that segregated
with disease in the family. It was shown to be dominantly inherited
and appeared de novo in the mother of the proband. ALPK1 has not
been previously associated with retinal or ophthalmic disorders.
There are no known similar cases elsewhere in the literature. The
proband and her affected sister have different fathers. We, therefore,
conclude that this is a dominantly inherited mutation that arose
spontaneously in the mother of the proband.
Conclusions: The discovery of ALPK1 as the likely disease-causing
mutation in this syndrome is a success in using WES to identify the
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
genetic basis of a new ocular and systemic disorder. These data may
yield new insights into function of the alpha protein kinase family of
proteins, the molecular function of ALPK1 in photoreceptors, and a
new type of genetic interaction between ocular and hematologic
disorders.
Commercial Relationships: Lloyd B. Williams, None; Chad D.
Huff, None; Denise Morgan, None; Rosann Robinson, None;
Margaux A. Morrison, None; Krista Kinard, None; George
Rodgers, None; Kathleen B. Digre, None; Margaret M.
DeAngelis, None
Program Number: 3368 Poster Board Number: C0024
Presentation Time: 11:00 AM - 12:45 PM
Genetic diagnostic testing in a large cohort of retinitis
pigmentosa patients using panel-based next generation
sequencing
Julia Mohr1, Nicola Gloeckle1, Susanne Kohl2, Tim Scheurenbrand1,
Andrea Sprecher1, Antje S. Bernd3, Eberhart Zrenner3, Bernd
Wissinger2, Konstanze Hörtnagel1. 1CeGaT GmbH, Tübingen,
Germany; 2Centre for Ophthalmology, Molecular Genetics
Laboratory, University of Tübingen, Tübingen, Germany; 3Centre for
Ophthalmology, University of Tübingen, Tübingen, Germany.
Purpose: Worldwide, a total of two million patients are affected by
retinitis pigmentosa (RP). We used panel-based next generation
sequencing (NGS) to study the underlying genetic heterogeneity of
autosomal recessive and sporadic RP cases, and to evaluate its
applicability in a diagnostic setting.
Methods: A cohort of 150 patients with sporadic or suspected
autosomal recessive RP was selected. Using a customized enrichment
panel including 105 genes involved in retinal disease, the samples
were analyzed by NGS on a SOLiD 5500xl platform. Sequence reads
analyzed by LifeScope software were mapped to the human reference
genome. Annotation of the variants generated by LifeScope software
package was performed using the Ensembl database, dbSNP and inhouse variant databases. Indel calling was also supported. Variants
were selected for further analysis, if the global minor allele frequency
(dbSNP and Exome Variant Server) was below 5%. Validation of all
mutations identified by NGS as well as analysis of underrepresented
regions (<10 reads per base) was performed using Sanger sequencing.
To ensure an ideal and fast analysis, all genes currently known to be
associated with autosomal recessive RP were analyzed first. In
unsolved cases the analysis was expanded to all remaining genes of
the panel.
Results: In 53% of patients we were able to identify the causative
mutation(s), 12% of cases were inconclusive and 35% remained
unsolved. We observed mutations in 39 different genes, with
USH2A, EYS and RPGR being the most prevalent mutated genes. In
5 cases (3%) and in 11 cases (7%) we found causative mutations in
X-linked RP genes and in autosomal dominant RP genes,
respectively.
Conclusions: Like in other populations, we observed large genetic
heterogeneity among our cohort of RP patients. Our study
demonstrates that panel-based NGS is capable to uncover the
principle genetic cause in more than 50% of cases with apparently
sporadic and autosomal recessive RP. Interestingly, about 10% of
cases showed a pathogenic mutation that is most likely incompatible
with autosomal recessive inheritance. Therefore, genes associated
with an autosomal dominant or X-linked form of RP must also be
taken into account in patients with apparently sporadic RP which is
of major relevance for genetic counseling.
Commercial Relationships: Julia Mohr, CeGaT GmbH (E); Nicola
Gloeckle, CeGaT GmbH (E); Susanne Kohl, None; Tim
Scheurenbrand, CeGaT GmbH (E); Andrea Sprecher, CeGaT
GmbH (E); Antje S. Bernd, None; Eberhart Zrenner, Retina
Implant AG (F), Retina Implant AG (I), Retina Implant AG (C),
Retina Implant AG (P), QLT Inc (C), Servier, Paris (C), Steinbeis
GmbH&CoKG, Stuttgart (I), Steinbeis GmbH&CoKG, Stuttgart (C),
Neurotech, USA (C), Pfizer, USA (C); Bernd Wissinger, None;
Konstanze Hörtnagel, CeGaT (E)
Program Number: 3369 Poster Board Number: C0025
Presentation Time: 11:00 AM - 12:45 PM
Highly efficient genetic diagnostic testing in patients with
inherited retinal dystrophies using Panel-based Next Generation
Sequencing
Nicola Gloeckle1, Susanne Kohl2, Julia Mohr1, Tim Scheurenbrand1,
Andrea Sprecher1, Saskia Biskup1, Wolfgang Berger3, 4, Bernd
Wissinger2, John Neidhardt3. 1CeGaT GmbH, Tuebingen, Germany;
2
Centre for Ophthalmology, Molecular Genetics Laboratory,
University of Tuebingen, Tuebingen, Germany; 3Institute of Medical
Molecular Genetics, University of Zurich, Zurich, Germany; 4Zurich
Center for Integrative Human Physiology (ZIHP), University of
Zurich, Zurich, Germany.
Purpose: Genetic heterogeneity is a well known feature in Retinal
dystrophies (RD). Therefore next generation sequencing (NGS)
technology is the most promising tool to identify mutations in RD. To
date NGS is not routinely used in genetic diagnostic testing of RD
patients. Our aim was to establish a diagnostic pipeline for RD
patients using NGS.
Methods: 105 genes associated with RD were selected from
literature or databases. The genes were subdivided into 8 subpanels
according to the clinical diagnosis and the suspected mode of
inheritance. For analysis we performed a customized target-insolution-enrichment for exonic and flanking intronic region. The
SOLiD 5500xl platform was used for NGS. Sequence reads were
mapped to the human reference genome GRCh37/hg19. The variant
calls generated by LifeScope were annotated using Ensembl, dbSNP
and in-house variant databases. Indel calling was also supported.
Variants with a global minor allele frequency below 5% (based on
dbSNP and Exome Variant Server) were selected for further
investigation. All mutations were validated by conventional Sanger
sequencing. Regions with less than 10 reads per base were examined
by Sanger sequencing.
Results: We analyzed 160 patients with different forms of RD. The
majority of cases were diagnosed with retinitis pigmentosa (RP),
where we obtained a diagnostic detection rate of 58%. Within the
other monogenic forms of RD we detected 50% of solved cases. In
the syndromic forms of RP (Usher and Bardet-Biedl syndome) we
detected homozygous or compound heterozygous mutations in 74%.
Among all analyzed patients the genes USH2A, EYS, ABCA4 and
RPGR were more frequently affected. Furthermore, we found several
mutations in x-linked and dominant genes among the cases with
sporadic RD. These results imply consequences for counseling of
patients and families.
Conclusions: We have developed a diagnostic NGS pipeline which
allows us to detect the causative mutations in 50 to 74% of patients
with RD. Thus, the NGS-based gene diagnostics is a reliable and cost
efficient tool in genetically heterogeneous diseases.
Commercial Relationships: Nicola Gloeckle, CeGaT GmbH (E);
Susanne Kohl, None; Julia Mohr, CeGaT GmbH (E); Tim
Scheurenbrand, CeGaT GmbH (E); Andrea Sprecher, CeGaT
GmbH (E); Saskia Biskup, None; Wolfgang Berger, None; Bernd
Wissinger, None; John Neidhardt, None
Program Number: 3370 Poster Board Number: C0026
Presentation Time: 11:00 AM - 12:45 PM
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
Inherited eye disease gene chip study
Liping Yang. Peking university third hospital, Beijing, China.
Purpose: At present, more than 300 genes have been described in
association with inherited eye disease, including retinitis pigmentosa,
Leber congenital amaurosis, Glaucoma, Corneal dystrophy et al.
Which made genetic diagnosis of these diseases difficult and costly.
The aim of this study was to evaluate an inherited eye disease gene
chip (including 378 inherited eye disease causing genes) for its
application to the molecular diagnosis of inherited eye disease in Han
Chinese patient.
Methods: We analyzed 64 unrelated Han Chinese families with
retinitis pigmentosa. Among those 28 were diagnosed as autosomal
dominant retinitis pigmentosa, 12 were diagnosed as autosomal
recessive retinitis pigmentosa, 24 were diagnosed as X linked retinitis
pigmentosa. All mutations found were further confirmed with Sanger
sequencing.
Results: In the autosomal dominant retinitis pigmentosa families, the
gene chip detected the disease causing mutation in 16 of the 28
families, and RHO was found to be the most frequently mutated gene
in these families (14.29% of the adRP families). In the autosomal
recessive retinitis pigmentosa families, the gene chip detected the
disease causing mutation in 5 of the 12 families, with different
mutation in different families. In the X linked retinitis pigmentosa
families, the gene chip detected the disease causing mutation in 10 of
the 24 families, and RPGR was found to be the most frequently
mutated gene in these families (20.84% of the xlRP families). The
rate of false positives (microarray results not confirmed with
sequencing) was less than 5%.
Conclusions: The inherited eye disease gene chip is a quick, costefficient first step in the molecular diagnosis of Han Chinese patient
with inherited eye disease.
Commercial Relationships: Liping Yang, None
Support: NSFC 81170877
Program Number: 3371 Poster Board Number: C0027
Presentation Time: 11:00 AM - 12:45 PM
Carriers of degenerative retinal diseases can be easily identified
using target capture next generation sequencing
Shirel R. Weiss1, 4, Eran Eyal2, Mali Salmon-Divon2, Yoram Cohen4,
5
, Nitza Goldenberg-Cohen1, 3. 1The Krieger Eye Research
Laboratory, Felsenstein Medical Research Center- Tel Aviv
University,, Petah Tiqwa, Israel; 2Bioinformatics Laboratory, Cancer
Research Center, Chaim Sheba Medical Center, Tel HaShomer,
Ramat Gan, Israel; 3Pediatric Ophthalmology Unit, Schneider
Children’s Medical Center of Israel, Petach Tiqwa, Israel; 4Sackler
Faculty of Medicine, Tel Aviv University, Petach Tiqwa, Israel;
5
Department of Gynecology, The Gynecology Research Laboratory,
Chaim Sheba Medical Center, Tel HaShomer, Ramat Gan, Israel.
Purpose: Retinal degeneration ranks high among the many genetic
causes of blindness.
Mutations in multiple genes are responsible for a wide variety of
retinal dystrophy phenotypes, such as autosomal recessive Stargardt
disease, cone-rod dystrophy and retinitis pigmentosa. Here, we report
on the development of a new test to find all possible diseaseassociated variants in the coding sequences of 25 recessive
degenerative retinal diseases, based on target enrichment and next
generation sequencing (NGS).
Methods: 372 exonic regions from 25 target genes were enriched by
hybrid capture, sequenced by NGS to a depth of up to 0.36 gigabases,
and assessed with stringent bioinformatic filters.
Results: Two DNA samples of control subjects not known to suffer
from retinal degeneration or any other eye disease were investigated,
and 1 mutation diagnosed Leber Congenital Amuorosis (LCA)
patient. An average target coverage of 179x (ranging from 151-218x)
was achieved. 66.6% (45-93%) of nucleotides had at least 7x
coverage, and 59.6% (39-86%) had at least 20x coverage. The
enrichment factor which is the ratio of the coverage of the targeted
region versus the coverage of the genome outside the target region
was found to be 2331 (1768-3068). In the initial experiment we
found, 191 (153-229) SNPS and 8 (5-11) indels ,138 (136-141) with
median coverage > 37x, 95.5% of these (151-230) have been
previously reported in dbSNP (v.132). 21 (19-24) aberrations were
found in coding regions, of them 10 (9-12) resulted in changes of the
protein sequence.
Using the more sensitive blast alignment algorithm, we detected
10bps deletion in the CRB1 gene of the LCA patient located in a
deeply covered region.
Conclusions: Given the difficulties in clinical diagnosis of similar
phenotypes, this method proved efficient. It revealed the specific
genetic mutations underlying retinal diseases, potentially lowering
the costs of testing, and enabling broad screening for carriers and
affected patients. If made available to the general population, NGS
may be an economical and superior method of diagnosis, genetic
consultation and treatment for patients.
Commercial Relationships: Shirel R. Weiss, None; Eran Eyal,
None; Mali Salmon-Divon, None; Yoram Cohen, None; Nitza
Goldenberg-Cohen, None
Support: The Zanvyl and Isabelle Krieger Fund, Baltimore ,
Maryland
Program Number: 3372 Poster Board Number: C0028
Presentation Time: 11:00 AM - 12:45 PM
A dual approach for comprehensive genetic testing of ABCA4 in
Stargardt disease
Miriam Bauwens1, Caroline Van Cauwenbergh1, Sarah De Jaegere1,
Steve Lefever1, Barbara D'haene3, Filip Pattyn1, Bart P. Leroy1, 2,
Elfride De Baere1, Frauke Coppieters1. 1Center for Medical Genetics
Ghent, Ghent University, Ghent, Belgium; 2Department of
Ophthalmology, Ghent University Hospital, Ghent, Belgium;
3
Biogazelle, Ghent, Belgium.
Purpose: Stargardt disease (STGD1) is one of the most frequent
autosomal recessive retinal dystrophies, with a prevalence of
~1/8000. Manifestation of STGD1 occurs in the 1st-2nd decade and
causes a rapid decrease in visual function, ultimately resulting in
legal blindness.
So far, over 600 mutations have been found in the 7 kb coding region
of the disease gene ABCA4. Genetic testing is mostly limited to chip
analysis or Sanger sequencing of the coding region. To overcome the
high cost and limitations of this approach, we designed a
comprehensive molecular test based on quantitative PCR (qPCR) and
massive parallel sequencing (MPS) to screen for single nucleotide
and copy number variation (CNV).
Methods: First, 50 assays were designed to cover all ABCA4 exons
and intron-exon boundaries. Following qPCR amplification (LC480,
Roche), ligation and shearing, amplicon pools were sequenced on a
Miseq run (Illumina). Twelve STGD1 patients were included.
Previous pre-screening (Asper chip and Sanger sequencing of the
coding region) revealed a molecular diagnosis in 4 patients and a
single heterozygous mutation in 3 patients.
CNV screening comprised 50 qPCR assays performed in 48 STGD1
patients with 1 heterozygous (18) or no mutation (30) in ABCA4
following ABCA4 chip and/or sequencing, and 27 controls (qBase
Plus, Biogazelle).
Results: MPS of 50 ABCA4 amplicons resulted in a minimal
coverage of 20x for 98% of the amplicons. A variant allele frequency
threshold of 25% allowed detection of 99% of the previously
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
identified variants in sufficiently covered regions. In addition, a
previously undetected heterozygous mutation was identified
(p.L2033R), resulting in a complete molecular diagnosis for one
patient.
CNV analysis of ABCA4 revealed a novel heterozygous deletion in a
patient heterozygous for a splice site mutation. The deletion covers
minimally 1.8 kb and maximally 5.6 kb, spanning exon 20-22.
Breakpoint delineation is currently ongoing.
Conclusions: MPS for ABCA4 proves to be a cost-efficient
alternative to Sanger sequencing and chip analysis. In addition, qPCR
screening allowed to identify a novel ABCA4 deletion. Although
standard ABCA4 molecular testing does not include CNV analysis,
this might be useful as in up to 30% of STGD1 patients only 1 or no
coding mutation can be detected by regular ABCA4 testing.
Nonetheless, the detection of only 1 CNV in 48 pre-screened cases
might suggest variations in non-coding regions.
Commercial Relationships: Miriam Bauwens, None; Caroline
Van Cauwenbergh, None; Sarah De Jaegere, None; Steve Lefever,
None; Barbara D'haene, None; Filip Pattyn, None; Bart P. Leroy,
None; Elfride De Baere, None; Frauke Coppieters, None
Support: Research Foundation Flanders (FWO) - Funds for Research
in Ophthalmology (FRO) - BOF Ghent University
Program Number: 3373 Poster Board Number: C0029
Presentation Time: 11:00 AM - 12:45 PM
High-coverage next-generation sequencing (NGS) for retinal
dystrophies and Usher syndrome: High diagnostic yield, CNV
detection, novel disease mechanisms and therapy targets
Hanno J. Bolz1, 2, Christine Neuhaus1, Markus N. Preising3, Arif O.
Khan4, Martin Gliem5, Peter Charbel Issa5, Uwe Wolfrum6, Andreas
Gal7, Birgit Lorenz3, Tobias Eisenberger1. 1Center for Human
Genetics, Bioscientia, Ingelheim, Germany; 2Institute of Human
Genetics, University Hospital of Cologne, Cologne, Germany;
3
Department of Ophthalmology, Justus-Liebig-University Giessen,
Universitätsklinikum Giessen and Marburg GmbH, Giessen Campus,
Giessen, Germany; 4Division of Pediatric Ophthalmology, King
Khaled Eye Specialist Hospital, Riyadh, Saudi Arabia; 5Department
of Ophthalmology, University of Bonn, Bonn, Germany; 6Cell and
Matrix Biology, Institute of Zoology, Johannes Gutenberg University
of Mainz, Mainz, Germany; 7Institute of Human Genetics, University
Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Purpose: Comprehensive genetic analysis for genetically
heterogeneous retinal dystrophies (RD): RP, early-onset severe RD
(EOSRD/LCA), macular dystrophies (MD), Usher syndrome (USH).
Methods: With NGS on the Illumina MiSeq system, we targeted the
exons of 31 autosomal recessive (ar)RP (413), 23 autosomal
dominant (ad)RP (248), 16 EOSRD (215) and 10 USH genes (373)
and analyzed 126 RD and 57 USH patients. A bioinformatic pipeline
was established for quantitative analysis of NGS data to detect copy
number variations (CNVs). NGS was subsequent to Sanger
sequencing (SaS) and MLPA in USH2A-negative USH2, and the
initial analysis in atypical USH or USH1.
Results: High coverage of Ø300-400 reads per exon was obtained,
and mutations identified in most RD patients: arRP: 72%; adRP:
81%; MD: 57%; EOSRD: 53%. CNVs were confirmed by MLPA
analysis. Heterozygous CNVs unmasked apparently homozygous
mutations as hemizygous. CNVs and point mutations in non-coding
exon 1 of EYS were identified in trans to coding mutations.
Heterozygous PRPF31 CNVs in sporadic patients shifted diagnosis
from arRP to adRP with incomplete penetrance. RP1 was the major
arRP gene. Truncating C-terminal RP1 alleles appeared to be nonpathogenic in ar families with biallelic mutations in secondary genes.
SaS and NGS identified mutations in 95% of USH2 and 94% of
atypical/USH1. CNVs accounted for 11% of USH2A alleles. We
show PTC124-induced read-through for a novel USH2A hot spot
mutation (23% of alleles), p.Trp3955*. Combined homozygous
mutations in OTOA and NR2E3 phenocopied USH1 in one patient.
Conclusions: NGS of RD genes dramatically increases the diagnostic
yield, improving genetic counseling. CNV detection from NGS data
highlights the method’s potential beyond sequencing. It avoids
pitfalls of SaS in consanguineous families with hemizygous
mutations, identifies the “missing hit” in recessive RD with
monoallelic mutations and uncovers novel candidate disease exons,
illustrating the benefit of 5’-UTR inclusion in NGS of disease gene or
exome panels. The discovery of non-pathogenic truncating RP1
alleles in RDs due to mutations in other genes underscores the
necessity to consider the variant load of all genes to avoid false
interpretation. A new USH2A hot spot mutation was accessible to
PTC124-induced readthrough and represents a major therapy target
for USH2.
Commercial Relationships: Hanno J. Bolz, Bioscientia (E);
Christine Neuhaus, Bioscientia (E); Markus N. Preising, None;
Arif O. Khan, None; Martin Gliem, Heidelberg Engineering,
Germany (F), Carl Zeiss Meditec, Germany (F), Optos, UK (F);
Peter Charbel Issa, Heidelberg Engineering (F); Uwe Wolfrum,
None; Andreas Gal, None; Birgit Lorenz, Optos (F); Tobias
Eisenberger, Bioscientia GmbH (E)
Program Number: 3374 Poster Board Number: C0030
Presentation Time: 11:00 AM - 12:45 PM
A Comparison of Two Commercially Available Genetic Tests for
Age-Related Macular Degeneration
Nancy M. Holekamp1, Mathew MacCumber2, Arghavan Almony3.
1
Ophthalmology, Pepose Vision Institute, Saint Louis, MO;
2
Ophthalmology, Rush University, Chicago, IL; 3Ophthalmology,
Carolina Eye Associates, Southern Pines, NC.
Purpose: To report on a series of patients in which two commercially
available genetic tests for age-related macular degeneration (AMD)
were performed on each subject.
Methods: One hundred and three consecutive patients were
prospectively enrolled in this multi-center, IRB-approved study.
Patients presented with no clinical evidence of AMD or early,
intermediate, or advanced AMD. Color fundus photographs, smoking
history, family history, weight and height were obtained. Two
commercially available genetic tests that identify alleles associated
with AMD were performed on each patient: Macula Risk (Arctic Dx,
Toronto) and RetnaGene (Sequenom CMM, San Diego).
Results: Out of 103 patients, the company-provided risk calculations
for advanced AMD resulting from the two tests were discordant 61
times. In all but one of the discordant cases, the RetnaGene results
predicted higher risk than Macula Risk. The CFH haplotype
identification between the two tests was discordant 62 times. Macula
Risk never produced H3, a moderate risk allele, and appeared to misidentify it as H4, a protective allele.
Conclusions: Two commercially available genetic tests for
prediction of advanced AMD produced discordant results about 60%
of the time. This may have been due, in part, to a technical problem
with Macula Risk distinguishing between H3 and H4 CHF alleles.
Commercial Relationships: Nancy M. Holekamp, Sequenom (C),
Sequenom (R), Sequenom (F), Arctic Dx (F), Allergan (C), Alimera
(C), Notal Vision (C), Notal Vision (F), Regeneron (C), Regeneron
(R), Genentech (C), Genentech (R); Mathew MacCumber,
Genentech (C), Regeneron (C), Allergan (C), Thrombogenics (C),
Optos (C), Sequenom (C), ArcticDx (C); Arghavan Almony, None
Support: Lifelong Vision Foundation
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
Program Number: 3375 Poster Board Number: C0031
Presentation Time: 11:00 AM - 12:45 PM
Evaluation of Genetic Testing Outcomes for a 4-year Interval of
Service Provided to Southwest Eye Registry Participants
Kaylie D. Webb, Dianna K. Wheaton. SW Eye Registry, Retina
Foundation of the Southwest, Dallas, TX.
Purpose: The Southwest Eye Registry (SER), a regional database of
patients with inherited eye diseases, is composed of clinical and
genetic data. The registry collects blood samples for genetic testing,
genotype-phenotype studies and to advance new gene discovery; an
overarching goal is to identify the genetic cause of disease for each
registry participant. Successful genotyping can differ from expected
disease-causing mutation detection rates and said rates vary
according to clinical diagnosis (e.g., ~65% of autosomal dominant
RP cases [Bowne IOVS, 2011], ~80% of x-linked RP cases [Sharon
Am J Hum Genet, 2003], and ~79% of Stargardt cases [Downes Arch
Ophthalmol, 2012]). Described herein is a 4-year interval of practicebased genetic testing outcomes.
Methods: The total database size is 4,241 affected and at-risk
relatives; ~200 new participants are added per year. DNA is prepped
onsite and sent for genetic testing at collaborating clinical diagnostic
and research labs according to disease diagnosis. The SER attempts
to genotype registry participants using resources available even in
cases with phenotypic ambiguity. The majority of samples (98%) are
submitted under research protocols rather than fee-for-service
submission. A 4-year interval (2008 - 2012) of genetic testing
services was evaluated to ascertain how many participants received
services, how many DNA samples were submitted for testing, and
whether a successful genetic testing outcome was achieved.
Results: During the study interval 803 participants were added to the
registry. DNA samples from 709 individuals were sent to one of six
centers for genotype analysis; mutation results were returned for 53%
of the submitted samples. A gene mutation was identified in 81% of
achromatopsia, 74% of choroideremia, 65% of x-linked RP, 50% of
autosomal dominant RP, 49% of Stargardt disease, and 24% of
Lebers congenital amaurosis. Observed mutation detection rates were
lower than expected (z>2.15, p<0.025) except for achromatopsia
(z>0.322, ns). Factors influencing deviations in rates included:
indeterminate phenotype, limited testing options, cost of testing, and
genotyping method.
Conclusions: Improved knowledge of genotype-phenotype
relationships and advances in mutation detection/gene discovery
techniques will improve genetic testing success rates for registry
participants.
Commercial Relationships: Kaylie D. Webb, None; Dianna K.
Wheaton, None
Support: Foundation Fighting Blindness C-CL-0812-0594-RFSW02
Program Number: 3376 Poster Board Number: C0032
Presentation Time: 11:00 AM - 12:45 PM
Positive and Unlabeled Learning for Prioritizing Candidate
Variants in Retinal Degenerative Diseases
Alex H. Wagner1, 2, Kyle Taylor2, Adam P. DeLuca2, Thomas
Casavant2, Edwin M. Stone2, Robert F. Mullins2, Todd E. Scheetz2,
Terry A. Braun2. 1Genetics, University of Iowa, Iowa City, IA;
2
Ophthalmology and Visual Sciences, University of Iowa, Iowa City,
IA.
Purpose: Exome sequencing experiments are useful in identifying
genetic variants that are causative of rare diseases with extreme
genetic heterogeneity, such as retinal degenerative diseases.
However, the number of plausible disease-causing variations
identified in such studies are often overwhelming, even after applying
expert-guided filtering steps. Thus, we seek to prioritize lists of
candidate variants identified in exome sequencing studies by their
likelihood to contribute to a retinal degenerative disease phenotype.
Methods: We leveraged publicly available data to quantitatively
describe all genes in the context of relevant features. Specifically, we
selected microarray data of gene expression in 10 tissues of the
human eye, RNA-seq data in human retinal tissue and 16 other body
tissues, and ChIP-seq data profiling CRX binding sites across the
genome. We developed a novel method (Positive and Unlabeled
Learning for Prioritization—PULP) to rank candidate disease-causing
variants, and trained it on these data.
Results: Using a Monte Carlo simulation, PULP was shown to
perform significantly better than random gene ordering (p < 1 x 106). Thirteen published RP linkages, all with identified diseasecausing genes, were used to evaluate our system. The causative gene
was prioritized at the top of the list 62% of the time, and within the
top 5 results 77% of the time. These results were highly significant (p
= 6 x 10-6). In addition, we demonstrate that our algorithm
outperforms ENDEAVOUR, a current state-of-the-art technique in
this field, which had successfully prioritized an RP causative variant
in a familial study on Usher’s Syndrome. Our system also
successfully prioritized the RP-causing DHDDS variant as the #1
candidate from a list of 20 from a recent exome study, and prioritized
a recently identified RP-causing variant in MAK to #4 in a list of 348
candidates.
Conclusions: The PULP retinal degenerative disease model
represents a huge step forward in the identification of retinal
degenerative disease variants from exome sequencing studies.
Moreover, this technique represents an unbiased, generalizable
approach to integrating disease-specific quantitative genetic features
for the purpose of disease-associated rankings in candidate lists.
Commercial Relationships: Alex H. Wagner, None; Kyle Taylor,
None; Adam P. DeLuca, None; Thomas Casavant, None; Edwin
M. Stone, None; Robert F. Mullins, Alcon Research Ltd (F); Todd
E. Scheetz, None; Terry A. Braun, Alcon Research, LTD (F)
Support: NIGMS Predoctoral Training Grant T32GM082729
Program Number: 3377 Poster Board Number: C0033
Presentation Time: 11:00 AM - 12:45 PM
A whole exome variant filtering software for identification of
disease causing variants
Bruno Maranhao1, 2, Pooja Biswas2, Gabriel A. Silva1, 2, John R.
Heckenlively3, S. Amer Riazuddin4, 5, Pauline Lee2, Radha Ayyagari2.
1
Bioengineering, University of California, San Diego, La Jolla, CA;
2
Ophthalmology, University of California, San Diego, La Jolla, CA;
3
Ophthalmology and Visual Sciences, University of Michigan
Medical School, Ann Arbor, MI; 4National Centre for Excellence in
Molecular Biology, Lahore, Pakistan; 5Center for Corneal Genetics,
The Wilmer Eye Institute, John Hopkins University School of
Medicine, Baltimore, MD.
Purpose: To develop and validate a software program that can
efficiently analyze sequence changes identified by whole exome
and/or whole genome sequencing to detect causal variants.
Methods: exomeSuite is a freely available multi-platform application
we have designed to analyze exomes of both single familial cases and
large cohorts consisting of multiple pedigrees or a group of unrelated
individuals. The software identifies candidate genes that exhibit
sequence variants consistent with the predicted pattern(s) of
inheritance. The candidate gene list can be further filtered for any
number and combination of user specified criteria which are limited
only by the information provided in the input files (e.g. gene minor
allele frequency, absence or presence in dbSNP, and 1000 genomes,
etc). Furthermore, we have integrated into the software the ability for
users to filter the data by the annotation of the missense mutation
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
predicted by PolyPhen, and the EST profile in the NCBI UniGene
Database. Exome variants of three pedigrees with retinal
degeneration were analyzed using exomeSuite.
Results: exomeSuite software filters data for sequence variations
(SNVs and indels) following either dominant, recessive homozygous,
or compound heterozygous pattern of inheritance for the monogenic
disease of interest. This software can also be used for homozygosity
mapping and for the identification of variants associated with genetic
traits in a population. In addition, the user may define a list of “genes
of interest” to filter results; the software by default includes a list of
RetNet genes. Additionally, exomeSuite can build databases of
sequenced exomes and filter results based on allele frequency within
the database.
Results will vary between pedigrees and based on the number of
exomes sequenced, the inheritance pattern filtered for, and on user
defined filters. Typically we have filtered over 50,000 variant calls to
fewer than 10, in some instances just one or two.
Analysis of the three pedigrees used to validate exomeSuite led to the
identification of causative mutations in the USH2A, RPE65 and
RBP4 genes.
Conclusions: exomeSuite rapidly enables users with little to no
computer programming and/or bioinformatics experience to filter
large datasets for variants segregating with disease, allowing virtually
anyone to capitalize on the ever increasing throughput and decreasing
cost of next generation sequencing technologies.
Commercial Relationships: Bruno Maranhao, None; Pooja
Biswas, None; Gabriel A. Silva, None; John R. Heckenlively,
None; S. Amer Riazuddin, None; Pauline Lee, None; Radha
Ayyagari, None
Support: FFB, RPB, RO1- EY013198, RO1-EY021237, P30EY22589.
Program Number: 3378 Poster Board Number: C0034
Presentation Time: 11:00 AM - 12:45 PM
Detection of sample contamination in clinical next-generation
sequencing
Todd E. Scheetz1, 2, Adam P. DeLuca2, Edwin M. Stone1, Terry A.
Braun1, 2. 1Ophthalmology, University of Iowa, Iowa City, IA;
2
Biomedical Engineering, University of Iowa, Iowa City, IA.
Purpose: To detect contamination of genomic DNA samples used in
next-generation sequencing applications. To take advantage of the
abundance of third party sequencing solutions, it is important to be
able to ensure that any variations detected result from the correct
patient sample. Known genotype fingerprints can help validate
sample identity. But additional quantitative measures are required to
ensure sample integrity.
Methods: The Genome Analysis Toolkit (GATK) from the Broad
Institute was used to call variations. The relative number of
supporting reads (supporting / cover) was calculated for each
variation. The distribution of the relative number of reads supporting
each variant was compared to a distribution derived from a cohort of
control samples. Contamination was detected as an increase in
variations with a relative number of supporting reads below 35%.
Results: We have developed and implemented a systematic approach
for identifying contamination in samples used in next-generation
sequencing experiments. The distribution of relative supporting reads
for a few dozen exomes is shown in Figure 1 below. Noncontaminated samples are shown with solid black lines.
Contamination presents as a substantial and distinctive increase in the
fraction of variations found below 50%. Two samples (large dashed
lines) are clearly contaminated, and two other samples (small dashed
lines) exhibit an indication of potential contaminated. We are actively
evaluating exomes from several large whole-exome sequencing
projects. Together with our collaborators we will be validating
samples that appear contaminated to evaluate our algorithm’s
specificity and sensitivity.
Conclusions: We have developed a simple method for identifying
contaminated samples in exome sequencing experiments. Further
research in this area is needed to determine the power of this method
in identifying and quantifying the extent of contamination, and the
amount of contamination that can be tolerated without compromising
accuracy.
The distribution of variations by the fraction of reads supporting the
variation. Heterozygous variations are expected at 50%, and
homozygous variations at 100%.
Commercial Relationships: Todd E. Scheetz, None; Adam P.
DeLuca, None; Edwin M. Stone, None; Terry A. Braun, Alcon
Research, LTD (F)
Support: Foundation Fighting Blindness Grant BR-GE-0608-0457UIA
Program Number: 3379 Poster Board Number: C0035
Presentation Time: 11:00 AM - 12:45 PM
Towards Comprehensive Registration of DNA Sequence Variants
Associated with Inherited Retinal Diseases in Leiden Open
Variation Databases
Frans P. Cremers1, 2, Johan T. den Dunnen3, Muhammad Ajmal1, 2,
Alamdar Hussain2, Muhammad I. Khan1, 2, Markus N. Preising5,
Stephen P. Daiger6, Raheel Qamar2, 4. 1Department of Human
Genetics, Radboud University Medical Centre, Nijmegen,
Netherlands; 2Biosciences, COMSATS Institute of Information
Technology, Nijmegen, Pakistan; 3Center for Human and Clinical
Genetics, Leiden University Medical Center, Leiden, Netherlands;
4
Shifa College of Medicine, Shifa Tameer-e-Millat University,
Islamabad, Pakistan; 5Department of Ophthalmology, Justus-Liebig
University, Giessen, Germany; 6Human Genetics Center, University
of Texas Health Science Center, Houston, TX.
Purpose: Inherited retinal diseases (RD) display an impressive
degree of allelic and genetic heterogeneity as nearly 10,000 mutations
in >190 genes have been identified. Mutations in these genes account
for 30% to 90% of cases, depending on the type of disease.
Comprehensive genotyping of persons with inherited RD improves
genetic counseling and the accuracy of disease prognoses. Moreover,
genotyping identifies persons who are eligible for novel therapies.
We are entering an era of routine testing for RD-associated defects,
both in academic and non-academic centers. The identified known
and novel variants are not published or deposited in open access
databases. Sharing sequence variants and their associated phenotypes
are at the heart of DNA diagnostics and it therefore is of utmost
importance to register this information in publicly available
databases.
Methods: The structure and use of RD-mutation databases need to
meet the following criteria: 1). Web-based open access; 2).
Registration of all published sequence variants; 3). Easy upload of
new variants; 4). Accurate assessment of mutation data; 5). Regular
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
updating. We propose the implementation of Leiden Open Variation
Databases (LOVDs) for all RD genes in the next five years. LOVDs
were previously created for 10 Usher syndrome-associated genes. A
team of BS students and staff members in Islamabad, will collect all
published sequence variants for the remaining RD genes, scrutinize
them for their proper annotations, and upload them in gene-specific
LOVDs. World-wide curators will check the new entries.
Results: ‘Empty’ LOVDs were created for all RD associated genes,
and all published variants were registered for AIPL1, LCA5, RDH5,
SEMA4A, and TULP1. Other mutation repositories previously were
created for CEP290, NDP, and Bardet-Biedl syndrome-associated
genes. These will be taken up in LOVDs. In 2013, variants of another
25 RD-associated genes will be deposited in LOVDs and the existing
LOVDs will be updated every year.
Conclusions: The long-term success of this endeavor relies on a
robust organization of sequence variant updating, proper curation,
database maintenance, and a sound financial basis. It will also be
vital to introduce compulsory deposition of sequence variants prior to
publication submissions, and the compliance of diagnostic facilities
worldwide to deposit unpublished variants in LOVDs.
Commercial Relationships: Frans P. Cremers, None; Johan T.
den Dunnen, None; Muhammad Ajmal, None; Alamdar Hussain,
None; Muhammad I. Khan, None; Markus N. Preising, None;
Stephen P. Daiger, None; Raheel Qamar, None
Program Number: 3380 Poster Board Number: C0036
Presentation Time: 11:00 AM - 12:45 PM
Challenges of Microbiome Research on the Ocular Surface
Diane L. Smith1, 2, Suzanne Kennedy3, Richard A. Gibbs4, 5, Dan B.
Jones6, Cintia S. De Paiva6, Stephen C. Pflugfelder6, Joseph F.
Petrosino2, 7. 1Interdepartmental Program in Translational Biology
and Molecular Medicine, Baylor College of Medicine, Houston, TX;
2
Molecular Virology and Microbiology, Baylor College of Medicine,
Houston, TX; 3MO-BIO Laboratories, Carlsbad, CA; 4Human
Genome Sequencing Center, Baylor College of Medicine, Houston,
TX; 5Molecular and Human Genetics, Baylor College of Medicine,
Houston, TX; 6Ophthalmology, Baylor College of Medicine,
Houston, TX; 7Center for Metagenomics and Microbiome Research,
Baylor College of Medicine, Houston, TX.
Purpose: To apply culture-independent techniques for the
characterization of bacterial communities colonizing the ocular
surface.
Methods: Samples were collected from the right inferior tarsus of
three subjects with the Opia EYEPRIM® membrane. Genomic DNA
extraction was performed with a modified protocol for the MO-BIO
PowerLyzer PowerSoil DNA Isolation Kit on each sample as well as
on a blank membrane. Amplification of variable regions V1-V3 of
the 16S rRNA gene was performed with the 27F and 534R primers
used for the Human Microbiome Project (HMP). The BCM Human
Genome Sequencing Center performed Roche 454 pryosequencing
on pooled amplicons. Data analysis was performed using mothur.
Results: Working with MO-BIO Laboratories, we developed a
method for extracting genomic DNA from low-biomass samples
without measureable background. Furthermore, the yield was high
enough that whole genome amplification (WGA) was not necessary
prior to sequencing. 16S rRNA gene sequencing from EYEPRIM®
membranes collected from three subjects produced 130 reads on
average that were normalized to 22 reads per sample and classified
into 5 phyla (Actinobacteria, Bacteroides, Fusobacteria,
Proteobacteria, and Firmicutes). Proteobacteria was the most
abundant phylum in all 3 subjects, comprising at least 50% of the
microbiota. Sequences classified to the genus level revealed genera
such as Propionibacterium, Escherichia, and Staphylococcus, which
were previously cultured from the ocular surface.
Conclusions: 16S rRNA gene sequencing without WGA identified
bacterial genera that include those cultured from the ocular surface.
Furthermore, additional genera were also identified as members of
the ocular surface microbiota and will be confirmed through
longitudinal sampling. Though we were able to perform 16S rRNA
sequencing to characterize the microbiota of the ocular surface, the
sequencing output was not as robust as with other sample types.
Pending whole genome shotgun sequencing will maximize the use of
these low-yield samples and characterize species and gene content in
the ocular surface microbiome.
Commercial Relationships: Diane L. Smith, None; Suzanne
Kennedy, None; Richard A. Gibbs, None; Dan B. Jones, None;
Cintia S. De Paiva, Glaxo Smith Kline (C), Baylor College of
Medicine (P); Stephen C. Pflugfelder, Allergan (C), Glaxo Smith
Kline (C), Bausch and Lomb (C), Baylor College of Medicine (P);
Joseph F. Petrosino, None
Support: NEI grant T32-EY07001-36
Program Number: 3381 Poster Board Number: C0037
Presentation Time: 11:00 AM - 12:45 PM
Utilizing Golden Helix SVS Software to Facilitate in the
Identification of Genes for Ocular Diseases
Khanh-Nhat Tran-Viet1, Elizabeth St.Germain1, Greta L. Peterson2,
Autumn Laughbaum2, Vincent J. Soler3, Tammy Yanovitch4, Steve
Rozen5, Terri L. Young1, 5. 1Center for Human Genetics, Duke
University, Durham, NC; 2Golden Helix, Bozeman, MT; 3UMRS
563, Centre de Physiopathologie de Toulouse Purpan, Université Paul
Sabatier, Toulouse, France; 4The Department of Ophthalmology,
University of Oklahoma/Dean McGee Eye Institute, Oklahoma City,
OK; 5Duke-National University of Singapore Graduate Medical
School, Singapore, Singapore.
Purpose: Technological advances in next generation sequencing
provide clinicians and researchers with more effective methods to
identify pathogenic gene mutations for heritable diseases. To date,
the National Eye Institute Bank lists over 450 genes associated with
eye-related disorders. There are a plethora of applications that can be
used to determine causal gene variants ranging from targeted
resequencing, exome sequencing, and whole genome sequencing
strategies. Analytical processing of the large data sets generated can
be cumbersome for all parties involved and some issues that can
cause inefficiencies include learning programming languages and
reliance on inconsistent freeware. Herein, we demonstrate the ability
to utilize a commercially available software tool to help identify
known and novel genes for various ocular diseases.
Methods: 84 DNA samples from patients with various ocular
phenotypes underwent whole exome sequencing. Sequencing results
were aligned, and cleaned. Variants were called using BurrowsWheeler Aligner (BWA), SAMtools, and Genome Analysis Toolkit
(GATK), respectively. Variant calling files generated were imported
into the Golden Helix SNP & Variation Suite (SVS) v7.6.11 program
for analysis. Multiple filtering strategies were employed based on
mode of inheritance. Current public databases and internal exome
controls were used in conjunction to assist in the filtering process.
Variant(s) of interest were validated using Sanger Sequencing,
bioinformatics, and functional studies.
Results: The following mutations were identified: a novel nonsense
mutation in a Stickler/Wagner phenotype, compound heterozygous
mutations in CYP1B1 in a family with heritable primary congenital
glaucoma, a novel premature stop codon in a high-grade myopia
family, and a de novo mutation for a rare corneal intraepithelial
dyskeratosis.
Conclusions: Appropriate systematic filtering techniques using SVS
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
efficiently identified an array of mutations. SVS provides the enduser with a simple, transparent method to identify mutations in ocular
phenotypes. Clinicians and researchers now have tools with simple
click-based filtering methods to analyze, identify, and perform
complex analyses, eliminating the need to rely heavily on
bioinformatics infrastructure. Filtering and validation of variants are
currently underway for other ocular phenotypes.
Commercial Relationships: Khanh-Nhat Tran-Viet, Golden Helix
(R); Elizabeth St.Germain, None; Greta L. Peterson, Golden
Helix, Inc. (E); Autumn Laughbaum, Golden Helix (E); Vincent J.
Soler, None; Tammy Yanovitch, None; Steve Rozen, None; Terri
L. Young, National Institutes of Health (F)
Support: National Institutes of Health Grant R01 EY014685, the
Lew Wasserman Award from Research to Prevent Blindness Inc.,
Chicago, Illinois, and a Duke-National University of Singapore core
grant to Terri Young
Program Number: 3382 Poster Board Number: C0038
Presentation Time: 11:00 AM - 12:45 PM
Vision Variation Database (VVD)
Adam P. DeLuca1, Sean Ephraim1, Todd E. Scheetz2, 1, Edwin M.
Stone2, 3, Terry A. Braun1. 1Biomedical Engineering, University of
Iowa, Iowa City, IA; 2Department of Ophthalmology and Visual
Sciences, University of Iowa, Iowa City, IA; 3Howard Hughes
Medical Institute, Iowa City, IA.
Purpose: Genetic testing has dramatically changed with the
introduction of clinical exome and targeted exon sequencing. The
burden has shifted from generating genotypes to interpreting
variations. There are vast amounts of publically available data to aid
this interpretation. However, currently there are no centralized
resources to capture and annotate variations observed in patients with
inherited eye diseases. To address these issues in clinical genetic
testing of retinal disease genes, we have developed the Vision
Variation Database (VVD) that incorporates 1) reported and
unpublished disease-causing variants, 2) publically available allele
frequency data from control and diseased populations, and 3)
pathogenicity prediction scores.
Methods: A manually curated list of retinal disease-causing variants
has been collected from the literature and from unpublished data to
provide interpretation for clinical genetic testing. Allele frequencies
were obtained from the snp135 table in the UCSC genome database
and the NHLBI GO Exome Sequencing Project. Pathogenicity
predictions from SIFT, Polyphen2, MutationTaster, and LRT for
coding single nucleotide variations were acquired from dbNSFP.
Sequence reads from the 1000 genomes project, the Ciliopathies
Exome Sequencing Initiative (phs000288.v1.p1), and local exome
samples were aligned using BWA. Variants were called using the
GATK UnifiedGenotyper and annotated. Summary data from these
sets are provided including allele frequencies and frequencies of
compound heterozygotes. To reduce false positives in interpreting
exome results minimal filtering is performed on these recalled
datasets.
Results: Access to these data is provided via a public website
available at: VVD.eng.uiowa.edu The interface allows users to
navigate to a variant by gene and an API is provided. The interface
allows users to view pathogenicity predictions, and allele frequencies
for disease-causing variants and polymorphisms in retinal disease
genes. The site currently lists 1522 causative variants in 49 retinal
disease genes.
Conclusions: We have created a public resource to catalogue and aid
in the interpretation of variants in retinal disease genes. As more
exome and genome sequence data becomes available, data on specific
disease causing alleles and variants will accumulate and will enable
the refinement of disease sub-types.
Commercial Relationships: Adam P. DeLuca, None; Sean
Ephraim, None; Todd E. Scheetz, None; Edwin M. Stone, None;
Terry A. Braun, Alcon Research, LTD (F)
Program Number: 3383 Poster Board Number: C0039
Presentation Time: 11:00 AM - 12:45 PM
The Ocular Tissue Database
Terry A. Braun1, 5, Alex H. Wagner4, 5, Adam P. DeLuca3, 5, Thomas
Casavant3, 5, Todd E. Scheetz2, 7, Abbot F. Clark8, 9, Robert F.
Mullins2, 7, Edwin M. Stone6, 7. 1Ophthal/Biomed Eng, Univ of Iowa,
Iowa City, IA; 2Ophthalmology and Visual Sciences, Univ of Iowa,
Iowa City, IA; 3Biomedical Engineering, Univ of Iowa, Iowa City,
IA; 4Genetics, Univ of Iowa, Iowa City, IA; 5Center for
Bioinformatics and Computational Biology, Univ of Iowa, Iowa City,
IA; 6Howard Hughes Medical Institute, Univ of Iowa, Iowa City, IA;
7
Institute for Vision Research, Univ of Iowa, Iowa City, IA;
8
Department of Cell Biology and Anatomy, Univ North Texas Health
Science Center, Fort Worth, TX; 9North Texas Eye Research
Institute, Univ North Texas Health Science Center, Fort Worth, TX.
Purpose: Evaluating gene expression in ocular tissues is useful for
identifying genes and pathways involved in normal vision and
inherited eye disease. In addition, tissue specific expression and
alternative splicing can be observed by profiling multiple ocular
tissues. The purpose of our study was to develop a public database of
exon expression in ten different human ocular tissues.
Methods: Ten tissues were obtained from each of six human donors:
retina, optic nerve head (ONH), optic nerve (ON), ciliary body (CB),
trabecular meshwork (TM), sclera, lens, cornea, choroid/RPE and
iris. RNA from each tissue was extracted and pooled. Affymetrix
human Exon 1.0 ST arrays were used to profile gene expression in
these tissues at a resolution of individual exons. The Affymetrix
Power Tools (APT) probeset-summarize utility was used for
background estimation and expression level quantification.
Results: The estimated values of expression for genes and their
exons are now available on a public website at:
https://genome.uiowa.edu/otdb/. Expression by tissue, gene or
individual probes on the 1.0 ST array are accessible. Probeset-level
detection above background estimates and alternate splicing graphs
are also accessible. We compared the expression results versus retina
RNAseq and other microarray and EST data in various tissues of the
eye. We demonstrate the utility of profiling pooled RNA from
multiple ocular tissues on a single platform.
Conclusions: We have created a public resource for evaluating
normal expression of genes in 10 ocular tissues. Evaluation of genes
known to be highly expressed in specific tissues demonstrates the
value of this resource. We anticipate that this resource will be
valuable for studies of changes in the transcriptome between tissues
of the eye and to identify tissue specific alternative splicing.
Commercial Relationships: Terry A. Braun, Alcon Research, LTD
(F); Alex H. Wagner, None; Adam P. DeLuca, None; Thomas
Casavant, None; Todd E. Scheetz, None; Abbot F. Clark, Alcon
Research, Ltd. (F); Robert F. Mullins, Alcon Research Ltd (F);
Edwin M. Stone, None
464 Advances in Ocular Genetics
Wednesday, May 08, 2013 2:45 PM-4:30 PM
TCC LL 4/5 Paper Session
Program #/Board # Range: 4971-4977
Organizing Section: Genetics
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
Program Number: 4971
Presentation Time: 2:45 PM - 3:00 PM
Transgenic TBK1 mouse develops signs of normal tension
glaucoma
John H. Fingert1, Kathy A. Miller1, Frances Solivan-Timpe1, Ben
Roos1, Alan L. Robin2, Robert F. Mullins1, Michael G. Anderson3.
1
Ophthalmology, University of Iowa, Iowa City, IA; 2Ophthalmology
and International Health, Johns Hopkins University, Baltimore, MD;
3
Physiology, University of Iowa, Iowa City, IA.
Purpose: To generate a transgenic mouse with the same TANKbinding kinase 1 (TBK1) mutations that cause normal tension
glaucoma (NTG) in humans and to characterize the features of
glaucoma in these transgenic mice.
Methods: Transgenic TBK1 mice were generated using a BAC
vector to integrate the human TBK1 gene (with native promoter and
intron sequences) into the mouse genome. Aged transgenic mice and
littermates were evaluated for features of glaucoma with tonometry,
optical coherence tomography (OCT), retinal ganglion cell counts,
and optic nerve axon counts.
Results: Baseline examinations, showed no elevation of IOP, and no
retinal abnormalities on clinical, OCT, or histological examiations.
At 7 months, transgenic mice (n=13) and wild-type littermates (n=13)
showed no elevation in intraocular pressure. Retinal ganglion cell
counts were reduced in the transgenic mice by 9.6% when compared
with littermates (p < 0.001). Preliminary counts of optic nerve axon
counts are also reduced but are still underway.
Conclusions: Copy number variations (gene duplication) in TBK1
have been associated by NTG. Here we report the first data showing
that a similar gene defect engineered in transgenic mice also produces
an NTG phenotype. Preliminary studies of transgenic TBK1 mice
show that they develop features of glaucoma (reduced retinal
ganglion cell counts) in the absence of elevated intraocular pressure.
These data suggest that this model system will be a useful resource
for investigating the causes of optic nerve damage in glaucoma.
Futhermore, these mice may also be used to test new diagnostic and
therapeutic approaches for human glaucoma. Larger cohorts of mice
are being aged further and will provide even more definitive data to
support these conclusions.
Commercial Relationships: John H. Fingert, None; Kathy A.
Miller, None; Frances Solivan-Timpe, None; Ben Roos, None;
Alan L. Robin, merck (C), merck (R), Aerie (C), Aerie (I), Sucampo
(E), Glaukos (C), Glaukos (I), Allergan (R); Robert F. Mullins,
Alcon Research Ltd (F); Michael G. Anderson, None
Support: American Glaucoma Society Mid Career Award, NIH
R01EY018825
Program Number: 4972
Presentation Time: 3:00 PM - 3:15 PM
Dominant-negative RBP4 mutations cause congenital eye
malformations through a maternal-fetal nutritional interaction
Tom M. Glaser1, 2, Chris Chou2, Sue A. Tarle2, Jon Pribila4, Tanya
Bardakjian5, Adele S. Schneider5, Christine C. Nelson3, Tom M.
Glaser1, 2. 1Cell Biology & Human Anatomy, Univ of California,
Davis School of Medicine, Davis, CA; 2Human Genetics, Univ
Michigan, Ann Arbor, MI; 3Ophthalmology, Univ Michigan, Ann
Arbor, MI; 4Pediatric Ophthalmology, Park Nicollet Hospital,
Lincoln Park, MN; 5Genetics, Albert Einstein Medical Center,
Philadelphia, PA.
Purpose: Vitamin A (retinol) is an essential nutrient for retinal
physiology and eye morphogenesis. Gestational vitamin A deficiency
poses a risk for ocular malformations. We determined the molecular
basis of autosomal dominant microphthalmia, anophthalmia and
coloboma (MAC) disease in a large pedigree.
Methods: We performed linkage mapping and DNA sequence
analysis in familial and sporadic cases, and biochemical and cellular
assays to characterize the functional effects of coding mutations.
Results: We identified novel missense mutations in RBP4 (p.A73T
and p.A75T), in three unrelated families with autosomal dominant
MAC. This trait exhibits low penetrance and a maternal parent-oforigin effect. The RBP4 gene encodes serum retinol binding protein
(RBP), a 21 kD circulating lipocalin responsible for mobilizing
hepatic vitamin A stores and transporting retinoids to peripheral
tissues, including the placenta. At target tissues, RBP binds the
STRA6 transmembrane receptor for cellular uptake of vitamin A
(Kawaguchi et al. 2007). Previously reported RBP4 mutations
(p.G93D and p.I59N) are associated with recessive night blindness
(Biesalski et al. 1999). We systematically compared biochemical and
functional properties of dominant and recessive RBP4 alleles,
including cellular secretion, transthyretin (TTR) interaction, 3Hretinol and STRA6 receptor binding. The RBP4 p.A73T and p.A75T
alleles encode stable, circulating forms that mimic wild-type protein
but bind retinol poorly, consistent with predicted structural effects on
the ligand pocket, whereas the recessive alleles grossly alter folding
and destabilize RBP in the circulation.
Conclusions: The dominant alleles compete with wild-type RBP for
STRA6 binding. This dominant-negative mechanism creates
sequential “bottlenecks” that limit vitamin A delivery, at the
maternal-fetal interface and fetal tissues, causing congenital eye
malformations and explaining the unique maternal inheritance
pattern. Our findings reveal a novel mechanism for genetic disease
involving mimicry by a defective plasma cargo protein, a unique
gene x environment interaction controlling penetrance, and a new
explanation for maternal origin effects, which may apply broadly to
other congenital diseases.
Commercial Relationships: Tom M. Glaser, None; Chris Chou,
None; Sue A. Tarle, None; Jon Pribila, None; Tanya Bardakjian,
None; Adele S. Schneider, None; Christine C. Nelson, None; Tom
M. Glaser, None
Support: EY19497
Program Number: 4973
Presentation Time: 3:15 PM - 3:30 PM
Non-exomic and synonymous variants in ABCA4 are an
important cause of Stargardt disease
Edwin M. Stone1, 2, Budd A. Tucker1, Terry A. Braun1, Robert F.
Mullins1, Alex H. Wagner1, Samuel G. Jacobson3, Artur V.
Cideciyan3, Byron L. Lam4, Gerald A. Fishman5. 1Department of
Ophthalmology, Institute for Vision Research, University of Iowa,
Iowa City, IA; 2Howard Hughes Medical Institute, Iowa City, IA;
3
Department of Ophthalmology, Scheie Eye Institute, University of
Pennsylvania, Philadelphia, PA; 4Department of Ophthalmology,
Bascom Palmer Eye Institute, University of Miami, Miami, FL;
5
Chicago Lighthouse for People Who Are Blind or Visually
Impaired, Chicago, IL.
Purpose: Many patients with clinical features of ABCA4-associated
retinal disease have one or no disease-causing mutations found after
screening the entire ABCA4 coding sequence. The purpose of this
experiment was to identify as many non-exomic disease-causing
ABCA4 mutations as possible.
Methods: We hypothesized that mutations near minor splice
junctions in ABCA4 might increase the probability of missplicing at
these sites. We performed next generation sequencing of RNA
extracted from normal eye bank eyes to identify and characterize
minor ABCA4 transcripts. We screened 15 patients who had clear
clinical evidence of recessive Stargardt disease but only one coding
sequence mutation in ABCA4 for mutations at 15 of the most
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
convincing alternate splice junctions suggested by the RNA
sequencing data. Stable cell lines were derived from patients who
harbored novel variants at these junctions and RNA from these cells
was used to assess splicing of ABCA4. A second cohort of 79
Stargardt patients was used to validate these results.
Results: Four of 15 individuals in the initial cohort (and none of 412
controls) were found to harbor a single novel intronic mutation that
increased the predicted strength of a cryptic splice recognition
sequence. Analysis of RNA obtained from stable cell lines prepared
from 2 of these patients revealed improper splicing at this site.
Screening of the second Stargardt cohort revealed 4 additional
patients with this mutation. An additional intronic variant affecting
splicing of the same exon was also identified and confirmed by RNA
analysis. Finally, we noted that one of the most abundant minor
ABCA4 transcripts in human retina contains an improperly spliced
exon 46. A rare synonymous codon variant at this position
(Val2114Val), previously thought to be a non-disease-causing
polymorphism, accentuates missplicing at this locus and inactivates
the allele. Collectively, these three new mutations account for 12 of
94 (12.7%) previously undetectable disease-causing ABCA4 variants.
Conclusions: Each new disease-causing ABCA4 variant that is
discovered improves the sensitivity of clinical testing for Stargardt
disease and other ABCA4-associated retinal diseases. It also increases
the accuracy of genetic counseling that can be given to patients and
increases the number of individuals eligible for clinical trials of genereplacement and other therapies for these diseases.
Commercial Relationships: Edwin M. Stone, None; Budd A.
Tucker, None; Terry A. Braun, Alcon Research, LTD (F); Robert
F. Mullins, Alcon Research Ltd (F); Alex H. Wagner, None;
Samuel G. Jacobson, None; Artur V. Cideciyan, None; Byron L.
Lam, None; Gerald A. Fishman, None
Support: Howard Hughes Medical Institute, Foundation Fighting
Blindness-C-GE-0711-0527-UIOW1, NIH-EY016822
Program Number: 4974
Presentation Time: 3:30 PM - 3:45 PM
Application of Whole-Exome and Retinal-Capture NextGeneration DNA Sequencing to Identify Disease-Causing
Mutations in Families with a Diagnosis of Autosomal Dominant
Retinitis Pigmentosa
Stephen P. Daiger1, Lori S. Sullivan1, Sara J. Bowne1, George M.
Weinstock2, Daniel C. Koboldt2, Rui Chen3, John R. Heckenlively4,
Kari E. Branham4, David G. Birch5, Dianna K. Wheaton5. 1Human
Genetics Center, School of Public Health, The Univ. of Texas Health
Science Center, Houston, TX; 2The Genome Institute, Washington
University, St. Louis, MO; 3Dept. of Molecular and Human Genetics,
Baylor College of Medicine, Houston, TX; 4Kellogg Eye Center,
Univ. of Michigan, Ann Arbor, MI; 5Retina Foundation of the
Southwest, Dallas, TX.
Purpose: To detect disease-causing mutations in families with a
diagnosis of autosomal dominant retinitis pigmentosa (adRP),
focusing on genes known to cause inherited retinal diseases, using
whole-exome next-generation sequencing (NGS) and retinal-capture
NGS.
Methods: Families were selected from a cohort of 260 with a
diagnosis of adRP and 3 affected generations and affected females, or
two generations with male-to-male transmission. The families were
previously screened for common mutations by Sanger sequencing.
Coding exons and flanking intron-exon junctions of all known
RetNet genes and candidate genes were targeted for NGS. Testing
was either 1.) whole-exome NGS using Agilent SureSelect liquidcapture probes and the Illumina platform, focusing analysis on 190
retinal disease genes, 2.) targeted liquid-capture and NGS of coding
regions of 160 retinal disease genes, or 3.) both. Families tested
included positive controls with known mutations and families without
a previously-identified mutation. Mutations detected by NGS were
confirmed by Sanger sequencing.
Results: Probands and additional family members from 87 families,
including controls, were tested by one or both methods. Of 22
families tested by whole-exome NGS, mutations were found in 7
(32%). Of 18 control families with known mutations tested by
retinal-capture NGS, mutations were found in 17. (A large deletion in
PRPF31 was not detected). Of 69 families without known mutations
tested by retinal-capture NGS, mutations were found in 10 (15%). In
summary, of a total of 76 families whose mutations were not known
prior to NGS, mutations were found in 17 (22%). Several of these
mutations are in genes not currently associated with autosomal
dominant disease.
Conclusions: Targeted retinal-gene capture or focused retinal-gene
analysis following whole-exome NGS, are reliable, efficient methods
for detecting disease-causing mutations in families with dominant
retinitis pigmentosa. Based on a variety of methods, mutations in
coding exons or intron-exon junctions of known retinal disease genes
(RetNet genes) account for at least 72% of families in our cohort with
dominant RP.
Commercial Relationships: Stephen P. Daiger, None; Lori S.
Sullivan, None; Sara J. Bowne, None; George M. Weinstock,
None; Daniel C. Koboldt, None; Rui Chen, None; John R.
Heckenlively, None; Kari E. Branham, Arctic DX (P); David G.
Birch, Acucela (C), QLT (C), Neurotech, USA (C); Dianna K.
Wheaton, None
Support: NIH grant EY007142 and grants from the Foundation
Fighting Blindness
Program Number: 4975
Presentation Time: 3:45 PM - 4:00 PM
Rare and common variants in extracellular matrix gene fibrillin
2 (FBN2) are associated with inherited and age-related macular
degeneration, respectively
Anand Swaroop1, Rinki Ratna Priya1, Xiaowei Zhan2, Robert Fariss1,
Kari E. Branham2, Emily Y. Chew1, Dwight Stambolian4, Shomi S.
Bhattacharya3, John R. Heckenlively2, Goncalo Abecasis2. 1N-NRL,
Bldg 6, National Eye Institute, Bethesda, MD; 2University of
Michigan, Ann Arbor, MI; 3Institute of Ophthalmology, London,
United Kingdom; 4University of Pennsylvania, Philadelphia, PA.
Purpose: Macular degenerative diseases are a major cause of mostly
incurable central vision loss in humans and pose substantial
healthcare burden. Different forms of macular degeneration
encompass monogenic early-onset to complex late-onset forms like
age-related macular degeneration (AMD), with considerable clinical
and genetic heterogeneity. However, both early and late forms share
clinical features and identification of causative genes in Mendelian
early-onset forms can provide important insights into complex
pathobiology of AMD.
Methods: We sequenced all the protein-coding region of the genome
(the “exome”) in four members of a two-generation family with an
autosomal dominant form of early onset macular degeneration. We
screened additional patients for rare variants in patients with early
onset maculopathies. We further tested for an association between a
functional FBN2 variant rs154001 (p. Val965Ile) and AMD in over
4000 cases and controls.
Results: We identified a novel segregating mutation, p.Glu1144Lys
in FBN2, a cysteine-rich glycoprotein, which forms a principal
component of the elastin-rich extracellular matrix (ECM).
Sequencing analysis identified additional rare variants in early onset
maculopathies and AMD patients. Genes of ECM component have
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
been implicated in both early and late macular degenerations and
recent genomewide association studies have identified variants in
extracellular/collagen matrix pathway genes (TIMP3, COL8A1,
COL10A), conferring susceptibility in AMD patients. This prompted
us to explore whether common variants in FBN2 are associated with
AMD, where we identified a non-synonymous (Val965Ile) SNP,
rs154001, that was found to be associated with AMD (p value=
1.03×10-4). Immunofluorescence studies in human and monkey eye
localized the FBN2 protein in Bruch’s membrane, choroid and sclera.
Conclusions: We have identified both rare and common variants in
FBN2 in patients with macular degeneration. Abundant expression of
FBN2 in sclera, choroid and Bruch’s membrane advocates the role of
this novel ECM gene FBN2 in macular disease pathogenesis. More
significantly, this study establishes an important link between rare
and common forms of macular degenerative diseases.
Commercial Relationships: Anand Swaroop, None; Rinki Ratna
Priya, None; Xiaowei Zhan, None; Robert Fariss, None; Kari E.
Branham, Arctic DX (P); Emily Y. Chew, None; Dwight
Stambolian, None; Shomi S. Bhattacharya, None; John R.
Heckenlively, None; Goncalo Abecasis, University of Michigan (P),
Illumina (R), Affymetrix (R)
Support: NIH Intramural research program
Program Number: 4976
Presentation Time: 4:00 PM - 4:15 PM
Exome sequencing in the mid-western Amish to identify rare
variation influencing AMD
Jessica N. Cooke Bailey1, Laura D'Aoust1, Lan Jiang1, Renee Laux1,
Anita Agarwal1, William K. Scott2, Margaret A. Pericak-Vance2,
Jonathan L. Haines1. 1Center for Human Genetics Research,
Vanderbilt University Medical Center, Nashville, TN; 2Hussman
Institute of Human Genomics, Miller School of Medicine, University
of Miami, Miami, FL.
Purpose: Age-related macular degeneration (AMD) is a progressive
neurodegenerative disease that is the leading cause of blindness in
elderly individuals in developed countries. Risk for AMD is mediated
by genetic and environmental factors; known major risk loci include
ARMS2/HTRA, CFH, C2/CFB, and C3. These loci harbor common
genetic variants that influence risk for AMD; rare variation has not
been extensively examined in these regions. We hypothesized that
rare variants contribute to AMD susceptibility.
Methods: We evaluated individuals from the Amish communities of
Ohio and Indiana, an isolated population useful for genetic studies
due to the relatively few founders, limited heterogeneous
environmental exposure, and the presence of detailed records on their
multigenerational pedigrees. Genetic information from this
population can be used to further the knowledge of diseases know to
have genetic and environmental components, including AMD.
Exome sequencing was performed in five members of a small nuclear
Amish family with AMD who lack the common risk alleles at the
major AMD risk loci. Follow-up genotyping and association analysis
using MQLS was performed in a cohort of 929 additional Amish
individuals including 95 with self-reported AMD (15 confirmed);
self-report of AMD is a reliable proxy for AMD disease status in this
cohort.
Results: Exome sequencing identified a variant (P503A) in CFH that
is not present in dbSNP or 1000Genomes and is predicted to be
damaging by Polyphen2. Further, the variant has a GERP score of
3.64, indicating the base pair is strongly conserved across species.
Three additional rare variants were detected in MFN2, RIC8B, and
LRP4, genes which are not known contributors to AMD, but which
warrant further follow-up to determine potential undetected
involvement. Analysis of 900 additional members of an Amish cohort
revealed that the four variants were associated (P<0.005) with AMD,
with P503A being the most significantly associated (P<1x10-6).
P503A was absent when evaluated in a cohort of 700 elderly nonAMD, non-Amish controls.
Conclusions: Exome sequencing of five members of an Amish
family lacking the common AMD risk alleles identified four novel
variants that appear to contribute to AMD. Identification of these
novel risk variants provides evidence that rare variants remain to be
detected in AMD and that these variants can be identified through
exome sequencing of major AMD risk loci.
Commercial Relationships: Jessica N. Cooke Bailey, None; Laura
D'Aoust, None; Lan Jiang, None; Renee Laux, None; Anita
Agarwal, Vanderbilt University (P); William K. Scott, Duke
University/ArcticDx (P); Margaret A. Pericak-Vance, None;
Jonathan L. Haines, Arctic Dx (I), AMD genes (P)
Support: NIH grant 5R01EY012118-12; NIH grant
5R01AG019085-10
Program Number: 4977
Presentation Time: 4:15 PM - 4:30 PM
Chipping Away At The Genetics of Age Related Macular
Degeneration
Goncalo Abecasis1, Matthew C. Schu3, Xiaowei Zhan1, Sivakumaram
Arumugam4, Jennifer Bragg Gresham1, Lars Fritsche2. 1University of
Michigan, Ann Arbor, MI; 2Institute of Human Genetics, University
of Regensburg, Regensburg, Germany; 3Boston University School of
Medicine, Boston, MA; 4Case Western Reserve University,
Cleveland, OH.
Purpose: Age-related macular degeneration (AMD) is a common
form of blindness in the elderly. Disease predisposition is complex
and influenced by a variety of environmental and genetic risk factors.
To extend understanding of AMD genetics and biology, we set out to
examine the association between common and rare genetic variation
in a large set of AMD cases and controls.
Methods: Through international collaboration, we set out to
assemble a sample of large sample of AMD cases and matched
controls. We designed a custom genotyping array including 250,000
common and rare coding variants discovered in large scale
sequencing experiments (and enriched for variants discovered in
sequencing experiments targeting individuals with AMD) and an
additional set of 250,000 common variants distributed evenly across
the genome. Working in collaboration with the National Institutes of
Health Center for Inherited Disease Research, we then set out to
genotype all available samples using this custom array.
Results: We successfully assembled and organized an international
research team focused on age-related macular degeneration genetics.
We centrally curated and organized DNA samples for >48,000
individuals (48.25% macular degeneration cases and 51.75%
controls). Among cases, 46.5% had neovascular disease, 14.7% had
geographic atrophy, and 9.0% had geographic atrophy in one eye and
neovascular disease in the fellow eye (resulting in 70.2% of cases
with advanced disease). In remaining cases, 16.5% had large drusen
and 13.3% had earlier signs of disease. We successfully designed and
manufactured a low cost custom genotyping array, especially
designed for studies of AMD - including ~250,000 coding variants
(approximately 50% of these with frequency <0.1%) and a grid of
additional variants allowing us to tag or impute common variants
with frequency >5% across the genome. We are expecting the first set
of samples to be genotyped by January 2013 and all 48,000 samples
to be genotyped by Spring 2013.
Conclusions: Our experiment illustrates how collaborative teams
with a shared research interest can organize. Pooling resources and
effort, we were able to study a very large number of individuals, as
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
will be needed for studies of rare coding variants. We expect 80%
power to detect variants with a frequency of >0.1% that lead >2.45fold increase in disease risk. At the ARVO meeting, we will review
results from our initial rounds of analysis.
Commercial Relationships: Goncalo Abecasis, University of
Michigan (P), Illumina (R), Affymetrix (R); Matthew C. Schu,
None; Xiaowei Zhan, None; Sivakumaram Arumugam, None;
Jennifer Bragg Gresham, None; Lars Fritsche, ArcticAx Inc.,
Toronto, Ontario (C)
Support: NIH Grant EY022005, HG006934
541 Genetic Risk Factors for Common Eye Disorders with
Complex Inheritance
Thursday, May 09, 2013 10:30 AM-12:15 PM
Exhibit Hall Poster Session
Program #/Board # Range: 6165-6215/C0050-C0100
Organizing Section: Genetics
Contributing Section(s): Visual Neuroscience
Program Number: 6165 Poster Board Number: C0050
Presentation Time: 10:30 AM - 12:15 PM
Is AMD a Disease or a Disorder? A Systems Biology Attempt to
Subtype AMD on the Basis of Gene Expression
Mones S. Abu-Asab1, Chi-Chao Chan2, 1. 1Histopathology Core,
National Eye Institute, Bethesda, MD; 2Laboratory of Immunology,
National Eye Institute, Bethesda, MD.
Purpose: Genetic pathways underlying the initiation and progression
of age-related macular degeneration (AMD) have not been yet
sufficiently revealed, and whether AMD’s phenotypes represent a
single disease or a disorder with an assemblage of diseases is still
awaiting resolution. We attempt to tackle both problems with a
systems biology analytical paradigm called parsimony phylogenetics.
Methods: We profiled genome wide gene expression data of macular
and extra-macular specimens of retinas (55 normal, 13 pre-AMD, and
47 AMD) and retinal pigment epithelium (RPE)-choroids (96 normal,
21 pre-AMD, and 60 AMD). The AMD specimens encompassed dryAMD without geographic atrophy (GA), choroidal
neovascularization (CNV), and GA (GEO DataSets: GSE29801).
Pre-AMD and AMD gene expression values of retinal and RPEchoroidal specimens were polarized separately against their
respective normal specimens, and the new polarized data matrices
were processed through MIX, a parsimony program of the PHYLIP
package, to produce phylogenetic cladograms for both sets of data.
Gene lists extracted from the cladograms nodes were processed in
Genomatix GePS to generate pathway networks.
Results: Cladograms indicated heterogeneous gene-expression
profiles within phenotypic subtypes commonly classified with
conventional diagnostic systems. AMD phenotypes were not distinct
according to their transcriptome profiles of the retina or RPE-Choroid
suggesting more clinical subtypes than those currently recognized.
Macular and temporal extra-macular tissues of the same patient
separated in most of the retinal and RPE sets but some clustered
together.
Conclusions: The topology, groupings (clades), and nodal
synapomorphies of our cladograms do not support classical AMD
phenotypes as valid transcriptomal subtypes. The majority of the
dysregulated gene expressions are specimen-specific or clade-specific
(occurring in a small group of specimens) suggesting multiple
pathway involvement in conventional clinical subtypes. Gene lists
defined by cladogram nodes showed that the AMD-related
dysregulations occurring in the neural retina are different from those
in RPE-choroidal tissue. Our analysis indicates a complex
transcriptional profile of the phenotypes that requires the study of
much earlier stages of the disease to elucidate the initial events of
AMD.
Commercial Relationships: Mones S. Abu-Asab, None; Chi-Chao
Chan, None
Program Number: 6166 Poster Board Number: C0051
Presentation Time: 10:30 AM - 12:15 PM
Population Attributable Risk of Known AMD Genetic Risk
Factors Based on the 1000 Genomes Project
Joe M. Butler1, Yit C. Yang2, Luminita I. Paraoan1. 1Eye and Vision
Science, Institute of Ageing and Chronic Disease, University of
Liverpool, Liverpool, United Kingdom; 2Ophthalmology,
Wolverhamptom Med Inst-New Cross, Wolverhamptom, United
Kingdom.
Purpose: This analysis aims to dissect the contribution of known
genetic risk factors for age-related macular degeneration (AMD).
Using data from the 1000 Genomes Project we gain insights into the
contribution of each genetic risk factor across different populations.
Methods: Five non-synonymous SNPs with known risk to AMD
were considered, these were: CFH (Y402H), ARMS2 (A69S), C3
(R102G), C2 (E186D,E318D) and CST3 (A29T). The allele
frequencies in African, Asian, European and American populations
were estimated using the Variation Pattern Finder from the 1000
Genomes Project. For each SNP the effect size was evaluated from
previously reported data; the odds ratio is assumed to be the same
throughout all four populations. The population attributable risk
(PAR) was calculated based on the estimates of allele frequencies,
odds ratio and disease prevalence. AMD prevalence in each of the
four populations was informed by a previous epidemiology study. To
determine which allele is ancestral and which is derived, the
chimpanzee reference sequence was compared with the human
reference sequence (GRCh37/hg19). The PAR was calculated for the
derived allele as opposed to the ancestral allele. If the frequency of
the derived allele is higher in cases than controls then it is a risk
allele; if it is higher in controls it is protective.
Results: In the population of European descent CFH had the largest
(25.6%) attributable risk, followed by ARMS2 (22.5%), then C3
(9.1%) and CST3 (5.8%). The derived allele of C2 has a protective
effect with the highest value in the European population (6.2%). In
all other populations the risk allele in the ARMS2 locus is the major
contributor to risk, followed by CFH. In African and Asian
populations CST3 takes precedence over C3 as the third strongest
contributor to AMD risk.
Conclusions: This analysis provides evidence that the contribution of
genetic risk factors towards AMD varies across African, American,
Asian and European populations. For instance the PAR profile for
Asian population is dominated by ARMS2. However the CST3 locus,
encoding cystatin C, is a risk factor which contributes consistently
throughout all four populations. The C2 locus is unique within this
gene set in that the derived allele is protective against developing
AMD; it is rare in all populations studied and may offer valuable
therapeutic potential.
Commercial Relationships: Joe M. Butler, None; Yit C. Yang,
Novartis (R); Luminita I. Paraoan, None
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
Program Number: 6167 Poster Board Number: C0052
Presentation Time: 10:30 AM - 12:15 PM
Association of PTX3 polymorphisms with age-related macular
degeneration and polypoidal choroidal vasculopathy
Jianhuan Chen1, 2, Haoyu Chen1, Weiqi Chen1, Mingzhi Zhang1, Chi
Pui Pang1, 2. 1Joint Shantou International Eye Center, Shantou
University & the Chinese University of Hong Kong, Shantou, China;
2
The Chinese University of Hong Kong, Hong Kong, China.
Purpose: Long pentraxin-3 (PTX3) is a key modulator of
complement factor H, an associated gene in exudative age-related
macular degeneration (AMD). We investigated the association of
PTX3 with AMD and its subtypes, typical AMD (tAMD) and
polypoidal choroidal vasculopathy (PCV).
Methods: A total of 568 unrelated subjects were recruited at
Shantou, China, including 346 controls and 222 AMD patients. Five
tag single nucleotide polymorphisms (SNPs) of PTX3 including
rs9289983, rs2305619, rs1840680, rs3845978, and rs4680367 were
genotyped using Taqman assays. Disease association was analyzed
by logistic regression controlled for sex and age.
Results: Association with AMD was implicated in the SNP
rs1840680 in the recessive model (odds ratio [OR] = 1.75, P =
0.047). Further subtype analysis revealed that enrichment of the
rs1840680 AA genotype was significant in PCV (OR = 2.40, P =
0.017) but not in tAMD (OR = 1.39, P = 0.341). Furthermore,
rs9289983-rs3845978 haplotypes were associated with AMD (P =
0.001), and with its subtypes, tAMD and PCV (P = 0.037 and 8.4*104 respectively). Notably the A-T haplotype conferred high disease
risk (AMD: OR = 12.34, P = 0.004; tAMD: OR = 10.99, P = 0.003;
PCV: OR = 19.67, P = 0.008). In accord with this, a decay of linkage
disequilibrium in the PTX3 genomic region was found in AMD and
the two subtypes when compared to controls.
Conclusions: In the current study PTX3 polymorphisms was
associated with AMD and its two subtypes, with larger contribution
to PCV than to tAMD.
Commercial Relationships: Jianhuan Chen, None; Haoyu Chen,
None; Weiqi Chen, None; Mingzhi Zhang, None; Chi Pui Pang,
None
Support: National Natural Science Foundation of China (No.
81000397, 30901646 and 81170853), Science and Technology
Planning Project of Guangdong Province, China (No.
2010B031600130 and 2011B031300013) and Joint Shantou
International Eye Center, Shantou University/The Chinese University
of Hong Kong (No. 10-020, 10-021 and 10-022).
Program Number: 6168 Poster Board Number: C0053
Presentation Time: 10:30 AM - 12:15 PM
Genetic determinants of Age-related Macular Degeneration in
Diverse Populations: the Population Architecture using
Genomics and Epidemiology (PAGE) Study
Nicole Restrepo1, Tiana Garrett2, Petra Buzkova3, Richard A.
Jensen3, Barbara E. Klein4, Ronald Klein4, Tien Yin Wong5, E
Shyong Tai6, Dana Crawford1. 1Center for Human Genetics
Research, Vanderbilt University, Nashville, TN; 2Department of
Epidemiology, University of North Carolina, Chapel Hill, Chapel
Hill, NC; 3Department of Biostatistics, University of Washington,
Seattle, WA; 4Office of Population Genomics, National Human
Genome Research Institute, Bethesda, MD; 5Department of
Ophthalmology and Visual Sciences, University of Wisconsin,
Madison, WI; 6Singapore Eye Research Institute, Singapore national
Eye Centre, Singapore, Singapore.
Purpose: Substantial progress has been made in identifying
susceptibility variants for AMD. The most widely replicated loci are
Complement Factor H (CFH) Y402H and Age-related Maculopathy
Susceptibility-2 (ARMS2),which were discovered in European
populations. To date, little data exist for these variants in diverse
populations. A major goal of the Population Architecture using
Genomics and Epidemiology (PAGE) study is to describe the
underlying genetic architecture of common, complex diseases such as
AMD across diverse populations.
Methods: The PAGE study consists of the National Health and
Nutrition Examination Surveys, Atherosclerosis Risk in Communities
Study,and the Cardiovascular Health Study. Included are the
Singapore Prospective Study Programme and Singapore Malay Eye
Study. Targeted genotyping was performed for AMD-associated
SNPs in CFH, CFI, ARMS2, VEGF,and lipid trait loci. AMD cases
were >60 years and presented with early/late AMD, determined by
fundus photography. A total of 830, 95, 107,and 21 cases and 5,710,
1,172, 863,and 206 controls from European Americans, African
Americans, Malays, and Chinese, respectively, were available for
study. We performed a meta-analysis following logistic regression
assuming an additive genetic model performed at each study site and
adjusted for age, sex, body mass index, smoking status,and HDL.
Results: In European Americans, rs1061170 (CFH) and rs10490924
(ARMS2) replicated at p=3.7x10-10 (OR= 1.61, 95% CI= 1.85and
1.41) and p=1.8x10-5 (OR = 1.49, 95% CI=1.78 and 1.25),
respectively. None of the CFH or ARMS2 SNPs tested generalized to
African Americans (p>0.05). Interestingly, the HDL associated
variant CETP rs1800775 (OR=0.55; 95% CI=0.86 and 0.35;
p=0.009) was associated with AMD in African Americans but not
European Americans or Chinese suggesting a potential risk modifier
in lipid pathways to AMD in this population.
Conclusions: Further studies are needed to determine if lack of
generalization in major CFH and ARMS2 variants is due to statistical
power or differences in linkage disequilibrium and allelic distribution
across these diverse populations.
Synthesis view plot of meta-analysis results. P- values transformed
by -log10, with the threshold (p = 0.05) marked by a red line. Arrows
show direction of effect (beta). P-values, beta’s, and coded allele
frequencies plotted by race/ethnicity.
Commercial Relationships: Nicole Restrepo, None; Tiana
Garrett, None; Petra Buzkova, None; Richard A. Jensen, None;
Barbara E. Klein, None; Ronald Klein, None; Tien Yin Wong,
Allergan (C), Bayer (C), Novartis (C), Pfizer (C), GSK (F), Roche
(F); E Shyong Tai, None; Dana Crawford, None
Support: NIH Ocular Genomics Training Grant: 1T32EY021453-01
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
Program Number: 6169 Poster Board Number: C0054
Presentation Time: 10:30 AM - 12:15 PM
Association between δ-sarcoglycan gene (SGCD) polymorphisims
and age-related macular degeneration (AMD) in Mexican
patients
Javier F. Estrada1, Alexandra B. Luna-Angulo1, Lilliana G. CortesBallinas1, Ramón Coral-Vázquez2, Alvaro Rendon4, Dalia C.
Guadarrama5, Héctor Rangel-Villalobos3, Juan C. Zenteno5.
1
Biochemistry and Molecular Biology, Universidad Panamericana,
México, Mexico; 2seccion de estudios de posgrado e
investigacion.Escuela de Medicina IPN, IPN, Mexico, Mexico;
3
genetica, Unioversidad de Guadalajara, Guadalajara, Mexico;
4
Centre de Recherche, Institut de la Vision, Paris, France; 5Genética,
Instituto de oftalmología Conde de Valenciana, Mexico, Mexico.
Purpose: We searched for an association between the rs931798 and
rs970476 polymorphisms of the SGCD gene and the occurrence of
AMD, especially the advanced form. rs931798 and rs970476 are
significant SNP’s (0.000369 and 0.0000720 p values, respectively)
according to a re-analysis of a genome-wide association study
(GWAS) to AMD. Furthermore, we studied the rs75334195,
rs140616 and rs140617 SNP’s near to rs931798 and we will studied
rs75722607, rs76902439 and rs78951425 SNP’s near to rs970476 in
order to explore a possible AMD association.
Methods: The polymorphisms were determined with DNA from
peripheral blood lymphocytes from 113 patients (68% wet form and
32% dry form) and 95 controls by automated sequencing. The
significance of the polymorphisms was assessed by multiple logistic
regression, producing odds ratios (ORs) and 95% confidence
intervals (CIs). Haplotype analysis will be determined to look for
association with AMD.
Results: Until now we have worked with the region containing
rs931798, rs75334195, rs140616 and rs140617 SNP’s in patients and
controls samples. The SNP variant rs140616 A/A showed risk of dry
AMD (OR=11.96 and p=0.02) . The others SNP's not showed
associated to AMD and rs75334195 has not showed be polymorphic.
Conclusions: Genetic variation in SGCD significantly alters
susceptibility to dry AMD. SGCD would be a new target of study in
this significant ocular affection.
Commercial Relationships: Javier F. Estrada, None; Alexandra
B. Luna-Angulo, None; Lilliana G. Cortes-Ballinas, None; Ramón
Coral-Vázquez, None; Alvaro Rendon, None; Dalia C.
Guadarrama, None; Héctor Rangel-Villalobos, None; Juan C.
Zenteno, None
Support: Escuela de Medicina, Universidad Panamericana
Program Number: 6170 Poster Board Number: C0055
Presentation Time: 10:30 AM - 12:15 PM
Association of Family History with Genetic Risk Factors in
Patients with Age-Related Macular Degeneration
Nicole T. Saksens1, Yara T. Lechanteur1, Sanne K. Verbakel1,
Joannes M. Groenewoud2, Camiel J. Boon1, 3, Anneke I. Den
Hollander1, 4, Carel B. Hoyng1. 1Ophthalmology, Radboud University
Nijmegen Medical Center, Nijmegen, Netherlands; 2Epidemiology,
Biostatistics and Health Technology Assessment, Radboud
University Nijmegen Medical Center, Nijmegen, Netherlands;
3
Oxford Eye Hospital, John Radcliffe Hospital, University of Oxford,
Oxford, United Kingdom; 4Human Genetics, Radboud University
Nijmegen Medical Center, Nijmegen, Netherlands.
Purpose: This study aims to determine the effect of family history on
the association of genetic risk factors in patients with age-related
macular degeneration (AMD).
Methods: A cohort of 486 patients retrieved from the European
Genetic Database (EUGENDA) underwent an ophthalmic
examination, and were interviewed with a questionnaire about
smoking behaviour, body mass index (BMI), and family history.
DNA was isolated from venous blood samples and analyzed for 21
single nucleotide polymorphisms (SNPs) known to be associated with
AMD. We calculated odds ratios (ORs), adjusted for age, sex,
smoking, and BMI with binary logistic regression analysis for the
risk allele frequencies in familial AMD patients, using unaffected
control individuals as a reference. Furthermore, ORs in familial
AMD patients were compared to those in AMD patients without a
positive family history (sporadic AMD).
Results: The cohort consists of 93 (19.1%) unrelated AMD patients
with a reported positive family history (familial AMD), 230 (47.3%)
sporadic AMD patients and 163 (33.5%) control individuals without
AMD.
Risk alleles in the complement factor H (CFH Y402H) and agerelated maculopathy susceptibility 2 (ARMS2 A69S) genes were
significantly associated with sporadic and familial AMD as compared
to control individuals (CFH OR 1.65 and OR 2.96, respectively.
ARMS2 OR 2.71 and OR 2.11, respectively).
In patients with familial AMD, the risk allele frequency of CFH
Y402H was significantly higher (64.1%) than in the sporadic AMD
patients (53.7%, OR 1.55). The risk allele frequency of ARMS2 A69S
was not significantly different between familial and sporadic AMD
patients.
In the familial AMD patients we investigated the number of affected
family members. In 10 families at least 4 family members are
affected.
Conclusions: Although CFH and ARMS2 risk alleles are associated
with both sporadic and familial AMD, the allelic association with the
Y402H variant in the CFH gene appears to be stronger in familial
AMD. Large, densely affected AMD families are currently being
collected to examine the total AMD-associated SNP load and to
determine the role of rare highly penetrant variants, using exome
sequencing in these families.
Commercial Relationships: Nicole T. Saksens, None; Yara T.
Lechanteur, None; Sanne K. Verbakel, None; Joannes M.
Groenewoud, None; Camiel J. Boon, None; Anneke I. Den
Hollander, None; Carel B. Hoyng, None
Support: Foundation Fighting Blindness USA center grant C-GE0811-0548-RAD04
Program Number: 6171 Poster Board Number: C0056
Presentation Time: 10:30 AM - 12:15 PM
Interaction analysis of exogenous estrogen in age-related macular
degeneration (AMD) finds novel associations within the VEGF,
Complement, and TGFB pathways
Monique D. Courtenay1, William Cade1, Stephen G. Schwartz2,
Jaclyn L. Kovach2, Anita Agarwal3, Gaofeng Wang1, Jonathan L.
Haines4, Margaret A. Pericak-Vance1, William K. Scott1. 1Hussman
Institute of Human Genomics, University of Miami, Miami, FL;
2
Bascom Palmer Eye Institute, University of Miami, Miami, FL;
3
Ophthalmology, Vanderbilt University, Nashville, TN; 4Center for
Human Genetics Research, Vanderbilt University, Nashville, TN.
Purpose: AMD’s multifactorial etiology includes genetic and
environmental risk factors. Recently the AMD Gene Consortium
identified new genes that implicated the Complement, VEGF, and
TGFB pathways for further analysis. Also, it has been shown that
women who take estrogen in the form of hormone replacement
therapy (HRT) or birth control pills (BCPs) have reduced risk of
AMD. The purpose of this study was to test the association of these
pathways with AMD while accounting for main effects and estrogen
use.
Methods: White female AMD cases (n=540) and controls (n=239)
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
were genotyped using Affymetrix 6.0 chipsets. History of estrogen
use was collected with a self-administered questionnaire. SNPs
within 20kb of each gene in the 3 pathways were chosen. Gene and
pathway-based test statistics were calculated as the mean of all
independent SNP (r2<0.8) chi-square statistics derived from Kraft’s 2
degree of freedom joint test of genetic and environmental factors.
The joint test utilized a likelihood ratio test comparing a logistic
regression model with SNP and estrogen main effects, a pairwise
interaction term, age, and smoking status, and a model excluding the
SNP and interaction terms. The analysis was repeated 10,000 times,
permuting phenotypes with the Biased Urn sampling procedure to
generate an empirical p-value.
Results: Four known (CFH, CFB, C2, and VEGFA) and 30 novel
genes were associated with AMD (p<0.05) when accounting for
estrogen. Fifteen were not significant in tests of main effects
suggesting their effects are moderated by estrogen. The VEGF
pathway was significantly associated with neovascular AMD but only
when accounting for BCP use (P= 0.013). Complement was also
associated with AMD, but its results were driven solely by the main
effect of CFH. The TGFB pathway was not associated with AMD
when accounting for estrogen use or main effects only.
Conclusions: Our pathway results suggest that estrogen modifies the
genetic effect of SNPs within the VEGF pathway. This pathway and
all novel genes warrant closer examination for AMD association in
the context of estrogen usage. These results illustrate the utility of
exploring pathways of previously associated genes to find novel
associations. This study also demonstrates the importance of
incorporating environmental exposures in tests of genetic association,
at the SNP, gene, or pathway level.
Commercial Relationships: Monique D. Courtenay, None;
William Cade, None; Stephen G. Schwartz, Alimera (C), Bausch +
Lomb (C), Eyetech (C), IC Labs (P), ThromboGenics (C); Jaclyn L.
Kovach, None; Anita Agarwal, Vanderbilt University (P); Gaofeng
Wang, None; Jonathan L. Haines, Arctic Dx (I), AMD genes (P);
Margaret A. Pericak-Vance, None; William K. Scott, Duke
University/ArcticDx (P)
Support: NIH Grant 7R01EY012118, NIH Center Grant P30EY014801, and by an unrestricted grant to the University of Miami
from Research to Prevent Blindness, New York, NY
Program Number: 6172 Poster Board Number: C0057
Presentation Time: 10:30 AM - 12:15 PM
Genetic interaction mapping of AMD identifies potential genedisease associations
Lee Kiang, Jillian Huang, Ryan E. Tsuchida, Kanishka T.
Jayasundera. Kellogg Eye Center, University of Michigan, Ann
Arbor, MI.
Purpose: Systems biology networks provide a way to rapidly
visualize, analyze and model gene functions, interactions and
expression. Using a network-driven approach for Age-Related
Macular Degeneration (AMD), we sought to identify key regulatory
genes of the disease network, potential biomarkers and therapeutic
targets, and to assess the genetic interactions underlying differential
response to therapy and environmental modifers such as smoking.
Methods: We used visANT, a web-based program, to extract
molecular networks based on physical and functional relationships to
individual genes. The initial network was created by inputting 16
‘seed’ genes or nodes confirmed to be associated with AMD
(identified in >2 GWAS studies) and querying for internal
connections among nodes. We defined ‘hub’ genes as those with
highest degree of interaction with other network genes and ‘inferred’
genes as nodes through which seed genes are connected. To
determine key biological processes of the network, we clustered
genes based on a hierarchical algorithm that evaluates degree
centrality and betweenness of nodes based on the shortest path
between node pairs. We queried each cluster for the most significant
GO terms, and examined biological pathways linked to the network
through KEGG pathways. Because smoking is a well-established risk
factor for AMD, we mapped a set of 5 genes that are overexpressed
in smokers compared to controls, onto our gene network, in order to
infer intermediate genes which might link smoking to AMD.
Results: Ten inferred genes link seed genes well-established to play a
role in AMD by one degree of separation (ALB, LRP1, UBC, C4B,
HNF4A, EP300, GRB2, THBS1, ONECUT1, EFEMP2). Five
inferred genes (ABL1, SMAD3, KPNA1, CTNNB1 and KDR) link
smoking to AMD. Several inferred genes have been implicated in
AMD pathogenesis, such as ABL1 (required for apoptosis of
lipofuscin-laden RPE in an experimental model). Cluster analysis
revealed our gene sets were involved in immune response, cholesterol
metabolism, angiogenesis, and apolipoprotein binding.
Conclusions: Via genetic interaction mapping, we inferred genes
known to play a role in AMD, as well as genes not previously
associated with the disease. Network mapping is valuable in
revealing key players in AMD, with the potential of identifying
integral hub genes, which may include novel biomarkers for disease
diagnosis, surveillance and progression, and new therapeutic targets.
Commercial Relationships: Lee Kiang, None; Jillian Huang,
None; Ryan E. Tsuchida, None; Kanishka T. Jayasundera, None
Program Number: 6173 Poster Board Number: C0058
Presentation Time: 10:30 AM - 12:15 PM
Cfh genotype interacts with dietary glycemic index to modulate
early age-related macular degeneration-like features in mice
Sheldon Rowan1, Karen Weikel1, Min-Lee Chang1, Barbara Nagel2,
Jeffrey Thinschmidt3, Maria B. Grant3, Steven J. Fliesler4, 5, Donald
Smith1, Allen Taylor1. 1Human Nutrition Research Center on Aging,
Tufts University, Boston, MA; 2Department of Pathology, Saint
Louis University, Saint Louis, MO; 3Department of Pharmacology
and Therapeutics, University of Florida, Gainesville, FL; 4Research
Service, VAWNYHS, Buffalo, NY; 5Departments of Ophthalmology
& Biochemistry, SUNY-Buffalo & SUNY Eye Institute, Buffalo,
NY.
Purpose: Age-related macular degeneration (AMD) is a leading
cause of visual impairment in the aging population. Both genetics and
diet contribute to the relative risk for developing AMD. Common
variants in the Complement Factor H (CFH) gene confer
susceptibility to or protection from developing AMD. Consuming
higher glycemic index (GI) diets has been associated with increased
AMD risk as well as with insulin resistance and CNS inflammation.
Here, we explored the effects of dietary GI manipulation on
development of early AMD-like features (eAMD-f) and changes to
CNS inflammation in the Cfh-null mouse model of AMD.
Methods: Starting at 12-weeks of age, wildtype C57Bl6/J (WT) or
Cfh-null mice were fed high or low GI diets for 33 weeks. At 10months of age, mice were evaluated for eAMD-f in the neural retina
and retinal-pigmented epithelium (RPE) by light (LM) or electron
(EM) microscopy. Brains were analyzed for Iba-1
(macrophage/microglia) immunostaining, an indicator of
inflammation.
Results: At 10-months of age, WT mice showed no eAMD-f on
either diet, consistent with prior observations. Cfh-null mice,
however, showed distinct eAMD-f in the RPE when fed a low GI
diet, including vacuolation, disruption of basal infoldings, and
increased numbers of basal laminar deposits (by EM). Cfh-null mice
fed either diet also showed some thinning of the photoreceptor inner
segment layer (by LM). The number of Iba-1+ macrophages was
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
increased in brains of Cfh-null mice relative to WT mice, but there
was no diet effect.
Conclusions: The presence of eAMD-f by 10-months of age in Cfhnull mice fed a low GI diet precedes the overt retinal phenotypes
previously described in 2-year-old Cfh-null mice. This surprising
result is in contrast to the apparent protection from eAMD-f observed
in older (17- and 23-month) WT mice fed lower GI diets. Modulation
of dietary GI did not appear to influence CNS inflammation. Our
findings highlight the need to consider genetic/diet interactions when
developing therapeutic approaches to treat AMD.
Commercial Relationships: Sheldon Rowan, None; Karen Weikel,
None; Min-Lee Chang, None; Barbara Nagel, None; Jeffrey
Thinschmidt, None; Maria B. Grant, None; Steven J. Fliesler,
None; Donald Smith, None; Allen Taylor, None
Support: This work was supported by intramural funds from the
HNRCA (SR, KW, M-LC, DS, AT), by NEI RO1 EY021212 (AT),
NEI RO1 13250 (AT), and by an RPB Unrestricted Grant (SJF)
Program Number: 6174 Poster Board Number: C0059
Presentation Time: 10:30 AM - 12:15 PM
Overexpression of HtrA1 and Smoking Evokes Choroidal
Neovascularization and Retinal Deposit in Aged Mice
Mao Nakayama, Daisuke Iejima, Masakazu Akahori, Takeshi Iwata.
National Institute of Sensory organs, Nat'l Hosp Org Tokyo Med Ctr,
Tokyo, Japan.
Purpose: Previous studies have shown that Complement Factor H
(CFH) and ARMS2/HtrA1 genes significantly associate with agerelated macular degeneration (AMD). In our study, stronger
association with ARMS2/HtrA1 compared to CFH in Japanese wet
AMD was observed (Goto, Akahori et al., JOBDI 2009). Based on
the original report that HtrA1 transcription is elevated in AMD
patient (Dewan et al., Science 2005), we generated HtrA1 transgenic
mouse (Tg) and observed fundus changes by fluorescein angiography
(FA), indocyanine green angiography (IA), optical coherence
tomography (OCT) and pathological changes at over 12 month after
birth. We also examined the effects of cigarette smoke for these Tg
mice to evaluate the progression of the disease by environmental risk
factor.
Methods: Chicken actin promoter (CAG) was used to drive human
HtrA1 expression in entire mouse body and maintained for one year.
Fundus observation including FA (Micron III, Phoenix Research), IA
and OCT (Spetralis HRA+OCT, Heidelberg Engineering) was
performed. The eyes were embedded and sectioned for H&E staining
and immunohistochemistry. Smoking exposure to mice was
performed using Natural American Spirit cigarette and smoking
chamber (INH06-CIGR02A, MIPS). The Tg and control mice were
exposed to smoking for 30 min/day and 5 days/week for 12 weeks.
After 12 weeks, fundus photo and pathological analysis were
performed.
Results: HtrA1 Tg mice at 12 month exhibited hyperfluorescent
lesion on FA and low fluorescent on IA. In this lesion choroidal
neovascularization (CNV) was observed by OCT. CNV was observed
in radial formation spreading from choroid through the RPE into the
retina. Immunohistochemistry of CD31/CD34, an endothelial cells
marker, and fibronectin, a proliferation marker, were positive in this
region. No change was observed for control mice. Our result showed
that approximately 18.8% of HtrA1 Tg mice appeared retinal
disorders such as CNV. Smoking exposure for three month enhanced
CNV by approximately 7.7% for control and 30.0% for Tg mice. In
addition, abnormal deposit was observed between photoreceptor and
retinal pigment epithelium exclusively for Tg mice exposed to
smoking.
Conclusions: Overexpression of HtrA1 in mice induced CNV in
mice. Exposure to smoking enhance CNV rate in Tg mice and also in
control mice. Our result suggests overexpression of HtrA1 alone can
evoke CNV and smoking as strong risk factor for wet AMD.
Commercial Relationships: Mao Nakayama, None; Daisuke
Iejima, None; Masakazu Akahori, None; Takeshi Iwata, None
Support: Japan Society for the Promotion of Science, Smoking
Research Foundation
Program Number: 6175 Poster Board Number: C0060
Presentation Time: 10:30 AM - 12:15 PM
Contribution of CFH Y402H polymorphism and CFHR3/CFHR1
deletion to age-related macular degeneration in Brazil
Monica B. Melo1, Daniela D. Sacconi1, Galina Ananina1, Fabio E.
Hirata2, Priscila H. Rim2, Andrea M. Torigoe2, Marcio J. Silva1, Jose
Paulo C. Vasconcellos2. 1CBMEG, University of Campinas,
Campinas, Brazil; 2Ophthalmology, University of Campinas,
Campinas, Brazil.
Purpose: Age-related macular degeneration (AMD) is the leading
cause of irreversible blindness in developed countries and is
considered a multifactorial disorder. Several risk factors are involved
in its etiology and undoubtedly genetics plays an important role.
Complement factor H (CFH) gene, involved in the regulation of the
alternative pathway of the complement cascade, was the first
significant gene to be related with the pathogenesis of AMD.
Subsequently, other variants of complement pathway-associated
genes have been studied and also implicated in the susceptibility or
protection for the development of AMD, including complement
factor H-related 1 and 3 (CFHR3 and CFHR1). The purpose of this
study was to evaluate the role of the Y402H coding variant
(rs1061170) in the CFH gene and the CFHR3/CFHR1 deletion in the
etiology of AMD in a sample of Brazilian patients.
Methods: Ninety-eight unrelated AMD patients and 70 matched
controls were evaluated for the rs1061170 SNP and CFHR3/CFHR1
deletion through PCR/direct sequencing and multiplex PCR,
respectively. AMD patients were diagnosed according to the
International ARM Epidemiological Study Group.
Results: The C allele frequency of the rs1061170 SNP was
significantly higher in AMD patients than in controls (p=0.0006)
with an odds ratio (OR) of 2.187 (95%CI; 1.371-3.488). Genotype
distribution was also significantly different when cases and controls
were compared (p=0.006). For the comparison between CT versus
TT genotype (p=0.0209), the OR for heterozygotes was 2.114
(95%CI; 1.082-4.132) and for the comparison between CC versus TT
(p=0.0027) the OR for CC homozygotes was 4.933 (95%CI; 1.65614.7). On the other hand the frequency of the CFHR3/CFHR1
deletion was significantly higher in controls when compared to AMD
cases (p=0.0330).
Conclusions: These results are in accordance with the literature and
support the role of the C allele of the CFH gene in AMD
susceptibility as well as the contribution of the CFHR3/CFHR1
deletion as a protective factor against the development of the disease
in this sample of Brazilian AMD patients.
Commercial Relationships: Monica B. Melo, None; Daniela D.
Sacconi, None; Galina Ananina, None; Fabio E. Hirata, None;
Priscila H. Rim, None; Andrea M. Torigoe, None; Marcio J. Silva,
None; Jose Paulo C. Vasconcellos, None
Support: CNPq
Program Number: 6176 Poster Board Number: C0061
Presentation Time: 10:30 AM - 12:15 PM
Novel CFH variants in patients with age-related macular
degeneration
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
Maheswara R. Duvvari1, Johannes P. van de Ven1, Elena B.
Volokhina2, Nicole T. Saksens1, Camiel J. Boon1, Lies H. Hoefsloot3,
Lambert P. van den Heuvel2, Carel B. Hoyng1, Anneke I. Den
Hollander1, 3. 1Ophthalmology, Radboud University Nijmegen
Medical Centre, Nijmegen, Netherlands; 2Pediatrics, Radboud
University Nijmegen Medical Centre, Nijmegen, Netherlands;
3
Human Genetics, Radboud University Nijmegen Medical Centre,
Nijmegen, Netherlands.
Purpose: Age-related macular degeneration (AMD) is associated
with common variants in the complement factor H (CFH) gene. In
addition, rare pathogenic CFH variants have been reported in AMD
and in patients with basal laminar drusen (BLD), a clinical subtype of
AMD. The goal of this study was to assess the frequency of CFH
mutations in patients with BLD, and to validate their functional effect
on the CFH protein.
Methods: We analyzed a panel of 156 probands who were diagnosed
with BLD. All exon and intron-exon boundaries of the CFH gene
were analyzed by Sanger sequencing. Prediction algorithms were
used to predict the impact of missense changes on protein function.
The frequency of identified variants was checked in public databases
(1000 Genomes, ESP5400, dbSNP).
Results: We identified 13 rare, heterozygous CFH variants in 15
(9.6%) patients, and three polymorphisms: Val62Ile in 35 (22.4%),
His402Tyr in 124 (79.4%), and Glu936Asp in 32 (20.5%) patients.
Seven variants (Leu3Val; Phe170Cys; Ala173Gly; Arg175Gln;
Ser193Leu; Ile216Thr; Ala301AsnfsX25) are novel, three variants
(Gln408X; Arg1078Ser; Arg1210Cys) were previously reported in
AMD or BLD, and three variants (Gln400Lys; Gln950His;
Thr956Met) were previously reported in atypical haemolytic uremic
syndrome (aHUS). Six variants affect the N-terminal SCR domains
(1-4) of CFH, which play a role in complement regulatory activity.
Two variants affect the C-terminal SCR domains (18-20), which
mediate surface binding and target recognition. Two variants affect
SCR7 domain, necessary for binding heparin and CRP. One variant
was found in SCR16 domain, the precise function is not known.
Conclusions: Rare CFH variants were detected in 9.6% of patients
with the BLD subtype of AMD. Functional studies are in progress to
assess the ligand binding and cofactor activity for all identified
missense variations.
Commercial Relationships: Maheswara R. Duvvari, None;
Johannes P. van de Ven, None; Elena B. Volokhina, None; Nicole
T. Saksens, None; Camiel J. Boon, None; Lies H. Hoefsloot, None;
Lambert P. van den Heuvel, None; Carel B. Hoyng, None;
Anneke I. Den Hollander, None
Support: Netherlands Organization for Scientific Research, Vidi
Innovational Research Award 016.096.309
Program Number: 6177 Poster Board Number: C0062
Presentation Time: 10:30 AM - 12:15 PM
Targeted Sequencing, Augmented with Public Resources,
Identifies a Rare C3 Allele Associated with Large Risk of Agerelated Macular Degeneration
Xiaowei Zhan1, 2, David E. Larson3, 4, Robert S. Fulton3, 4, Chaolong
Wang5, Dwight Stambolian6, Emily Y. Chew7, Elaine Mardis3, 4,
Anand Swaroop7, Goncalo Abecasis1, 2. 1Biostatistics, University of
Michigan, Ann Arbor, MI; 2Center of Statistical Genetics, University
of Michigan, Ann Arbor, MI; 3The Genome Institute, Washington
University, St. Louis, MO; 4Department of Genetics, Washington
University, St. Louis, MO; 5Department of Biostatistics, Harvard
University, Boston, MA; 6Department of Ophthalmology and Human
Genetics, University of Pennsylvania, Philadelphia, PA; 7National
Eye Institute, Bethesda, MD.
Purpose: Macular degeneration is one of the most common causes of
incurable blindness. Common alleles in >19 loci have now been
associated with disease. We set out to investigate whether rare
variants in the same loci were also associated with disease risk and to
compare the relative effect sizes of common and rare variants.
Methods: In collaboration with the Genome Sequencing Center at
Washington University in St. Louis, we designed a sequencing study
focused on 8 of the known AMD risk loci (CFH, ARMS2, C3,
C2/CFB, CFI, CETP, LIPC and TIMP3/SYN3) and 2 other candidate
regions (LPL and ABCA1). We re-sequenced these regions in 3,124
individuals (2,335 cases and 789 controls) to an average depth of
85x. To augment the number of controls available for analysis, we
designed an algorithm to identify previously sequenced samples with
good coverage of our regions of interest and similar genetic ancestry.
Finally, we investigated association between genetic variation in each
locus and risk of disease using both single variant and gene-level
burden tests.
Results: Across 967 kb of examined sequence, we discovered 41,202
high quality SNP variants in the 3,124 sequenced individuals (34,346
of these were novel, and not previously described in dbSNP). Among
these variants, we focused our attention on 1,800 nonsynonymous,
stop and splice variants. We estimated the ancestry of our sequenced
samples and of samples from the NHLBI exome sequencing project,
identifying an ancestry matched set of 2,268 cases and 2,268
controls. Individuals in this matched set had deep (minimum 10x)
coverage of coding regions in the 10 targeted loci. Association
analysis identified two strongly associated variants, one in the CFH
gene (control frequency = 0.02%, exact P-value = 2.91x10-6, OR =
23.11) and another in the C3 gene (control frequency = 0.40%, exact
P-value = 2.73x10-4, OR = 2.68). Replication efforts for these
findings are ongoing.
Conclusions: Through targeted sequencing efforts, augmented with
publicly available control data, we replicated a previously reported
rare variant association in the CFH gene and identify a new rare
variant signal in the C3 gene. In both instances, these rare variants are
associated with substantially larger odds ratios than common variants
in the same regions.
Commercial Relationships: Xiaowei Zhan, None; David E.
Larson, None; Robert S. Fulton, Orion Genomics (C); Chaolong
Wang, None; Dwight Stambolian, None; Emily Y. Chew, None;
Elaine Mardis, None; Anand Swaroop, None; Goncalo Abecasis,
University of Michigan (P), Illumina (R), Affymetrix (R)
Support: R01 EY022005
Program Number: 6178 Poster Board Number: C0063
Presentation Time: 10:30 AM - 12:15 PM
Three New Genetic Loci (R1210C mutation in CFH , SNPs in
COL8A1 and RAD51B) are Related to Progression to Advanced
Stages of Age-Related Macular Degeneration
Johanna M. Seddon1, 2, Robyn Reynolds1, Yi Yu1, Soumya
Raychaudhuri3, 4, Bernard Rosner5. 1Ophthalmic Epidemiology and
Genetics Service, New England Eye Center, Tufts Medical Center,
Boston, MA; 2Ophthalmology, Tufts University School of Medicine,
Boston, MA; 3Divisions of Rheumatology and Genetics, Brigham and
Women's Hospital, Harvard Medical School, Boston, MA; 4Program
in Medical and Population Genetics, Broad Institute of Harvard and
MIT, Boston, MA; 5Channing Laboratory, Boston, MA.
Purpose: There is a strong genetic component and an important
environmental influence on the development of age-related macular
degeneration (AMD). We discovered a novel rare variant (R1210C)
in CFH and new common variants were found based on analyses of
several genome-wide case-control association studies. We
determined the relative impact of these new variants on progression
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
to advanced stages, controlling for previously known genes, as well
as demographic and behavioral factors.
Methods: In this longitudinal study of 2743 individuals in AREDS,
775 subjects progressed to advanced AMD with geographic atrophy
or neovascular AMD during 12 years of follow-up. Cox proportional
hazards regression analyses were performed to calculate hazard ratios
(HR) for progression. Covariates included demographic and
environmental factors, 9 variants in 8 genes, baseline macular drusen
size, and presence and type of advanced AMD in one eye at baseline.
Results: In multivariate models, age, smoking, body mass index, and
common SNP’s in the known genes CFH, ARMS2/HTRA1, C3, C2,
CFB were independently associated with AMD progression. R1210C
in CFH was related to progression from early or intermediate to
advanced stages of AMD: HR 2.5, 95% confidence interval (CI) 1.25.2, P= 0.013. Common variants in the genes COL8A1 (HR 2.0, 95%
CI 1.1-3.4, P=0.016) and RAD51B (HR 0.8, 95% CI 0.60-0.99, P=
0.038) were also independently associated with progression.
Conclusions: We identified 3 new genes that add predictive power to
risk models for progression to advanced AMD, in addition to drusen
size, baseline AMD status, demographic and environmental factors.
These models will be useful for AMD surveillance and for designing
clinical trials.
Commercial Relationships: Johanna M. Seddon, Genentech (F),
Tufts Medical Center (P); Robyn Reynolds, None; Yi Yu, None;
Soumya Raychaudhuri, None; Bernard Rosner, None
Support: NEI R01 EY011309; FFB; RPB; Lion's; MVRF;
AMDF;Macular Degeneration Research Fund, Tufts Medical Center,
Boston, MA
Program Number: 6179 Poster Board Number: C0064
Presentation Time: 10:30 AM - 12:15 PM
Investigating Age-related Macular Degeneration in the Amish
Joshua D. Hoffman1, Laura D'Aoust1, Lan Jiang1, Renee Laux1, Anita
Agarwal3, William K. Scott2, Margaret A. Pericak-Vance2, Jonathan
L. Haines1. 1Ctr for Human Genetics Resrch, Vanderbilt Univ
Medical Center, Nashville, TN; 2Hussman Institute for Human
Genomics, University of Miami, Miami, FL; 3Ophthalmology,
Vanderbilt Univ Medical Center, Nashville, TN.
Purpose: Age-related macular degeneration (AMD) is the leading
cause of blindness among the adult population in the developed
world. To further the understanding of this disease, we have studied
the genetically isolated Amish population of Ohio and Indiana. The
Amish are more genetically homogeneous than outbred groups and
live a stricter lifestyle, thus reducing the variability of environmental
effects on AMD in this population. For this analysis we set out to
characterize how known AMD associated loci contribute to disease
risk in the Amish.
Methods: We performed genotyping using the Sequenom
MassARRAY platform for pools of SNPs previously shown to be
associated with AMD in the outbred population. In total we
genotyped 84 SNPs in 1022 Amish individuals (96 with self-reported
AMD). A previous study of 73 self-reported cases and controls was
conducted to compare self-report AMD status to clinical diagnosis.
The PPV and NPV, 89% and 90% respectively, provide evidence that
self-report status is a reliable phenotype. Case-control association
analysis with adjustment for relatedness based on the complete 13generation pedigree was carried out using MQLS (Thornton &
McPeek 2007). Parametric heterogeneity LOD (HLOD) scores were
computed for affecteds-only parametric and nonparametric 2-point
linkage using Merlin. A disease allele frequency of 10% was used.
Mean cumulative genetic risk scores were calculated using a
previously described weighting scheme and compared between cases
and controls.
Results: Our most significant uncorrected MQLS p-value observed is
0.0013 for SNP rs10490924 on chromosome 10 in the gene encoding
ARMS2. Additional signals are observed at rs2230199 (p-value =
0.0022) on chromosome 19, and rs3753394 (p-value = 0.0061) on
chromosome 1. In our cumulative genetic risk score analysis we
observe a mean risk score of 1.041 (95% CI [1.026, 1.055]) in our
controls and 1.099 (95% CI [1.053, 1.145]) in our cases. This mean
difference in risk scores is statistically significant at a p-value of
0.006.
Conclusions: In this analysis a subset of loci previously identified in
the general outbred population associate with AMD risk in the
Amish. We also observe a statistically significant increase in
cumulative genetic risk in our cases versus controls. As of this study,
only 96 self-reported cases of AMD had been ascertained within the
larger Amish study. We plan to ascertain additional cases to increase
power to detect associations.
Commercial Relationships: Joshua D. Hoffman, None; Laura
D'Aoust, None; Lan Jiang, None; Renee Laux, None; Anita
Agarwal, Vanderbilt University (P); William K. Scott, Duke
University/ArcticDx (P); Margaret A. Pericak-Vance, None;
Jonathan L. Haines, Arctic Dx (I), AMD genes (P)
Support: EY012118-12
Program Number: 6180 Poster Board Number: C0065
Presentation Time: 10:30 AM - 12:15 PM
Genetic Risk Score Predicts of Severity of Age-related Macular
Degeneration
Jonathan L. Haines1, Anita Agarwal1, Jessica N. Cooke Bailey1,
Joshua D. Hoffman1, J. Allie McGrath1, Lana M. Olson1, Jaclyn L.
Kovach2, Stephen G. Schwartz2, William K. Scott3, Margaret A.
Pericak-Vance3. 1Ctr Human Genetics Res-Med Ctr, Vanderbilt
University, Nashville, TN; 2Bascom Palmer Eye Institute, Naples,
FL; 3Human Genetics, University of Miami, Miami, FL.
Purpose: Impact of a genetic risk score based on 16 AMD risk loci
on asymmetric AMD severity & progression. We hypothesized that a
genetic risk score would contribute significantly to differences
between AMD progressors & non-progressors and severity of AMD.
Methods: Clinical & genotype risk factor data on two sets of patients
were evaluated. Patients who entered the study with nonexudative
AMD (Grades 1, 2, 3 or 4) in both eyes and followed annually were
in group 1. Group 2 contained patients who had exudative AMD
(grade 5) in one or both eyes when they entered the study. Evidence
of progression or non-progression of AMD grade in eyes with grade
4 or less was analyzed by evaluating serial fundus photographs.
Progressors were defined as those who advanced by one or more
grade or whose condition within a grade worsened clinically. Of 50
group 1 patients, 32 were progressors & 18 non-progressors. We
tested progression of AMD against a weighted genetic risk score
calculated based on 16 AMD risk variants. Age at initial exam,
smoking & gender were included as covariates and analyzed using
logistic regression. In group 2, 482 patients with grade 5 AMD in
both eyes were compared to 491 patients with grade 5 in one eye
only. An additional subset analysis was done on patients with Grade
5 in one eye & Grades 2, 3, or 4 in the fellow eye for determining the
influence of genetic risk score against disease severity.
Results: No significant association was detected when progressors
and non-progressors were compared in this small sample (group 1).
When group 2 patients with grade 5 AMD were stratified based on
unilateral (n=491) or bilateral disease (n=482), we detected a
significant effect from the risk score (P<0.0003; OR=4.85; 95% CI
2.69-8.76) when included along with gender & age at exam.
Including smoking as a covariate decreased our sample size to 726, &
significance remained trending. Comparing the unilateral grade 5
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
patients with grades 1, 2, 3 or 4 in the fellow eye and bilateral grade
5, the LSMEANS of the risk score trended significantly higher as the
AMD severity rose in the fellow eye - 1.06710658, 1.17629142,
1.20540778, 1.26111358 respectively, with the highest in bilateral
grade 5 patients - 1.28466396.
Conclusions: Cumulative genetic risk appears to predict severity of
AMD in a subset of individuals with Grade 5 in one versus both eyes,
and trended upwards as the severity of AMD grade increased in the
fellow eye.
Commercial Relationships: Jonathan L. Haines, Arctic Dx (I),
AMD genes (P); Anita Agarwal, Vanderbilt University (P); Jessica
N. Cooke Bailey, None; Joshua D. Hoffman, None; J. Allie
McGrath, None; Lana M. Olson, None; Jaclyn L. Kovach, None;
Stephen G. Schwartz, Alimera (C), Bausch + Lomb (C), Eyetech
(C), IC Labs (P), ThromboGenics (C); William K. Scott, Duke
University/ArcticDx (P); Margaret A. Pericak-Vance, None
Support: NIH/NEI 5R01 EY012118 -(Pericak-Vance, PI) Unifying
Genetics Epidemiology of Macular Degeneration.
Program Number: 6181 Poster Board Number: C0066
Presentation Time: 10:30 AM - 12:15 PM
The association of elastin gene variants with two angiographic
subtypes of polypoidal choroidal vasculopathy
Shigeru Honda, Suihou Yanagisawa, Akiko Miki, Wataru Matsumiya,
Akira Negi. Kobe University Graduate School of Medicine, Kobe,
Japan.
Purpose: To compare the association of elastin gene variants
between two different angiographic phenotypes of polypoidal
choroidal vasculopathy (PCV).
Methods: We included 155 Japanese patients with PCV and 203
controls. PCV was classified into two phenotypes according to the
presence (Type 1) or absence (Type 2) of the feeding vessels found in
indocyanine-green angiography. The single nucleotide
polymorphisms (SNPs) in the elastin region including rs3757584,
rs2301995 and rs2856728 were genotyped using the TaqMan assay.
Results: The minor allele frequency (MAF) of rs2301995 was
significantly different between Type 2 PCV (21%, n=82) and control
(17%) (p=0.0061), while no difference was found between Type 1
PCV (n=73) and control (p=0.25). Haplotype analysis revealed that
the association of the haplotype (A-T-C) at rs3757584, rs2301995
and rs2856728 differed significantly between Type 2 PCV (25%) and
control (17%) (p=0.027), while no difference was found between
Type 1 PCV (20%) and control (p=0.40).
Conclusions: There may be significantly different association in
genetic variants of elastin between two angiographic phenotypes of
PCV.
Commercial Relationships: Shigeru Honda, None; Suihou
Yanagisawa, None; Akiko Miki, None; Wataru Matsumiya, None;
Akira Negi, None
Support: Grant-in Aid (C) 23592567 from the Ministry of
Education, Science and Culture, Tokyo, Japan
Program Number: 6182 Poster Board Number: C0067
Presentation Time: 10:30 AM - 12:15 PM
Associations of C3 with neovascular age-related macular
degeneration and polypoidal choroidal vasculopathy
Ke Liu, Li Jia Chen, Pancy O.S. Tam, Chi Pui Pang. Department of
Ophthalmology and Visual Sciences, The Chinese University of
Hong Kong, Hong Kong, China.
Purpose: To investigate the associations of common single
nucleotide polymorphisms (SNPs) in the complement factor 3 (C3)
gene with neovascular age-related macular degeneration (AMD) and
polypoidal choroidal vasculopathy (PCV).
Methods: 708 unrelated study subjects were included, consisting of
200 neovascular AMD, 233 PCV and 275 controls. Eight tag SNPs in
C3 were selected from HapMap project database for Han Chinese.
Genotyping of each tag SNP was performed by TaqMan genotyping
assays (Applied Biosystems [ABI], Foster City, CA).
Results: None of the eight tag SNPs or the haplotypes showed
significant association with neovascular AMD or PCV. A significant
interaction between a SNP and gender was identified in PCV. After
stratified by gender, a risk effect of the minor allele of the SNP (p =
0.01, OR = 1.56) was observed in male PCV. A haplotype consist of
the major allele of the SNP showed a significant protection for PCV
(p = 0.01, OR = 0.64). The C3 SNPs or haplotypes were not
associated with PCV in female. Besides, no SNPs or haplotypes were
associated with neovascular AMD in either male or female.
Conclusions: Our results show significant association of a tag SNP
in C3 with PCV in male but not in female, suggesting a genderspecific effect of C3 in PCV.
Commercial Relationships: Ke Liu, None; Li Jia Chen, None;
Pancy O.S. Tam, None; Chi Pui Pang, None
Program Number: 6183 Poster Board Number: C0068
Presentation Time: 10:30 AM - 12:15 PM
Polymorphisms association of CFH, BF, C3, FHR 1-3 and
ARMS2 genes with geographic atrophy progression in AgeRelated Macular Degeneration
Sergio Recalde, Patricia Fernandez, Laura Garcia-Garcia, Jose M.
Caire, Ana I. Hernández, Vanessa Fernandez-Garcia, Maite MorenoOrduña, Alfredo Garcia-Layana. Ophthalmology, Universidad de
Navarra, Pamplona, Spain.
Purpose: Age related macular degeneration (AMD) is a leading
cause of blindness. AMD is a complex disorder caused by genetic
and environmental factors, in which single nucleotide polymorphisms
(SNPs) in the genes CFH, BF, C3, FHR 1-3, and ARMS2/HTRA1
have prognostic importance of major risk to develop advanced AMD.
To determine if genotype is associated with rate of growth of
geographic atrophy (GA) in eyes with AMD, we assessed the
relationship of GA progression with the previously mentioned SNPs
associated with AMD in a Spanish population.
Methods: We performed a prospective, controlled, multicenter study
in 8 Spanish Hospitals. Fundus autofluorescence photographs (FAF)
were taken at 0 and 24 months from patients with GA form of AMD
and changes in cumulative areas of GA were evaluated in 73 eyes of
73 patients. DNA samples were collected from donors to analyse
SNPs in CFH, BF, C3, FHR 1-3, and ARMS2 genes. Simultaneous
detection of SNPs was based on Multiplex PCR technology and
minisequencing. Logistic regression was used to analyse the
association of SNPs, Age, BMI, and smoking with GA progression.
Results: The logistic regression analysis for Rate of Progression (RP)
and Relative Growth (RG) showed a significant association between
RP and genetic factors [Y402H (p=0.036), I62V (p=0.039)] and
environmental factors [gender (p=0.025) and age (p=0.02) and
between RG and genetic factor [R32Q (p=0.043)].
Conclusions: This study confirms that variants in CFH and CB genes
and environmental risk factors confer significant risk for progression
in geographic atrophy of AMD in a Spanish population.
Commercial Relationships: Sergio Recalde, None; Patricia
Fernandez, None; Laura Garcia-Garcia, None; Jose M. Caire,
None; Ana I. Hernández, None; Vanessa Fernandez-Garcia,
None; Maite Moreno-Orduña, None; Alfredo Garcia-Layana,
None
Support: PI08/1705 and RETICS RD07/0062, Ministerio de Ciencia
e Innovación and PI11/00898 and RD11/0034, Ministerio Ministerio
de Competitividad, Spain
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
Program Number: 6184 Poster Board Number: C0069
Presentation Time: 10:30 AM - 12:15 PM
Genotype Analysis for Single Nucleotide Polymorphisms Profile
of Eyes with Vascularized Pigment Epithelial Detachment due to
AMD
Clement K. Chan1, 2, David Sarraf3, Prema Abraham4, Duy H.
Nguyen5, Steven G. Lin1, Maziar Lalezary1, Kang Zhang5, 6.
1
Vitreoretina, Southern California Desert Retina Consultants, Palm
Desert, CA; 2Department of Ophthalmology, Loma Linda University,
Loma Linda, CA; 3Retinal Disorders and Ophthalmic Genetics
Division, Jules Stein Eye Institute, University of California, Los
Angeles, Los Angeles, CA; 4Retina Section, Black Hills Regional
Eye Institute, Rapid City, SD; 5Department of Ophthalmology,
University of California, San Diego, San Diego, CA; 6Institute of
Genomic Medicine, Shiley Eye Center, University of California, San
Diego, San Diego, CA.
Purpose: To compare the single nucleotide polymorphisms (SNPs)
profile for eyes with a vascularized pigment epithelial detachment
(vPED) due to age-related macular degeneration (AMD) with 1)
Control 1: eyes without AMD (AREDS Severity Scale zero), and 2)
Control 2: eyes with drusen (AREDS Severity Scale 2), as well as 3)
to compare the SNPs profile of high responders with low responders.
Methods: Blood samples obtained from 39 eyes (39 patients) with
vPED treated with either 0.5 or 2.0 mg ranibizumab were sent to the
Institute of Genomic Medicine, Shiley Eye Center, UCSD for SNPsspecific genotype analysis. The analyses included: 1) Comparison of
variant allele frequencies (VAF) of 23 SNPs with known association
with AMD in 39 vPED eyes with the VAF in 184 Control 1 eyes, 2)
Comparison VAF of 23 SNPs associated with AMD in same 39
eyeswith the VAF in 85 Control 2 eyes, and 3) Comparison of SNPs
of high responders (≥ 50% decrease of PED height or ≥ 10 letter
gainers) (HR-50%) with low responders, and comparison of SNPS of
high responders (≥ 75% decrease of PED height or ≥ 10 letter
gainers) (HR-75%) with low responders. P ≤ 0.05 was considered
significant.
Results: 1) 1st analysis: Following SNPs were significantly
associated with vPED eyes vs. eyes without AMD: VEGF rs943080
(P=0.05), CFH rs1061170 (p=0.02), CFH rs2274700 (p=0.001),
APOE rs4420638 (p=0.005), HTRA1 rs10490924 (0.005), CFH
rs10737680 (p=0.002), and CFH rs10801555 (p=0.02). 2) 2nd
analysis: Following SNPs were significantly associated with vPED
eyes vs. eyes with drusen and dry AMD: COL15A1/TGFBR1
rs334353 (T/G) (p=0.002), APOE rs4420638 (A/G) (p=0.003), and
CFI rs4698775 (G/T) (p=0.008). 3) Comparisons for high vs. low
responders showed significant association of FRK rs3812111 (T/A)
with both HR-50 and HR-75 groups (p=0.03).
Conclusions: This study shows that 7 SNPs with prior known
association with AMD were more frequently associated with vPED
eyes when compared to non-AMD eyes, whereas 3 SNPs were more
frequently associated with vPED eyes when compared to eyes with
drusen. APOE rs4420638 was single SNP more frequently linked to
vPED eyes for both comparisons. One SNP [FRK rs3812111 (T/A)]
was also consistently associated with high responders. To our
knowledge, this is the first report on SNPs-specific profile of vPED
eyes associated with AMD.
Commercial Relationships: Clement K. Chan, Genentech (F),
Genentech (R), Alimera (C), Alimera (R), Allergan (R), Regeneron
(F), Regeneron (R), NEI (F), Thrombogenics (R), Valeant (C); David
Sarraf, Genentech (F), Regeneron (F), Allergan (F), Alcon (F),
DORC (F); Prema Abraham, None; Duy H. Nguyen, None; Steven
G. Lin, None; Maziar Lalezary, None; Kang Zhang, Acucela (F),
Genentech (F), Thrombogenics (F)
Support: Grant from Genentech-Roche, Protocol # FVF4332s
Clinical Trial: NCT00749021
Program Number: 6185 Poster Board Number: C0070
Presentation Time: 10:30 AM - 12:15 PM
Pharmacogenetic associations in neovascular age-related macular
degeneration. Results from the IVAN study
Andrew J. Lotery1, 2, Jane Gibson3, Angela J. Cree1, Susan M.
Downes4, Simon P. Harding5, Chris Rogers6, Barnaby C. Reeves6,
Sarah Ennis3, Usha Chakravarthy7. 1Clinical & Experimental
Sciences, Faculty of Medicine, University of Southampton,
Southampton, United Kingdom; 2Southampton Eye Unit, University
Hospital Southampton NHS Foundation Trust, Southampton, United
Kingdom; 3Human Development and Health, Faculty of Medicine,
University of Southampton, Southampton, United Kingdom; 4Oxford
Eye Hospital, Oxford University Hospitals NHS Trust, Oxford,
United Kingdom; 5Department of Eye and Vision Science, University
of Liverpool, Liverpool, United Kingdom; 6School of Clinical
Sciences, University of Bristol, Bristol, United Kingdom; 7The
Institute for Ophthalmology and Vision Science, The Queen’s
University of Belfast, Belfast, United Kingdom.
Purpose: To determine if pre-specified genetic polymorphisms
influence treatment responsiveness to VEGF inhibition in patients
with neovascular age-related macular degeneration (nAMD).
Methods: Participants enrolled in the IVAN clinical trial and
undergoing treatment for nAMD with either ranibizumab or
bevacizumab were classified as treatment responders or nonresponders based on the optical coherence tomography (OCT) metric
of total retinal thickness (TRT). We computed the change in TRT
from baseline to the latest time point for which OCT data was
available (3, 6, 9 or 12 months). Study eyes at or above the 75th
percentile for change were classified as responders and those at or
below the ≤25th percentile for change as non-responders.
Two analyses were performed using data for responders and nonresponders. Firstly, we tested for replication of three previously
reported pharmacogenetic associations of response to VEGF
inhibition in nAMD at the CFH, FZD4 and HTRA1/ARMS2 loci.
Secondly, we tested an additional 500 SNPs for pharmacogenetic
associations using a candidate gene approach.
These analyses compared single nucleotide polymorphism (SNP)
frequencies in responder and non-responder groups (2 x 2 tables), in
accordance with a prespecified analysis plan.
Results: 512 patients were available with both phenotype and
genotype information. Three were removed from further analysis due
to differing ethnicity. 423 had OCT data available at 12 months, 53 at
9 months, 20 at 6 months and 13 at 3 months. 255 were in the middle
50% and not analysed for the main “case/control” association tests,
126 were classified as non-responders and 128 as responders. SNP
rs10490924 in HTRA1/ARMS2 showed a borderline association after
Bonferroni correction (pc = 0.06; p = 0.02 without correction). For
the 500 additional SNPs tested for association, none were significant
after Bonferroni correction for multiple testing. The smallest p-value
was 0.0005 (pc =0.24) for rs9679290 in the EPAS1 (HIF2A) gene on
chromosome 2.
Conclusions: Our analysis tested pharmacogenomic association
using high quality phenotype data from a randomised controlled trial
of nAMD. SNPs investigated covered several biological pathways
which could be plausibly linked to nAMD. No replication of previous
associations or other significant associations was observed.
Commercial Relationships: Andrew J. Lotery, Novartis (F), Bayer
(R); Jane Gibson, None; Angela J. Cree, None; Susan M. Downes,
Novartis (F); Simon P. Harding, Novartis (F), Novartis (R); Chris
Rogers, Novartis (R); Barnaby C. Reeves, None; Sarah Ennis,
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
None; Usha Chakravarthy, Bayer (C), Novartis (F), Neovista (C),
Oraya (F)
Support: National Institute for Health Research (NIHR) Health
Technology Assessment (HTA) program (project number 07/36/01)
and by the Macular Disease Society. The trial was designed,
conducted, analyzed, and interpreted independently of the funding
sources. The views and opinions expressed are those of the authors
and do not necessarily reflect those of the HTA programme, NIHR,
the UK National Health Service or the Department of Health.
Clinical Trial: ISRCTN92166560
Program Number: 6186 Poster Board Number: C0071
Presentation Time: 10:30 AM - 12:15 PM
The Role of Genetics in Response to Anti-VEGF Therapy for Wet
AMD
Jaclyn L. Kovach1, Anita Agarwal2, Stephen G. Schwartz1, Milam A.
Brantley2, William Cade3, Jonathan L. Haines4, Margaret A. PericakVance3. 1Ophthalmology, Bascom Palmer Eye Institute, Naples, FL;
2
Ophthalmology, Vanderbilt Eye Insitute, Nashville, TN; 3Institute
for Human Genomics, University of Miami Miller School of
Medicine, Miami, FL; 4Human Genetics, Vanderbilt University
School of Medicine, Nashville, TN.
Purpose: To evaluate the relationship between high-risk genetic
polymorphisms and the response to anti-VEGF therapy in wet AMD.
Methods: Forty-three Caucasian patients with wet AMD (AREDS
grade 5) who were treated with bevacizumab, ranibizumab, and/or
aflibercept were followed for 1 year with complete eye exams and
OCT imaging Q1-3 months. Fundus photography and fluorescein
angiography were performed periodically. Individuals who gained at
least 2 lines of Snellen vision with a reduction or absence of
intraretinal/subretinal fluid at the end of 1 year were labeled “good
responders.” Those that lost at least 2 lines of Snellen vision with
persistent/increased intraretinal/subretinal fluid were classified as
“poor responders.” The remaining patients were categorized as
“maintainers.” Genomic DNA was extracted from whole blood and
analyzed. Patients were genotyped and two types of risk scores (RS)
were created that computed the number of high risk alleles for each
individual. The 16RS considered 16 single nucleotide polymorphisms
(SNP) identified as loci reaching genome-wide significance, and the
4RS considered CFH rs10737680, C2/CFB rs429608, ARMS2
rs10490924, and C3 rs2230199.
Results: Of 43 patients, 16 were good responders, 19 were
maintainers, and 8 were poor responders. A strong correlation existed
between the two types of risk scores. Maintainers had a higher 16RS
and 4RS than the other two groups. Average 16RS (p = 0.0197) and
4RS (p= 0.0186) were significantly lower in good responders
compared to maintainers, and individual risk allele frequencies for
the 4 major SNPs were lower in good responders compared to
maintainers. Both risk scores were slightly lower in poor responders
compared to maintainers, but the differences were not statistically
significant.
Conclusions: Individuals that maintained stable vision with antiVEGF therapy had greater genetic risks than those with good
responses. Further investigation of the role of genetics and
environmental factors in response to therapy in a larger sample size
of wet AMD patients may bring us closer to personalized treatment
for AMD.
Commercial Relationships: Jaclyn L. Kovach, None; Anita
Agarwal, Vanderbilt University (P); Stephen G. Schwartz, Alimera
(C), Bausch + Lomb (C), Eyetech (C), IC Labs (P), ThromboGenics
(C); Milam A. Brantley, None; William Cade, None; Jonathan L.
Haines, Arctic Dx (I), AMD genes (P); Margaret A. PericakVance, None
Support: NH Grant R01 EY012118-11
Program Number: 6187 Poster Board Number: C0072
Presentation Time: 10:30 AM - 12:15 PM
VEGF-A and VEGFR-2 Gene Polymorphisms and Response to
Anti-VEGF Therapy in the Comparison of AMD Treatments
Trials (CATT)
Stephanie A. Hagstrom1, 2, Gui-Shuang Ying3, Gayle J. Pauer1, Gwen
M. Sturgill-Short1, Jiayan Huang3, Maureen G. Maguire3, Daniel F.
Martin1. 1Cole Eye Institute, Cleveland Clinic, Cleveland, OH;
2
Ophthalmology, Cleveland Clinic Lerner College of Case Western
Reserve University, Cleveland, OH; 3Ophthalmology, University of
Pennsylvania, Philadelphia, PA.
Purpose: To evaluate the pharmacogenetic relationship of genotypes
of single nucleotide polymorphisms (SNPs) in vascular endothelial
growth factor (VEGF-A) and its main receptor (VEGFR-2) with
response to intravitreal injections of ranibizumab or bevacizumab for
neovascular age-related macular degeneration (AMD).
Methods: 835 (75%) of 1116 patients participating in the
Comparison of AMD Treatments Trials (CATT) were recruited
through 43 CATT clinical centers. Each patient was genotyped for
seven SNPs in VEGF-A (rs699946, rs699947, rs833069, rs833070,
rs1413711, rs2010963, rs2146323) and one SNP in the high-affinity
VEGF-A receptor, VEGFR-2 (rs2071559), using TaqMan SNP
genotyping assays. Genotypic frequencies were compared to clinical
measures of response to therapy at one year including visual acuity
(VA), change in VA, presence of fluid, retinal thickness, change in
lesion size and number of injections in the as needed groups.
Differences in response by genotype were evaluated with tests of
linear trend calculated from logistic regression models for categorical
outcomes and linear regression models for continuous outcomes. A
stepwise analysis was performed to examine the additive effects
based upon the total number of risk alleles from the eight SNPs.
Results: The genotypic frequencies for each SNP analyzed were
balanced across treatment groups. The effect of risk alleles on each
clinical measure did not differ by treatment group, drug or dosing
regimen (p >0.01). Therefore, we collapsed all treatment groups and
report our findings on the entire 835 patients as a single group. For
each of the measures of VA evaluated, there was no association with
any of the genotypes or with the number of risk alleles from the eight
SNPs. Four of the VEGF-A SNPs demonstrated an association with
retinal thickness (rs699947, rs833070, rs1413711, p=0.03 to 0.04;
rs2146323, p=0.006). However, none of these modest associations
were supported by any other anatomical measure. Among the
participants in the two PRN groups, no association was found in the
number of injections among the different genotypes for any of the
eight SNPs, or for the total number of risk alleles from the eight
SNPs.
Conclusions: There were no strong pharmacogenetic associations
between the studied VEGF-A
and VEGFR-2 SNPs and response to anti-VEGF therapy in patients
participating in CATT.
Commercial Relationships: Stephanie A. Hagstrom, None; GuiShuang Ying, None; Gayle J. Pauer, None; Gwen M. SturgillShort, None; Jiayan Huang, None; Maureen G. Maguire, Inspire
Pharmaceuticals (F), Amakem (F), IDx LLC (F), Merck (C); Daniel
F. Martin, None
Support: U10 EY017823, U10 EY017825, U10 EY017826, and U10
EY017828 from the National Eye Institute, National Institutes of
Health, Department of Health and Human Services
Clinical Trial: NCT00593450
Program Number: 6188 Poster Board Number: C0073
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
Presentation Time: 10:30 AM - 12:15 PM
Enhancer elements show enrichment for AMD-associated genetic
variants
Lakshmi P. Pulagam1, Nathan J. Morris1, Penny Benchek1, Peter
Scacheri2, Sudha K. Iyengar1, 2. 1Epidemiology and Biostatistics,
Case Western Reserve University, Cleveland, OH; 2Genetics and
Genome Sciences, Case Western Reserve University, Cleveland, OH.
Purpose: Enhancers are complex yet highly coordinate cis-regulatory
elements that regulate gene expression. Although age-related macular
degeneration (AMD)-associated variants at >12 loci have been
identified through GWAS, it is very difficult to identify
mechanistic/causal variants because they co-occur with nearby
markers that are in strong linkage disequilibrium (LD). The purpose
of this study was to determine whether AMD-associated variants lie
within enhancer elements.
Methods: We captured retina-specific enhancer profiles together
with expression abundance in primary Retinal Pigment Epithelium
(RPE) cells (HRPEpiC), using ChIP Seq for enhancer chromatin
marks, H3K4me1 and H3K27ac and RNA Seq for expression.
Paired-end sequenced data (2x100bp) was analyzed using BWA, and
ChIP Seq peaks were called using MACS, after using input DNA as
control. AMD-associated GWAS SNP(s) and RPE enhancer
colocalization profiles were plotted. At each locus, the local LD
profile surrounding the sentinel SNP was captured from HapMap2.
To determine the likelihood of these results being obtained by chance
alone, we randomly selected 1000 other sets of SNPs genome-wide
with a similar LD profile and frequency to the GWAS hit(s), and
conducted an identical test for each set.
Results: Peak analysis of the ChIP Seq data revealed that some of the
best reported AMD-association signals localize either within or in
proximity of enhancer peaks. For example, variants near
ARMS2/HTRA1 and CETP are either co-localized within or very
close to enhancer peaks (False Discovery Rate [FDR] = 0.22%, pvalue = 1.23 10-27, and FDR = 0.34%, p-value = 4.88 x 10-17
respectively). Among all loci, 35% of GWAS hits were either within
an enhancer peak, or were in LD (r2 ≥ 0.8) with a variant in the
enhancer. We calculated the percentage of SNP sets which were
either as enriched or more enriched for enhancer profiles. Among the
1000 simulated experiments genome-wide, only 3.7% had a result as
extreme or more extreme than the AMD GWAS-enhancer profiles.
Conclusions: Our results indicated that 35% of the AMD-associated
GWAS hits localize within enhancer profiles. Comparison with 17
other cell lines from ENCODE shows that this enhancer profile is
likely retina-specific. In summary, enhancer data supplemented with
other epigenetic marks from ENCODE is very useful in defining
mechanistic events to further understand the role of these variants in
the regulation of AMD pathogenesis.
Commercial Relationships: Lakshmi P. Pulagam, None; Nathan
J. Morris, None; Penny Benchek, None; Peter Scacheri, None;
Sudha K. Iyengar, None
Support: The International Retinal Research Foundation ;
CWRU/Cleveland Clinic CTSA Grant Number UL1 RR024989;
Genome and Transcriptome Sequencing Core, CWRU
Program Number: 6189 Poster Board Number: C0074
Presentation Time: 10:30 AM - 12:15 PM
Early Biomarkers of AMD (EBAMD) Study - Design and
Baseline Data: IRB-11-5677
Steven G. Pratt1, Stuart P. Richer2, Clifton Blake Perry1, 3, Grant
Ruteledge4, Carla Podella2. 1Scripps Health/Scripps Memorial
Hospital/Scripps Mericos Eye Institute/Scripps Clinical Research
Service, La Jolla, CA; 2Eye Clinic, Capt James A Lovell FHCC,
North Chicago, IL; 3Ophthalmology, University of Iowa, Iowa City,
IA; 4Biology, University of California, Irvine, CA.
Purpose: Children of AMD patients have a 45% lifetime risk of
developing AMD. EBAMD is a prospective exploratory study
evaluating baseline genetic risk against a battery of epigenetic serum,
nutritional & sub-clinical biomarkers in children of AMD patients.
Methods: Informed consent,staggered recruitment registry of (to
date) n=50 (35F/15M) children of AMD patients. Inclusion: 1) Age
40+; 2) mother and/or father w AMD; 3) no visible AREDS AMD
pathology or confounding ocular / systemic disease. Systemic : invivo skin carotenoids, BMI / waist circumference & hand strength
dynamometry. Genetics: Buccal swab categorized for total risk &
lifetime risk using a 5 genotype CFH, C3, ARMS2 & MT-ND2 8
SNP DNA array, www.njlabs.com & serum MTHFR
(methylenetetrahydrofate reductase) C677T & A1298C folate
mutations / hcyst metab. Epigenetics : 1) HS-n-3 Index array; 2) lipid
& apolipoprotein particle size, LP(a) & hs-CRP; 3) cell micronutrient
deficiency (B complex, AAs, metabolites, fatty acids, vitamins,
minerals, CHO metab, antiox index; cell prolif- immunidex index),
www.Spectracell .com; 4) serum carotenoids & inflamm marker
panels (IL-1a, IL-1B, IL-6, MCP-1,TNFa) www.pbrc.edu; 5) Food
Frequency Intake ,www.hsph.harvard.edu & 6) structure / function
SD-OCT retinal thickness / 1 deg macula pigment (QuantifEye) &
function (MDD2 glare recov (secs), Stereo Optical CSF (AUC) &
yellow VF scotoma (count).
Results: Subjects are 63.3 SD 8.8 yrs, 25.6 BMI, high skin
carotenoids 42,860 / 50,000 & hand grip strength R 82.6 SD 46 lb; L
75.5 SD 46.5 lb. Mean AMD pop genetic risk category = 2.15 SD
0.92 with total mean lifetime risk of 15.6 % SD 13. MTHFR
genotypes present in n=24 (16 HTZ/ 8 HOM) (C677T mutation) and
n=21 (17 HTZ / 4 HOM) (A1298c mutation). Of 70 serum factors,
the whole population had 2 abnormalities: ↑ Total sat fat & chol.
(Pop abn mean (SD) & ref range: ↑ Sat fat @ 43.4 SD 1.6 mg% (36.4
- 42.0 ref range); ↑ Total Chol @ 201.2 SD 33 (<200 NCEP
guidelines); none- the- less, the data revealed a variety of individual
baseline abnormalities awaiting correlation with genetics / structure
& visual function.
Conclusions: Minimal baseline abnormalities are consistent with this
well educated, well nourished, active and affluent population. By
identifying early individual AMD biomarkers in children &
instituting lifestyle changes/ nutrient repletion, EBAMD aims to
reduce the case-by-case clinical onset of AMD.
Commercial Relationships: Steven G. Pratt, None; Stuart P.
Richer, None; Clifton Blake Perry, None; Grant Ruteledge, None;
Carla Podella, None
Support: Scripps Memorial Hospital / Mericos Eye Institute /
Scripps Research Service, La Jolla, CA
Clinical Trial: 11-5677
Program Number: 6190 Poster Board Number: C0075
Presentation Time: 10:30 AM - 12:15 PM
Association of the SOD activity, rs1061170 in CFH and rs1531289
in VEGFR2 with protein levels in North Indian age related
macular degeneration patients
Neel Sharma1, Amod Gupta2, Sudesh Prabhakar1, Ramandeep
Singh2, Suresh Sharma3, akshay anand1. 1Neurology, Post Graduate
Institute of Medical Education and Research, Chandigarh, India;
2
Ophthalmology, Post Graduate Institute of Medical Education and
Research, Chandigarh, India; 3Statistics, Panjab University,
Chandigarh, India.
Purpose: Linkage has earlier been shown to the oxidative stress,
vascular endothelial growth factor 2 (VEGF2) gene, complementary
factor H (CFH) and age related macular degeneration (AMD). We
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
therefore, wanted to determine the levels of super oxide dismutase
(SOD) activity, polymorphism in CFH and VEGFR2 genes and their
serum levels in AMD patients in comparison to control subjects.
Methods: 176 subjects were enrolled in a case-control genetic study.
The SOD activity was analyzed using commercially available
superoxide dismutase assay kit. Real-Time PCR was used to analyze
the SNPs (rs1061170, rs1531289 and rs2305948) of CFH and
VEGFR-2 genes. ELISA was conducted to determine the levels of
CFH and VEGFR2. A non-parametric Mann-Whitney-U test was
applied for comparison of the ELISA levels and Pearson’s Chi-square
test was applied to study the association of polymorphism between
various groups.
Results: AG genotype (rs1531289) in VEGFR2 and TT (rs1061170)
genotype in CFH were significantly associated with AMD (OR=
2.13, p=0.047 and OR=16.5, p=0.0001 respectively). VEGFR2 levels
were found to be increased significantly and CFH levels were found
to be decreased in AMD patients as compared to controls. VEGFR2
levels were found to significant increased among wet AMD as
compared to dry AMD. We did not find any significant difference in
SOD activity between AMD and normal controls.
Conclusions: The increased levels of VEGFR2 and decreased levels
of CFH with polymorphism in our population may be associated with
AMD suggesting the role of VEGFR-2 and CFH in pathogenesis of
AMD.
Commercial Relationships: Neel Sharma, None; Amod Gupta,
None; Sudesh Prabhakar, None; Ramandeep Singh, None; Suresh
Sharma, None; akshay anand, None
Support: DST Grant F.No. SR/SO/HS-109/205 dated 1-05-2007
Program Number: 6191 Poster Board Number: C0076
Presentation Time: 10:30 AM - 12:15 PM
AMD-associated variants at chromosome 10q26 locus and
ARMS2/HTRA1gene expression
Gaofeng Wang1, Brenda Court1, Patrice Gay1, Sander R. Dubovy2,
Jaclyn L. Kovach2, Stephen G. Schwartz2, Anita Agarwal3, William K.
Scott1, Jonathan L. Haines4, Margaret A. Pericak-Vance1. 1Hussman
Inst for Human Genomics, Univ of Miami Miller Sch of Med, Miami,
FL; 2Bascom Palmer Eye Institute, University of Miami Miller
School of Medicine, Miami, FL; 3Ophthalmology, Vanderbilt
University, Nashville, TN; 4Center for Human Genetics Research,
Vanderbilt University, Nashville, TN.
Purpose: To analyze the relationship between age-related macular
degeneration (AMD) associated variants at the chromosome 10q26
locus and ARMS2/HTRA1 expression in human retinas and in vitro
systems.
Methods: ARMS2 minigenes with different genotypes at rs2736911
(R38X) and the 3’ UTR indel were constructed. Exogenous ARMS2
transcription from minigenes was assessed in mouse embryonic
fibroblasts (MEF) which do not express endogenous ARMS2. Dual
luciferase assays were applied to further evaluate the effect of the
indel on ARMS2 expression. Genotypes of retinal samples at
ARMS2 variants R38X and rs11200638 and HTRA1 variants
rs1049331 and rs2293870 were obtained by Taqman assays.
Genotypes at the indel of ARMS2 were obtained by PCR and gel
assay. RT-PCR and quantitative RT-PCR were applied to measure
endogenous ARMS2 or HTRA1 transcripts in human retinal samples
(n=82). HTRA1 protein was evaluated by Western blot.
Results: R38X significantly decreases exogenous ARMS2 transcript
levels from minigenes in MEF. In contrast, the indel does not
significantly change ARMS2 transcript levels . Dual luciferase assays
further show that the indel does not influence gene expression.
Although R38X appears to decrease the stability of exogenous
ARMS2 transcripts in cultured cells, genotypes at R38X, the indel
and rs11200638 do not significantly change the level of either
ARMS2 or HTRA1 transcripts in human retinas. Furthermore, two
synonymous variants rs1049331 and rs2293870 in HTRA1 do not
change its protein level either.
Conclusions: AMD-associated variants at chromosome 10q26 locus
do not significantly change the gene expression of either ARMS2 or
HTRA1 in vivo, suggesting that the influence of these variants on
risk of AMD may be through other biological mechanisms.
Commercial Relationships: Gaofeng Wang, None; Brenda Court,
None; Patrice Gay, None; Sander R. Dubovy, None; Jaclyn L.
Kovach, None; Stephen G. Schwartz, Alimera (C), Bausch + Lomb
(C), Eyetech (C), IC Labs (P), ThromboGenics (C); Anita Agarwal,
Vanderbilt University (P); William K. Scott, Duke
University/ArcticDx (P); Jonathan L. Haines, Arctic Dx (I), AMD
genes (P); Margaret A. Pericak-Vance, None
Support: NIH grant 2R01EY012118-11
Program Number: 6192 Poster Board Number: C0077
Presentation Time: 10:30 AM - 12:15 PM
Differential gene expression of RPE and choroid in old Cfh
transgenic mice
Cynthia X. Wang, Bogale Aredo, xiao chen, Rafael Ufret-Vincenty.
Ophthalmology, UTSW Medical Center, Dallas, TX.
Purpose: Our goal in this work is to determine how variants in
complement factor H (Cfh) and the levels of c-reactive protein (Crp)
affect the expression of RNA by RPE cells and/or their choroid as
mice age.
Methods: A transgenic mouse was generated which expresses
chimeric Cfh molecules consisting of human Cfh SCR6-8 flanked by
mouse Cfh SCR1-5 and SCR9-20 (under the ApoE promoter). These
mice were crossed to mCfhKO mice to eliminate mouse Cfh. We also
generated some lines by crossing the resulting CfhTg/mCfhKO mice
to hCrpTg mice, which express human Crp. Usinga method we
developed, we isolated RPE cells from mouse eyecups and isolated
RNA from RPE cells of 2 year old
CfhTg/mCfhKOvsCfhTgCrpTg/mCfhKO mice vs B6 mice. In
addition, we isolated RNA from the choroid/sclera. We ran Illumina
microarray chips on these samples, and performed a pathway analysis
on the resulting genes. We re-tested these genes using RT-PCR. We
also chose an independent group of candidate genes for RT-PCR
analysis.
Results: RPE cells were isolated from 2 year old CfhTg/mCfhKO
mice, CfhTg/hCrpTg/mCfhKO mice and age-matched B6 mice using
a new simple method. RNA was then extracted from the RPE cells
using the QiagenRNeasy micro kit. RNA was also extracted
(separately) from the residual choroid/scleral cup. Bioanalyzer testing
confirmed the high quality of the RNA. Analysis using Illumina
microarray chipsgenerated a list of genes that were differentially
expressed in CfhTg/mCfhKO mice or CfhTg/hCrpTg/mCfhKO when
compared to B6, either in the RPE cells or in the choroid. RT/PCR
analysis was used to confirm the results and look at additional
candidate genes.
Conclusions: Variants of Cfh, and the expression of Crp, both lead to
changes on the gene expression profile of RPE cells and choroid in
aging mice. Protein expression assays will be done in the future to
confirm these gene expression changes.
Commercial Relationships: Cynthia X. Wang, None; Bogale
Aredo, None; xiao chen, None; Rafael Ufret-Vincenty, None
Support: Research to Prevent Blindness(New Youk, NY, USA) and
NIH grant EY020799
Program Number: 6193 Poster Board Number: C0078
Presentation Time: 10:30 AM - 12:15 PM
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
GSTM1 and GSTM5 Polymorphisms and Expression in AMD
Allan A. Hunter, Zeljka Smit-McBride, Rachel Anderson, Esther Kim,
Susanna S. Park, Leonard M. Hjelmeland, Lawrence S. Morse.
Ophthalmology, University of California, Davis, Sacramento, CA.
Purpose: We have previously found reduced levels of mRNA and
protein of two antioxidant enzymes, GSTM1 and GSTM5, in AMD
retinas relative to age-matched controls. Herein, we correlate serum
RNA quantity to genomic copy number of both GSTM1 and GSTM5
in a study population to determine whether homozygous or
heterozygous deletions (-/-, +/-) could explain the reduction of
mRNA levels.
Methods: Cheek swab DNA and serum RNA samples were collected
from 50 AMD and 48 control patients. TaqMan gene copy number
assays were conducted in technical quadruplicate for relative
quantification of copy number variation in AMD vs. control patients.
TaqMan gene expression assays were conducted in technical
triplicate for relative quantification (RT-PCR).
Results: GSTM1 and GSTM5 copy numbers between AMD vs.
controls revealed no significant differences. GSTM5 copy number
was two in all but one sample, and GSTM1 copy number ranged from
0-4. Homozygous deletions were not correlated with AMD status
(mean copy number: AMD 1.21 vs. control 1.40, p = 0.31). A
subgroup analysis of heterozygous deletions with a single GSTM1
copy number did not correlate with AMD status, (number of samples:
AMD 6, control 8, p = 0.76). Relative quantification of serum
GSTM1 mRNA was not significantly different between AMD vs.
controls, (fold change AMD:Control, 0.91, p = 0.76). A subgroup
analysis comparing homozygous presence of genomic GSTM1 did
not show a significant difference in serum GSTM1 mRNA levels
between AMD vs. controls, (Fold Change AMD:Control, 0.93, p =
0.80). Likewise, comparing relative quantification of serum GSTM1
mRNA to genomic copy number(s) 1-4 did not show any significant
difference in AMD (r2 = 0.15) or controls (r2 = 0.12). Homozygous
genomic deletions lacked any detectable GSTM1 RNA quantity.
GSTM5 mRNA relative quantification did not change with AMD
status, (fold change 0.97, p = 0.89).
Conclusions: The reduced levels of both mRNA and protein of
GSTM1 and GSTM5 in AMD retinas does not appear to be due to
genomic deletion in this second study population. Genomic
heterozygous or homozygous present GSTM1 (+/-, +/+, copy number
3-4) does not alter serum levels of the mRNA to any significance.
The evidence presented suggests that the reduced amount of these
antioxidants in AMD retinas may be due to another form of
expression repression that is tissue specific. Diminished levels of
these enzymes in AMD retinas may increase susceptibility to
oxidative stress.
Commercial Relationships: Allan A. Hunter, None; Zeljka SmitMcBride, None; Rachel Anderson, None; Esther Kim, None;
Susanna S. Park, None; Leonard M. Hjelmeland, NeuroTech Inc.
(C); Lawrence S. Morse, Genentech, Inc. (C), Allergan (C), Iridex
(C)
Program Number: 6194 Poster Board Number: C0079
Presentation Time: 10:30 AM - 12:15 PM
Mitochondrial DNA Heteroplasmy in Retinas of AMD Patients
Hui Cai1, Peter L. Nagy2, Rando Allikmets1, 2. 1Ophthalmology,
Columbia University Medical Center, New York, NY; 2Pathology &
Cell Biology, Columbia University Medical Center, New York, NY.
Purpose: Accumulation of somatic genetic variation (heteroplasmy)
in mitochondrial genome has been implicated in age-related macular
degeneration (AMD). We used deep sequencing of mitochondrial
DNA samples from retinas (macular and peripheral regions) of donor
eyes from AMD patients and age-matched controls to assess the
extent of heteroplasmy acquired with age and disease.
Methods: In this pilot study we used genomic DNA isolated from
retina tissue of donor eyes of ten AMD patients and ten age-matched
controls. Samples were sequenced using mitochondrial DNA
(mtDNA) deep sequencing technology with a minimum of 2000X
coverage; i.e., the entire 16,569 bp of mitochondrial DNA in each
sample was covered by at least 2000 sequence reads. Sequence data
were annotated and genetic variation (heteroplasmy) was identified
and analyzed by NextGene (SoftGenetics) software. Identified
variants were compared to published mtDNA variants from the
control region and coding & RNA regions [MITOMAP.org: A
Human Mitochondrial Genome Database, 2012].
Results: The minimum depth of the reliably observed heteroplasmy
was set at 2%. In order to assess possible clinically meaningful
variants, the more stringent heteroplasmy threshold was set at 40%.
With these criteria seventy five variants were identified in 10 AMD
samples compared to sixty five in 10 control samples. Among these,
variants close to 99% heteroplasmy rates were seen at certain
positions in single samples in 37 out of 75 variants. More variants
were found in coding regions of mtDNA from AMD vs. control
samples (43 vs. 35). In AMD samples, thirteen variants in mtDNA
coding region were non-synonymous and were detected in NADH
subunits 1, 2, 4 and 5, CYTB, COX1, and ATP6 genes. These genes
are involved in the Krebs cycle and electron transport chain reaction
of aerobic respiration in mitochondria. Four mutations had not been
reported before and six mutations had been identified as associated
with human diseases, e.g., AMD, LHON, Alzheimer and Parkinson
Diseases.
Conclusions: Deep sequencing of mtDNA is an effective tool to
identify heteroplasmy. While our sample size in this pilot study was
small, we observed a real trend towards more heteroplasmy, both
qualitatively and quantitatively, in AMD samples as compared to
matched controls. Sequencing of a much larger cohort, which will
allow substantial statistical power for definitive conclusions, is under
way.
Commercial Relationships: Hui Cai, None; Peter L. Nagy, None;
Rando Allikmets, None
Support: NEI/NIH R01 EY13435, RPB
Program Number: 6195 Poster Board Number: C0080
Presentation Time: 10:30 AM - 12:15 PM
Comparison Between Haplogroup H and L Cybrids in ROS/RNS
Production and Gene Expression Further step in understanding
the pathogenesis of Age-related Macular Degeneration
Mohamed Tarek1, Javier Cáceres del Carpio1, Claudio A. Ramirez1,
Payam Falatoonzadeh1, Shari Atilano1, Deepika Malik1, S Michal
Jazwinski2, Miceli V. Michael2, Baruch D. Kuppermann1, Cristina M.
Kenney1. 1Gavin Herbert Eye Institute, Irvine, CA; 2Tulane Center
For Aging, Tulane University, New Orleans, LA.
Purpose: Mitochondrial DNA haplogroups can be classified
according to accumulation of specific single nucleotide
polymorphisms (SNPs). The H haplogroup is of European origin and
protective for AMD, the L haplogroup which is of African origin
shows only early AMD changes. Our work is to investigate the
differences in Reactive Oxygen/Nitrogen Species (ROS/RNS)
production and gene expression profiles of cybrids (cytoplasmic
hybrids) that have either the H haplogroup or the L haplogroup.
Methods: ARPE-19 cells lacking mitochondrial DNA (mtDNA)
(Rho0) were fused with platelets from individuals with either H or L
haplogroup mtDNA as determined by PCR analysis. After culture for
24 hours, ROS/RNS levels were determined with 2`, 7`dichlorodihydrofluorescein diacetate dye assay. Experiments were
repeated twice and performed in 8 wells per sample. RNA was
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
obtained from the H cybrids (n=3) and L cybrids (n=3) and the gene
expression patterns were determined using the Affymetrix Human
Genome U133 plus 2.0 array. The pathway profiles were analyzed
with the INGENUITY systems program. Quantitative PCR (Q-PCR)
was performed using primers for complement component 3 (C3) and
complement factor H (CFH) genes for the different H and L cybrids.
Results: Cybrids with the haplogroup L mtDNA produced
25.79±6.21 % lower levels of ROS/RNS compared to haplogroup H
cybrids (p=0.026). Complement pathway genes were expressed
differently, the haplogroup H cybrids showed higher expression
levels in C1S, C3, CFH, C4BPB, CFHR4, and ITGB2 genes by 2.9,
5.5, 2, 3.8, 17.8 and 3.4 folds respectively, while the C1QC, C4A,
C8B and CFP genes were expressed at higher levels in the
haplogroup L cybrids (3.2, 3.3, 6.2 and 3 folds respectively). By QPCR analyses, the L cybrids had a 0.66-fold expression level for the
CFH gene (p=0.03) and 0.17-fold level for the C3 gene (p=0.0004)
compared to the H cybrids.
Conclusions: Our findings demonstrate that cybrids which are
identical except for their mtDNA, have different levels of expression
for genes involved in the alternative complement pathway. Our
results are significant because they show that mtDNA variants
(haplogroups) can mediate not only energy production but also the
expression of genes for major cellular pathways involved in
inflammation.
Commercial Relationships: Mohamed Tarek, None; Javier
Cáceres del Carpio, None; Claudio A. Ramirez, None; Payam
Falatoonzadeh, None; Shari Atilano, None; Deepika Malik, None;
S Michal Jazwinski, None; Miceli V. Michael, None; Baruch D.
Kuppermann, Alimera (C), Allegro (C), Allergan (C), Genentech
(C), Glaukos (C), GSK (F), Novagali (C), Novartis (C), Ophthotech
(C), Pfizer (C), Regeneron (C), Santen (C), SecondSight (C), Teva
(C), ThromboGenics (C); Cristina M. Kenney, None
Support: Discovery Eye Foundation, Guenther Foundation,
Beckman Macualr Research Initiative, Polly and Michael Smith
Foundation, Max Factor Family Foundation, Skirball Foundation,
Lincy Foundation, Iris and B. Gerald Cantor Foundation, Gilbert
Foundation, Unrestricted grant from Research to Prevent Blindness,
National Institute For Aging
Program Number: 6196 Poster Board Number: C0081
Presentation Time: 10:30 AM - 12:15 PM
Genetic Testing for Age-Related Macular Degeneration in an
Armenian Population
Abraham Abraamyan1, Brent Zanke3, Preveen Ramamoorthy2, Kent
W. Small1. 1Macula & Retina Institute, Glendale, CA; 2Advanced
Diagnostics Laboratory, Denver, CO; 3Arctic Dx - Macula Risk,
Bonita Springs, FL.
Purpose: Age-related macular degeneration (ARMD) has a
significant genetic influence, especially in Caucasian groups. The
Armenian ethnogenesis dates back at least 3,000 years and is
considered to be a genetically isolated population. Additionally, the
Armenian genocide created a genetic bottleneck. Because of these
demographics, we hypothesized that the genetic involvement in
ARMD may be different than that of other Caucasian populations. To
our knowledge, this is the first reported evaluation of the genetic
contribution of AMRD susceptibility genes in an Armenian
population.
Methods: A retrospective review was performed of 38 Armenian
patients with wet (exudative) ARMD who had genetic analysis using
the commercially available Macula Risk genotyping method. We
obtained buccal cheek swabs which were sent to Advanced
Diagnostic Laboratories for routine clinical testing of previously
documented ARMD risk alleles: Complement Factor H (CFH) with 5
Single Nucleotide Polymorphisms, Complement Component 3 (C3),
Age-Related Maculopathy Susceptibility 2 (ARMS2) and
mitochondrially encoded NADH dehydrogenase 2 (MT-ND2). Each
genetic component adds to a specific portion of a Macula Risk score,
which predicts risk of developing advanced macular degeneration.
This was compared to the Macula Risk genetic database of a 786
person Caucasian population with wet ARMD. The data was
analyzed using Mann-Whitney U test to show differences in age and
Macula Risk score. Chi squared test with two degrees of freedom was
used to show differences in the genotypes (except MT-ND2, which
only required a 2x2 table).
Results: The Mann-Whitney U tests show no statistically significant
difference between the Armenian and Caucasian data sets for Macula
Risk score. The average age for Armenians (83.6) was higher than
Caucasians (79.8), p=0.04. The Chi squared tests show no
statistically significant difference between the genotypes.
Conclusions: In Armenian and Caucasian patients with wet ARMD,
there is no difference between genotypes and Macula Risk score.
Although we found no statistical significance, our data set is
relatively small and may not be sufficiently powered. Additional
analysis is necessary to evaluate the reasons contributing to the age
difference within these populations. In patients who present with wet
ARMD, we can find no genetic difference in the ARMD risk alleles
in the Armenian population compared to the Caucasian population.
Commercial Relationships: Abraham Abraamyan, Macula &
Retina Institute (F); Brent Zanke, ArcticDx (E), ArcticDx (I);
Preveen Ramamoorthy, ArcticDx (F); Kent W. Small, Valeant (C)
Program Number: 6197 Poster Board Number: C0082
Presentation Time: 10:30 AM - 12:15 PM
Global Review and Meta-analysis of Diabetic Retinopathy
Genetic Studies Highlight Gaps in the Pathogenesis between
Various Populations
Shi Song Rong, Pancy O.S. Tam, Chi Pui Pang, Li Jia Chen. Dept.
Ophthalmology & Visual Sciences, The Chinese University of Hong
Kong, Hong Kong, China.
Purpose: To identify the gene variants that are associated with
diabetic retinopathy (DR) by meta-analysis.
Methods: PubMed, EMBASE and EBM Cochrane Library were used
for comprehensive electronic search, which was supplemented by
hand-searching. All case-control (or cohort) studies were included.
Two reviewers independently screened all the reports and extracted
relevant data. Meta-analyses were conducted with ‘metafor’ package
in R (v2.15.0) and Stata (v11 StataCorp, USA). Tests for
heterogeneity, publication bias, small-study effects, and subgroup
analysis were used to clarify the potential bias.
Results: Totally 4724 reports on DR genetics were yielded, 347 were
genetic association studies. More than 200 genes and 430 variants
have been associated with DR with inconclusive results. Among
them, 56 reports were on 90 variants of 9 genes attributed to
inflammatory responses and oxidative stress, which have received
much attention in the past 5 years. The pooled data showed a single
nucleotide polymorphism (SNP; codominant, OR=0.43; 95% CI:
0.23-0.82) of the AGER gene was protective against DR. High-risk
alleles included variants in NOS2 (OR=0.16, 95% CI: 0.03-0.71),
SERPINF1 (OR=1.23, 95% CI: 1.01-1.49), and TNFB (OR=1.62,
95% CI: 1.05-2.49). After eliminating heterogeneity in subgroup
analysis, the AGER SNP showed opposite effects in
Chinese+Japanese (OR=0.25, 95% CI: 0.13-0.5) and Indian (OR=1.8,
95% CI: 1.31-2.48). A SNP of the ICAM1 gene was related to DR
only in the Chinese and Japanese populations (OR=2.59, 95% CI:
1.51-4.46).
Conclusions: Results of our meta-analyses support the involvements
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
of variants in AGER, SERPINF1, ICAM1 and TNFB in DR.
Substantial population differences in DR genetics are also indicated.
Commercial Relationships: Shi Song Rong, None; Pancy O.S.
Tam, None; Chi Pui Pang, None; Li Jia Chen, None
Program Number: 6198 Poster Board Number: C0083
Presentation Time: 10:30 AM - 12:15 PM
Relating risk genotypes for diabetic retinopathy with color vision
performance
Mirella Gualtieri1, Daniela M. Bonci2, Valéria D. Duarte Garcia2,
Stephen Juel3, Francisco Max Damico5, Maureen Neitz4, Dora F.
Ventura1, 2. 1Experimental Psychology, University of Sao Paulo, Sao
Paulo, Brazil; 2Neuroscience & Behavio, University of Sao Paulo,
Sao Paulo, Brazil; 3Minority Health and Health Disparities
International Research Training Program, Menphis, TN;
4
Ophthalmology, University of Washington, Seattle, WA;
5
Ophthalmology, University of Sao Paulo, Sao Paulo, Brazil.
Purpose: Three genetic markers of erythropoietin (EPO) expression
have been recently identified as either risk- or protective-factors for
the development of proliferative diabetic retinopathy (PDR). These
markers are located at the regions of single nucleotide
polymorphisms (SNP) rs1617640, rs507392, and rs551238.
Homozygous or heterozygous genotypes at these three locations are
significantly associated with a higher or lower risk of PDR in
diabetics (Tong et al 2008; Abhary et al 2010). We sought to analyze
how the presence of the different genotypes at these 3 SNP locations
relates to functional losses observed prior to retinopathy in a group of
type 1 and type 2 diabetics.
Methods: Visual function was assessed by color discrimination
thresholds measured along protan, deutan and a tritan axes, as well a
MacAdam ellipse (Cambridge Colour Test). The EPO markers were
identified after DNA extraction from blood cells by sequencing of
PCR products. The functional and the genetic assessments were
performed in a group of 29 (13 type 1; 16 type 2) diabetic patients
and a group of 17 healthy controls.
Results: Colour vision thresholds from the patients were higher than
controls’ for all three color confusion axes and the ellipse area. The
presence of the risk genotype coincided with worse color vision only
for the SNP rs1617640. Control subjects with the different rs1617640
genotypes did not have different color vision (figures 1 and 2). The
presence of different genotypes for the SNP rs507392, and rs551238
was not associated with different color vision results.
Conclusions: Diabetic patients who were carriers of the risk
genotype at the SNP location rs1617640 - a maker for the
development of PDR - were also the patients with worse color vision
thresholds The same association was not observed for the other EPO
gene SNP locations evaluated. The association between factors
related to vascular abnormality (EPO marker) and neural loss (color
vision) contribute to the intricate puzzle of the interaction between
vascular and neural abnormalities which is at the core of hypotheses
about the etiology of diabetic visual losses.
Color vision thresholds from the diabetics. Carriers of the allele T
have higher thresholds.
Color vision thresholds from controls. Thresholds did not change
across genotypes.
Commercial Relationships: Mirella Gualtieri, None; Daniela M.
Bonci, None; Valéria D. Duarte Garcia, FAPESP (F); Stephen
Juel, None; Francisco Max Damico, None; Maureen Neitz,
Genzyme (F), Alcon (F), Alcon (P); Dora F. Ventura, None
Support: FAPESP; CNPq, CAPES, MHIRT
Program Number: 6199 Poster Board Number: C0084
Presentation Time: 10:30 AM - 12:15 PM
MicroRNA-126 Regulates Heme Oxygenase-1-Mediated
Alterations in Diabetic Retinopathy
Jiawen Fan, Gezhi Xu, Tingting Jiang, Yaowu Qin, Xin Wang.
Ophthalmology Department, Eye and ENT Hospital of Fudan Univ,
Shanghai, China.
Purpose: Diabetic retinopathy (DR) is a progressive
neurodegenerative disease and a leading cause of blindness.
Overexpression of Heme Oxygenase-1(HO-1) by hemin induction
protected retinal ganglion cells in diabetic retinopathy through antiinflammatory, anti-apoptotic, and anti-proliferative effects. We
investigated microRNA (miRNA) alterations in DR after hemin
treatment with specific focus on miR-126, and its downstream target,
HO-1.
Methods: miRNA expression profiling microarray (Affymetrix
MicroRNA 2.0 Array) was used to examine the retinas of treated and
non-treated streptozotocin- induced diabetic rats. Expressions of
specific miRNAs were verified with PCR in the rat retina and in
glucose-exposed endothelial cells. A target search, based on sequence
complementarities, identified specific targets. We analyzed mRNA
levels and protein expression in endothelial cells from large vessels
and retinal capillaries and in the rat retina, with or without injection
of miR-126 mimic or antagomir. Localization of miR-126 and its
functional analysis in the rat retinas and vascular endothelial cells
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
were performed.
Results: Significant alteration of several miRNAs, including
downregulation of miR-126, were observed in the retina in nontreated diabetes. Such downregulation was validated in vascular
endothelial cells incubated in glucose. In parallel, HO-1 (target of
miR-126) mRNA and protein were degraded. The level of both miR126 and HO-1were elevated after hemin treatment in diacetic rats’
retina and vascular endothelial cells with high glucose. In the retina,
miR-126 was localized in glial and vascular elements. Transfection of
endothelial cells and intravitreal injection of miR-126 mimic
prevented diabetes-induced decreased HO-1 and increased VEGF
mRNA and protein. Also prevented were glucose-induced increased
permeability and angiogenesis. Furthermore, transfection of miR-126
antagonists (antagomir) led to decreased HO-1 and increased VEGF
production.
Conclusions: These studies show a novel mechanism involving miR126 in DR. Identification of such mechanisms may lead to the
development of novel miRNA-based therapy.
Commercial Relationships: Jiawen Fan, None; Gezhi Xu, None;
Tingting Jiang, None; Yaowu Qin, None; Xin Wang, None
Support: Supported by National Basic Research Grants of China
(81170857, 2011) and by National Science and Technology Major
Project (2011ZX09302-007-02).
Program Number: 6200 Poster Board Number: C0085
Presentation Time: 10:30 AM - 12:15 PM
Identification of Risk Alleles in the Erythropoietin gene for
Proliferative Diabetic Retinopathy in Type I and Type II
Diabetics
Stephen Juel3, Daniela M. Bonci1, 2, Mirella Gualtieri1, 2, Valéria D.
Duarte Garcia1, 2, Maureen Neitz4, Dora F. Ventura1, 2. 1Psicologia
Experimental, Universidade de São Paulo, São Paulo, Brazil; 2Núcleo
de Neurociências e Comportamento, Universidade de São Paulo, São
Paulo, Brazil; 3Minority Health International Research Training
Program, Christian Brothers University, Memphis, TN;
4
Ophthalmology, University of Washington, Seattle, WA.
Purpose: Genetic markers for the expression of the glycoprotein
erythropoietin (EPO) - a potent promoter of angiogenesis with
implications in diabetic microvascular proliferation - have been
identified. These markers, or SNPs, can manifest as either risk or
protective factors in relation to the development of proliferative
diabetic retinopathy (PDR). In this study we are seeking to
investigate the prevalence of EPO in pre-retinopathic diabetics of a
Brazilian population cohort.
Methods: We evaluated a total of 70 participants, neither of which
had developed PDR. The population consisted of 27 type 2 diabetics,
15 type 1 diabetics and an age matched control group of 28 subjects.
Participants were genotyped for 3 specific SNPs - locations
rs1617640, rs507392, and rs551238 - which have been found to be
associated with EPO expression, with the latter two SNPs as part of a
disease haplotype (TTA or GCC). The blood samples were collected
and the PCR for each SNP was performed after DNA extraction. The
PCR products were directly sequenced.
Results: Of the 70 individuals, controls and diabetics, that were
genotyped, 39 were homozygous for the T allele (57%) at the
location rs1617640, while only a small portion (N=7) were
homozygous for the aforementioned protective G allele (10%). The
remaining individuals (32%) were heterozygous (TG) (N=23).
Within the diabetic population subset (N=42), 24 patients were
homozygous for the T allele (60%). From 33 participants evaluated
for the disease haplotypes, 20 patients and 9 control subjects were
TTA and the GCC haplotype was present in only 1 patient and 3
controls subjects.
Conclusions: These results suggest a majority of individuals are
carriers of the EPO16 risk allele and the haplotype TTA described in
the literature. The correlation of these data with the patient visual
function can provide additional valuable tools for early detection and
clinical management of patients more susceptible to diabetic visual
damage.
Commercial Relationships: Stephen Juel, None; Daniela M.
Bonci, None; Mirella Gualtieri, None; Valéria D. Duarte Garcia,
FAPESP (F); Maureen Neitz, Genzyme (F), Alcon (F), Alcon (P);
Dora F. Ventura, None
Support: FAPESP, CAPES, CNPq, NIH 5T37MD001378 - 12
Program Number: 6201 Poster Board Number: C0086
Presentation Time: 10:30 AM - 12:15 PM
Expression Of VEGF-A Gene In Normal Retina And In PVR
Retina
Claudio Azzolini1, Ilaria S. Pagani2, Davide Borroni1, 2, Cristina
Pirrone2, Diana Pigni2, Muna Al Oum1, Riccardo Vinciguerra1,
Simone Donati1, Giovanni Porta2. 1Morphological and Surgical
Sciences, University of Insubria - Circolo Hospital, Varese, Italy;
2
Department of Clinical and Experimental Medicine, University of
Insubria, Varese, Italy.
Purpose: Vascular Endothelial Growth Factor-A, VEGF-A, is a key
angiogenesis protein in embryonic development and in adult tissues.
It is also involved in the pathogenesis of different human diseases
including eye disorders, like proliferative vitreoretinopathy (PVR).
Both in normal tissues expression and in PVR VEGF-A is regulated
by p53 family members. This protein induces cellular differentiation
regulating division in staminal cells, leading to asymmetric division,
while its loss of function leads to the symmetric division, resulting in
cellular growth.
Methods: We evaluated expression levels of p53, p63, p73 and
VEGF-A genes through quantitative Real-time reverse-transcriptase
PCR analysis in twelve retinal PVR samples, taken during necessary
retinectomies, and healthy human retina controls included in paraffin.
Results: We showed VEGF-A over-expression in samples affected
by a marked proliferative vitreoretinopathy compared with healthy
human retina. VEGF-A is less expressed in PVR tissues detached
from a long time from retina. Controls showed a basal levels of
VEGF-A expression. p53 and p73 seems to co-expresses with VEGFA while p63 is down-regulated in tissues with high levels of VEGFA.
Conclusions: The increase of VEGF-A levels in PVR tissues
promotes differentiation and cell survival and decreases apoptosis
suggesting its role in proliferation of retinal cells. These analysis
could be critical for the studies aimed to novel therapies for retinal
diseases.
Commercial Relationships: Claudio Azzolini, None; Ilaria S.
Pagani, None; Davide Borroni, None; Cristina Pirrone, None;
Diana Pigni, None; Muna Al Oum, None; Riccardo Vinciguerra,
None; Simone Donati, None; Giovanni Porta, None
Program Number: 6202 Poster Board Number: C0087
Presentation Time: 10:30 AM - 12:15 PM
Thrombophilic mutations and risk of retinal vein occlusion
Silvia R. Mendes, António F. Campos, Diana Beselga, Arminda
Neves, Joana Campos, Dulce Castanheira. Ophthalmology, Leiria,
Leiria, Portugal.
Purpose: The association between hereditary thrombophilia
abnormalities and retinal vein occlusion (RVO) is not well
established. Literature consists of small studies with controversial
results. The objective was to describe thrombophilic risk factors in
patients with diagnosed RVO
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
Methods: Consecutive patients with RVO were screened for
thrombophilic risk factors in our department. There was no group
control. Homocysteinemia, C677T methylenetetrahydrofolate
reductase (MTHFR) polymorphism,factor V 1691A (factor V
Leiden), PT 20210A and plasminogen activator inhibitor-1 mutation
(PAI1) were analysed. We excluded patients with renal disease,
cancer, taking vitamin supplements or folate B12/B6, and drugs
which influence the serum concentration of homocysteine as
estrogens, carbamazepine, antifolates, tricyclic antidepressants and
phenytoin.
Results: The study group included 36 eyes of 36 patients, 26 male,
10 female, diagnosed with RVO. Central RVO in 14 eyes and branch
RVO in 22 eyes. Mediam age was 65 (range 38-81), 13 patients were
less than 60 years old.
The MTHFR C677T polymorphism was found in 24 out of 36
(66,67%) patients with RVO (heterozygosity: 19 out of 36 and
homozygosity: 5 out of 36). Plasminogen activator inhibitor-1
mutation was found in 16 out of 36 patients (44,4%). One patient had
factor V Leiden and another presented PT 20210A mutation.
Homocysteinemia level was high in 21,4% of patients, in average
more 5μm/L on C667T MTHFR homozygosity than in other
polymorphisms. The prevalence of mutations in the younger group
was not statistically significant compared to older subjects.
Conclusions: The hereditary thrombophilia abnormalities tested in
our study are known as risk factors for venous thrombosis as well as
for arterial vascular disease. They were very common in patients
diagnosed with RVO, but a control group should be matched for
more consistent conclusions.
Commercial Relationships: Silvia R. Mendes, None; António F.
Campos, None; Diana Beselga, None; Arminda Neves, None;
Joana Campos, None; Dulce Castanheira, None
Program Number: 6203 Poster Board Number: C0088
Presentation Time: 10:30 AM - 12:15 PM
Association study of IL23R-IL12RB2 and IL10 gene
polymorphisms with susceptibility to Vogt-Koyanagi-Harada
disease in a Japanese population
Akira Meguro1, Tatsukata Kawagoe1, Masamitsu Nakahara1, 2,
Hideharu Fukasaku1, 2, Shigeaki Ohno3, Nobuhisa Mizuki1.
1
Department of Ophthalmology, Yokohama City Univ School of
Med, Yokohama, Japan; 2Fukasaku Eye Clinic, Yokohama, Japan;
3
Ocular Inflammation and Immunology, Hokkaido University
Graduate School of Medicine, Sapporo, Japan.
Purpose: Single nucleotide polymorphisms (SNPs) in the IL23RIL12RB2 and IL10 gene regions have been found to be associated
with several immune-mediated diseases. In this study, we
investigated whether the IL23R-IL12RB2 and IL10 gene
polymorphisms are associated with Vogt-Koyanagi-Harada (VKH)
disease in a Japanese population.
Methods: A total of 254 Japanese patients with VKH disease and
744 Japanese healthy controls were recruited. We genotyped 10 SNPs
in IL23R-IL12RB2 and 6 SNPs in IL10 using TaqMan genotyping
assays, and assessed the allelic and genotypic diversity among cases
and controls.
Results: All SNPs were in Hardy-Weinberg equilibrium among both
cases and controls. No significant differences in the frequency of
alleles and genotypes of the IL23R-IL12RB2 and IL10 SNPs in the
cases compared to the controls were detected. Stratification analysis
according to clinical features in the eyes, skin, ears, and central
nervous system did not show any association of the IL23R-IL12RB2
and IL10 SNPs with any clinical findings.
Conclusions: Our study showed that the IL23R-IL12RB2 and IL10
polymorphisms had no association with the risk of VKH disease,
suggesting that genetic variants in the IL23R-IL12RB2 and IL10
regions may not play an important role in the pathogenesis of VKH
disease.
Commercial Relationships: Akira Meguro, None; Tatsukata
Kawagoe, None; Masamitsu Nakahara, None; Hideharu
Fukasaku, None; Shigeaki Ohno, None; Nobuhisa Mizuki, None
Program Number: 6204 Poster Board Number: C0089
Presentation Time: 10:30 AM - 12:15 PM
Genetic Screening of TSPAN12, NDP and FZD4 Genes in Indian
Patients with Retinopathy of Prematurity
Inderjeet Kaur1, sonika rathi1, Ganeswara R. Musada1, Subhadra
Jalali2, Ramesh Kekunnaya3, Pramod Gaddam4, Subhabrata
Chakrabarti1. 1Kallam Anji Reddy Molecular Genetics Lab, LV
Prasad Eye Institute, Hyderabad, India; 2Smt.Kannuri Santhamma
Centre for Vitreo Retinal Diseases, LV Prasad Eye Institute,
Hyderabad, India; 3Jasti V Ramanamma Children’s Eye Care Centre,
LV Prasad Eye Institute, Hyderabad, India; 4Fernandez Hospital,
Hyderabad, India.
Purpose: Retinopathy of prematurity (ROP) is a vaso-proliferative
eye disease in prematurely born infants. The clinical manifestation of
this condition exhibits similarites with Familial Exudative Vitreoretinopathy (FEVR). Genes involved in the Wnt signalling pathway
(TSPAN12, NDP, FZD4) have been implicated in retinal
neovascularisation among FEVR patients. Thus, a similar molecular
mechanism could be envisaged underlying ROP based on its
phenotypic similarities with FEVR. In the present study, we aimed to
assess the role of TSPAN12, NDP, FZD4 genes in ROP.
Methods: The TSPAN12, NDP and FZD4 genes were screened in
ROP patients (n=200) and controls consisting of mature (n=127) and
premature babies devoid of ROP (n=147). Screening was
accomplished by resequencing using specific sets of primers to
amplify the entire coding and untranslated regions (UTR) in these
genes using the BigDye chemistry. The observed variants were
further validated by restriction digestion and characterized for
mutations or polymorphisms.
Results: The TSPAN12 gene screening led to the identification of a
heterozygous mutation in a patient with threshold ROP (L119V), a
novel SNP (c.*334A>T) and three reported SNPs (rs41623, rs41622,
rs189221112) in the study cohort. There were no significant
differences in the allele or genotype frequencies of these SNPs
between the ROP patients and controls. Two novel variations viz.,
IVS1+16A>G in the intron 1 and c.1503T>C in 3’UTR region and a
previosuly reported change in the 5’UTR region (c.171_184del14bp)
were observed in the NDP gene in 3 ROP patients. Extended
screening of the FZD4 also revealed two heterozygous variations
I192I and I360V and three reported SNPs (rs61735303, rs201168680,
rs61749246) in the study cohort. However, a formal genotypephenotype corelation could not be established due to the low
frequencies of the variant alleles in these genes.
Conclusions: These results suggest a minor involvement of
TSPAN12, NDP and FZD4 genes with ROP. Unlike FEVR, ROP may
be genetically more heterogeneous with multiple alleles with varying
magnitudes of effect.
Commercial Relationships: Inderjeet Kaur, None; sonika rathi,
None; Ganeswara R. Musada, None; Subhadra Jalali, None;
Ramesh Kekunnaya, None; Pramod Gaddam, None; Subhabrata
Chakrabarti, None
Support: Program Support grant on ROP, Department of
Biotechnology, India
Program Number: 6205 Poster Board Number: C0090
Presentation Time: 10:30 AM - 12:15 PM
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
Transcriptional regulation of synaptotagmin 11 in retinal
ganglion cells
Gillian C. Shaw1, 2, Cynthia A. Berlinicke1, Donald J. Zack1.
1
Ophthalmology, Johns Hopkins University, Baltimore, MD;
2
Molecular and Comparative Pathobiology, Johns Hopkins
University, Baltimore, MD.
Purpose: Gene regulation is essential for development and
maintenance of different cell types. Compared to other retinal cell
types, relatively little is known about transcriptional regulation in
retinal ganglion cells (RGCs). As a model for studying RGC
transcriptional regulation, we have been analyzing the cis-elements
and trans-factors that regulate the expression of the RGC-enriched
gene synaptotagmin 11 (Syt11).
Methods: 5’-upstream fragments from seven RGC enriched genes
(Syt11, Slc17a6, Sema6b, Sncg, Prph1 Nefm and Nrn1) were inserted
into reporter plasmids and transfected into primary rat RGCs and
dissociated whole retina. Deletion analysis of the Syt11 promoter
reporter followed by the bioinformatic analysis of the active regions
identified putative transcription factor (TF) binding sites (BSs). This
set of TFs was cross-referenced to microarray data of isolated rodent
RGCs. The predicted BSs of two RGC-expressed TFs, Deaf1 and
Sp1, were mutated in the promoter reporter construct and their
activity was compared to the unmutated construct. The effect of
Deaf1 and Sp1 knockdown on endogenous Syt11 expression was also
analyzed. Optimization of chromatin immunoprecipitation for Deaf1
and Sp1 is underway.
Results: Of the seven RGC enriched genes tested, the Syt11
construct showed the highest activity in RGCs compared to whole
retina. Promoter analysis showed that the region between -205 and 54bp contains important sequences for promoter activity in primary
RGCs. This region contains two overlapping predicted BSs for each
of the RGC-expressed TFs, Deaf1 and Sp1. Mutation of the Sp1 and
Deaf1 BSs caused the greatest decrease in promoter reporter activity.
Decreased expression of Deaf1, and to a lesser extent Sp1, was
achieved with siRNA knockdown, the effect of which on Syt11
expression is being analyzed. Confirmation of Deaf1 and Sp1 binding
to the promoter region of Syt11 in vivo is also being analyzed.
Conclusions: As a step in understanding transcriptional regulation in
RGCs, a system to analyze promoter reporters in cultured primary rat
RGCs was established. Using this system, we determined regions of
Syt11 that are important for its RGC expression. Bioinformatic
analysis of these regions identified BSs for Deaf1 and Sp1 and
mutating these sites revealed that they are important for promoter
activity. Analysis of the effect of Deaf1 and Sp1 knockdown and
confirmation of their binding to Syt11’s promoter is underway.
Commercial Relationships: Gillian C. Shaw, None; Cynthia A.
Berlinicke, None; Donald J. Zack, Alcon (C), Merck (F), Allergan
(C)
Support: NIH Grant EY015025
Program Number: 6206 Poster Board Number: C0091
Presentation Time: 10:30 AM - 12:15 PM
Systems Genetics of Intraocular Pressure—Mice to Humans: The
Use of BXD Murine Reference Panel for Identifying Novel
Genetic Modulators of Glaucoma and Bidirectional Translation
Monica M. Jablonski1, 2, Shankar Swaminathan1, Hong Lu1, Janey L.
Wiggs3, Robert W. Williams2, Lu Lu2. 1Hamilton Eye Institute, Univ
Tennessee Health Sci Ctr, Memphis, TN; 2Anatomy & Neurobiology,
The University of Tennessee Health Science Center, Memphis, TN;
3
Ophthalmology, Harvard Medical School MEEI, Boston, MA.
Purpose: To identify genetic modifiers of intraocular pressure (IOP)
using the enlarged BXD family of strains in combination with human
GWAS glaucoma cohorts. The BXD family consists of inbred
progeny strains of a cross between wildtype C57BL/6J and DBA/2J
that harbors mutations in both Tyrp1 and Gpnmb and develops
pigmentary dispersion glaucoma. We can exploit the known
segregation of these mutations among progeny and eliminate from
our analyses those that are mutant at both loci to remove any
influence of pigmentary dispersion glaucoma on IOP.
Methods: We acquired IOP estimates for parents and 68 progeny
free of mutations in Tyrp1 and Gpnmb at 1-2, 3-5, 6-9, 10-13 and >13
months-of-age, using an induction impact tonometer. Conventional
arrays and RNA-seq data were used to estimate gene expression from
eyes of parents and progeny. IOP datasets were mapped using
GeneNetwork (www.genenetwork.org). Candidate genes for high
IOP/POAG are now being evaluated using combinations of the
following criteria: (1) genes are located within confidence intervals
of murine QTLs; (2) genes have coding differences segregating
among progeny; (3) genes are expressed in the eye, and are
associated with cis-expression QTL (eQTL); (4) expression of
transcripts covaries with IOP; (5) genes have a plausible biological
link to IOP and glaucoma; and most importantly, (6) genes are close
to linkage peaks in the GLAUGEN and NEIGHBOR human GWAS
studies of POAG and IOP.
Results: We identify a robust eQTL in BXD mice strains aged 10-13
mo. Within this QTL, 21 candidates were nominated. The best SNP
in each candidate for each subset (HTG, NTG and POAG overall)
were identified using human data from the GLAUGEN/NEIGHBOR
meta-analyses. The top five candidates were selected based on the
Bonferroni-corrected p-value (0.0023). Top candidates were found to
play critical roles in ionic transport regulation, nitric oxide synthesis
and modulation, axon growth cone guidance regulation, and ciliary
body function—all of which could be important for elevated IOP and
possibly POAG.
Conclusions: In this study using the expanded BXD family and
human GWAS, we have identified five candidate genes that may
modulate IOP. These new findings could pave the way for novel
efficacious therapies for POAG using drug or gene delivery.
Commercial Relationships: Monica M. Jablonski, 8,092,825 (P);
Shankar Swaminathan, None; Hong Lu, None; Janey L. Wiggs,
None; Robert W. Williams, None; Lu Lu, None
Support: R01EY021200-02; R01EY015872-05S1; R01EY01912602S1; AA014425; AA017590; DA021131; UT-ORNL Governor's
Chair in Computational Genomics; UTHSC Neuroscience Institute;
Unrestricted Grant from Research to Prevent Blindness, New York,
NY
Program Number: 6207 Poster Board Number: C0092
Presentation Time: 10:30 AM - 12:15 PM
Genetic variation of superoxide dismutases in patients with
primary open-angle glaucoma
Dragana Celojevic1, Staffan Nilsson2, Anne Petersen1, Gunnar Tasa3,
Erkki Juronen3, Madeleine Zetterberg1. 1Institute of Neuroscience &
Physiology, University of Gothenburg, Gothenburg, Sweden;
2
Institute of Mathematical Sciences, Chalmers University of
Technology, Gothenburg, Sweden; 3Institute of General and
Molecular Pathology, University of Tartu, Tartu, Estonia.
Purpose: Oxidative stress has been described as an underlying
pathogenetic mechanism in retinal ganglion cell apoptosis, which is a
hallmark of primary open-angle glaucoma (POAG). Superoxide
dismutases (SODs) are enzymes involved in the protection against
oxidative stress by detoxification of superoxide. In this study, we
investigated a number of disease-associated single nucleotide
polymorphisms (SNPs) in the copper-zinc-containing SOD1 and
SOD3, and in the manganese superoxide dismutase SOD2, in POAG
patients.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
Methods: The study included 239 patients with POAG and 185
controls, all of Estonian origin, recruited at two ophthalmic clinics in
Tartu, Estonia. Eleven SNPs, either functional, disease-associated or
tag SNPs in SOD1, SOD2 and SOD3 were genotyped using TaqMan
Allelic Discrimination. Haplotype analysis was performed on the
SNPs in SOD2.
Results: Using binary logistic regression in an additive model, the
rs2842980 SNP in SOD2 was significantly associated with POAG
diagnosis (p=0.03) at a univariate level. None of the studied SNPs
showed an association with risk of POAG in a multivariate analysis,
including age and current smoking as covariates. Analysis of SOD2
haplotypes did not show any association with risk of POAG.
Conclusions: If oxidative stress is an important mechanism in
POAG-related retinal ganglion cell death, genetic variations in SOD1,
SOD2 and SOD3 are not major contributors in the pathogenesis.
Commercial Relationships: Dragana Celojevic, None; Staffan
Nilsson, None; Anne Petersen, None; Gunnar Tasa, None; Erkki
Juronen, None; Madeleine Zetterberg, None
Program Number: 6208 Poster Board Number: C0093
Presentation Time: 10:30 AM - 12:15 PM
COCH Variants are not Associated with Primary-open Angle
Glaucoma or Intraocular Pressure in Caucasians
Danyi Wang1, Baojian Fan1, Yutao Liu2, Michael A. Hauser2, R Rand
Allingham3, Jonathan L. Haines4, Janey L. Wiggs1. 1Ophthalmology,
Harvard Med Sch, Massachusetts Eye & Ear Infirmary, Boston, MA;
2
Ophthalmology & Medicine, Duke University Medical Center,
Durham, NC; 3Ophthalmology, Duke University Medical Center,
Durham, NC; 4Center for Human Genetics Research, Vanderbilt
University School of Medicine, Nashville, TN.
Purpose: COCH produces Cochlin, an extracellular matrix protein
that has been identified in glaucomatous TM tissue in both humans
and mice. Recently, a transgene carrying a copy of the human COCH
gene was shown to cause an increase in IOP in monkey organ
cultured anterior segments, suggesting that increased expression of
COCH in TM could lead to an increase in IOP. The human COCH
gene is located on chromosome 14q12, a genomic region that has
been implicated in POAG by genetic linkage studies. The purpose of
this study is to assess the association between single nucleotide
polymorphisms (SNPs) in the COCH gene region on chromosome
14q12 in relation to primary open angle glaucoma (POAG).
Methods: 539 POAG cases and 336 controls from the Massachusetts
Eye and Ear Infirmary (MEEI) were initially genotyped for 16 tag
SNPs capturing 100% of COCH alleles with mean r-square of 0.95.
Three SNPs of interest in the proximal gene region were genotyped
in a second independent group of 518 POAG cases and 445 controls
from Duke University Medical Center. Association analysis was
performed for POAG overall as well as subgroup analysis after
stratifying by IOP.
Results: A significant association was not found between any of the
COCH gene SNPs and POAG overall. In subgroup analysis after
stratifying by highest IOP, suggestive association between rs8015095
and IOP was found in both the MEEI [p= 0.038, OR 1.55 (1.032.33)] and Duke cohorts[(p=0.014, OR 2.01 (1.19-3.41)], and in a
meta-analysis (p=0.0011, OR 1.71), however a linear regression
analysis did not find an association between the risk allele and IOP
level.
Conclusions: Based on function and expression, COCH is an
excellent candidate gene for glaucoma. Our study however, did not
reveal statistically significant associations between POAG overall or
IOP with SNPs in the gene region in this population. It is possible
that rare variants in COCH gene may contribute to POAG and IOP
level.
Commercial Relationships: Danyi Wang, None; Baojian Fan,
None; Yutao Liu, None; Michael A. Hauser, None; R Rand
Allingham, New World Medical (C); Jonathan L. Haines, Arctic
Dx (I), AMD genes (P); Janey L. Wiggs, None
Support: NIH Grants R01EY015872 and P30EY014104, Research
to Prevent Blindness and The Massachusetts Lions Eye Research
Fund
Program Number: 6209 Poster Board Number: C0094
Presentation Time: 10:30 AM - 12:15 PM
Family-based Genome-wide Association Study in South Indian
Consanguineous Pedigrees confirms association between ZNF469
and central corneal thickness
Baojian Fan1, N. Soumittra2, Sarangapani Sripriya2, David S.
Friedman3, Vijaya Lingam4, Jonathan L. Haines5, Ronnie J. George4,
Janey L. Wiggs1. 1Dept of Ophthalmology, Harvard Med Sch
Massachusetts Eye & Ear Infirmary, Boston, MA; 2Vision Research
Foundation, Sankara Nethralaya, Chennai, India; 3Ophthalmology,
Johns Hopkins Medical School Wilmer Eye Institute, Baltimore, MD;
4
Medical Research Foundation, Sankara Nethralaya, Chennai, India;
5
Center for Human Genetic Research, Vanderbilt University School
of Medicine, Nashville, TN.
Purpose: Central corneal thickness (CCT) is a highly heritable
quantitative trait that can be a feature of primary open angle
glaucoma. Case-control and population-based genome-wide
association studies (GWAS) have identified several loci associated
with CCT. While these are effective approaches, large sample sizes
are required and population stratification can be a confounding factor.
Allelic homozygosity in consanguineous pedigrees may enhance
additive and/or subtractive effects of quantitative traits, creating
greater variation for quantitative trait loci mapping making it possible
to use a smaller sample for analysis. Additionally, family-based
GWAS are immune to confounding population stratification. The
purpose of this study is to use consanguineous pedigrees from South
India for family-based association studies for CCT.
Methods: 240 members of 16 Indian consanguineous pedigrees
underwent a comprehensive ophthalmic examination for ocular
quantitative traits such as intraocular pressure, central corneal
thickness and curvature, optic disc size, optic cup size, visual acuity,
and axial length. Genotyping was performed using the Illumina
HumanOmni2.5-8 platform. After data cleaning, 1,402,532 SNPs
were analyzed for association with CCT. A simple linear regression
of phenotype on genotype was performed with permutation testing to
account for family structure using QFAM in PLINK v1.07.
Results: SNPs located in the 16q24 genomic region near ZNF469
previously associated with CCT were nominally associated with CCT
in this population (top SNP rs4072556, p=5.4×10 -5). In addition,
novel loci on 5q11 (rs880944 near SNX18, p=3×10-6) and 20p12
(rs4813828, p=2×10-6) showed interesting potential association.
Conclusions: Using family-based genome-wide association analysis
and consanguineous families from South India, our results confirmed
the association between the 16q24 locus containing ZNF469 and
central corneal thickness in the South Indian population.
Additionally, novel loci on 5q11 and 20p12 were suggested by this
analysis, pending confirmation by further studies.
Commercial Relationships: Baojian Fan, None; N. Soumittra,
None; Sarangapani Sripriya, None; David S. Friedman, alcon (C),
bausch & lomb (C), merck (C), Pfizer (C), QLT, Inc (C); Vijaya
Lingam, None; Jonathan L. Haines, Arctic Dx (I), AMD genes (P);
Ronnie J. George, None; Janey L. Wiggs, None
Support: NEI Grants R21EY018149; P30EY014104; Research to
Prevent Blindness; Massachusetts Lions Eye Research Fund.
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
Program Number: 6210 Poster Board Number: C0095
Presentation Time: 10:30 AM - 12:15 PM
Fine mapping of the RXRA-COL5A1 locus for central corneal
thickness
Xiaoyi Gao1, Yutao Liu2, W. James Gauderman3, Mina Torres1, Talin
Haritunians4, Jane Z. Kuo4, Kent Taylor4, Jerome I. Rotter4, Rohit
Varma1. 1Department of Ophthalmology and Visual Sciences,
University of Illinois at Chicago, Chicago, IL; 2Department of
Medicine, Duke University, Durham, NC; 3Department of Preventive
Medicine, University of Southern California, Los Angeles, CA;
4
Medical Genetics Institute, Cedars-Sinai Medical Center, Los
Angeles, CA.
Purpose: Central corneal thickness (CCT) is associated with vision
disorders including glaucoma and keratoconus. Multiple studies have
reported the association of the RXRA-COL5A1 locus on
chromosome 9 with CCT. Here we describe in silico fine mapping,
followed by expression analysis.
Methods: We conducted this research using current GWAS data
from the Los Angeles Latino Eye Study (n = 1,800). All Latino
subjects were age 40 years and older. We carried out regional
genotype imputation over the RXRA-COL5A1 locus based on the
1000 Genomes Project reference panels and retained only SNPs of
high imputation quality with MACH Rsq greater than 0.80. We
performed single-marker and conditional analyses with adjustment
for age, sex and principal components of genetic ancestry. We also
performed expression analysis using human ocular tissues.
Results: We replicated several previously reported SNPs within the
RXRA-COL5A1 region and discovered a novel SNP, rs3118515 (P =
8.25E-10) in the uncharacterized LOC100506532 (gene type:
miscRNA), that better captures the association with CCT in Latinos.
Conditional analysis demonstrated that rs3118515 is responsible for
the association of the RXRA-COL5A1 locus. Multiple sources of
ENCODE evidence suggest that rs3118515 is in a regulatory region.
Reverse-transcription PCR products indicated that transcripts of
LOC100506532 surrounding rs3118515 were expressed in human
cornea.
Conclusions: Fine mapping and expression analysis improved the
localization and the annotation of SNPs that regulate CCT at the
RXRA-COL5A1 locus, indicating the involvement of
LOC100506532 and offering insights in the molecular mechanism of
CCT.
Commercial Relationships: Xiaoyi Gao, None; Yutao Liu, None;
W. James Gauderman, None; Mina Torres, None; Talin
Haritunians, None; Jane Z. Kuo, None; Kent Taylor, None;
Jerome I. Rotter, None; Rohit Varma, Allergan (C), AqueSys (C),
Genentech (C), Merck & Co. Inc (C), Replenish (C), Genentech (F),
National Eye Institute (F)
Support: NIH Grants 5U10EY011753 and 1R01EY022651.
Program Number: 6211 Poster Board Number: C0096
Presentation Time: 10:30 AM - 12:15 PM
Validation of APLP2 as Potential Candidate Gene for Human
Myopia Linked to Chromosome 11q24.3
Andrei V. Tkatchenko1, 2, Pawan Kumar Singh2, Ashok Kumar2, 1,
Tatiana V. Tkatchenko1. 1Anatomy & Cell Biology, Wayne State
University, Detroit, MI; 2Ophthalmology, Wayne State University,
Detroit, MI.
Purpose: We have recently identified several candidate genes for
human myopia in the monkey model of myopia using systems
genetics approach. One of these genes, APLP2, was overexpressed in
myopia, suppressed in hyperopia and localized within a critical
interval on chromosome 11q24.3 linked to human myopia. The
purpose of this study was to evaluate whether APLP2 may be the
gene responsible for the myopic phenotype in humans.
Methods: Normal refractive eye development and form-deprivation
myopia were analyzed in the Aplp2 knockout (Aplp2-/-), heterozygous
(Aplp2+/-) and wild-type (Aplp2+/+) mice using in situ hybridizations,
electroretinography (ERG) and photorefraction.
Results: We found that Aplp2 is co-expressed with Pax6, which
labels amacrine cells of the retina. Furthermore, adult Aplp2
knockout mice developed high degrees of hyperopia (+11.5 ± 2.2 D,
P35; +12.7 ± 1.5 D, P67) compared to both heterozygous (-0.8 ± 2.0
D, P35; -0.1 ± 1.7 D, P67; F(2, 98) = 24.83, p < 0.0001) and wildtype (+0.3 ± 2.2 D, P35; +0.6 ± 1.3 D, P67; F(2, 92) = 28.31, p <
0.0001) littermates, while refractive errors in heterozygous and wildtype animals were not significantly different (F(2, 114) = 0.20, p =
0.82). Knockout mice were significantly less susceptible to formdeprivation myopia compared to both wild-type and heterozygous
animals. Knockout mice developed -1.2 ± 0.6 D of myopia (p = 0.03)
in the deprived eyes compared to the contralateral control eyes,
whereas myopic refractive shift was -5.8 ± 1.0 D (p < 0.0001) in
heterozygous and -11.4 ± 0.8 D (p < 0.0001) in the wild-type
littermates. ERG analysis revealed that all animals independently of
the genotype had robust visual response; however, the amplitude of
the b-wave was significantly attenuated in the knockout animals
compared to both heterozygous and wild-type littermates. There was
no statistically significant difference in ERG between heterozygous
and wild-type animals.
Conclusions: APLP2 is a viable candidate gene for human myopia.
Reduced expression of Aplp2 resulted in the development of
hyperopia in mice and significantly decreased susceptibility to
myopia. In situ hybridizations and ERG also revealed that Aplp2 is
primarily expressed in the amacrine cells of the retina and that
reduced expression of Aplp2 affects visual response mediated by
inner retina in mice.
Commercial Relationships: Andrei V. Tkatchenko, None; Pawan
Kumar Singh, None; Ashok Kumar, None; Tatiana V.
Tkatchenko, None
Support: NIH Grant R21EY018902; NIH Grant P30EY004068
Program Number: 6212 Poster Board Number: C0097
Presentation Time: 10:30 AM - 12:15 PM
UV-Independent p53 Mutations in Sebaceous Carcinoma of the
Eyelid
Rehan M. Hussain1, Jared L. Matthews1, Sander R. Dubovy1,
Gaofeng Wang2. 1Ophthalmology, Bascom Palmer Eye Institute,
Miami, FL; 2Human Genetics, John P. Hussman Institute for Human
Genomics, Miami, FL.
Purpose: Sebaceous carcinoma (SC) is a potentially fatal neoplasm
that has a tendency to arise in the eyelid. UV exposure and mutations
of the p53 gene have been implicated in SC [1]. Kiyosaki et al
reported a high frequency (67%) of p53 mutations in SC from Asian
patients [2]. Most identified mutations of p53 in SC are point
mutations, which are distinct from tandem mutations caused by UV
exposure [1-3]. We analyzed p53 mutations in 14 cases to study the
relationship between the frequency of p53 mutations and SC of the
eyelid of Caucasians.
Methods: Sebaceous carcinoma of the eyelid resected from 14
Caucasian patients at Bascom Palmer Eye Institute from 1994 - 2010
were reviewed. An ophthalmic pathologist (SRD) performed
histologic evaluation of each specimen. Carcinoma tissues were cut
out by sterilized needles. Genomic DNA was extracted from paraffinembedded tissues. PCR primers were chosen from a published study
[4]. All 11 exons including flanking intronic regions were amplified
by PCR and sequenced in both directions. Sequencing traces were
analyzed. Sequence aberrations were confirmed by re-PCR and re-
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
sequencing.
Results: Of the 14 SC samples, 7 were found to have p53 mutations
(Table 1). Four specimens were found to have one p53 mutation.
Two samples carried two mutations. One sample was identified with
three missense mutations. We examined whether there is a possible
relationship between the severity of p53 mutations and pathological
features. We did not find any correlation between p53 mutations and
pathologic features, tumor size, stage, recurrence or metastasis.
Conclusions: A high frequency of p53 mutations have been reported
in SC of the eyelid from Asian patients [2]. Our data suggest that the
frequency of p53 mutation is also high (50%) in SC from Caucasian
patients. Mutations of p53 identified in previous studies are point
mutations. In addition to 7 novel point mutations, a 9 bp deletion and
a 6 bp insertion are also identified in our samples. No mutations are
tandem mutations, suggesting that aberrant p53 is likely UV
independent. There is no obvious correlation between severity of p53
mutations and pathological characterizations. The results suggest that
UV independent mutations in p53 could be a pathogenic factor for
SC of the eyelid.
Sebaceous carcinoma of the eyelid in a patient without p53
mutations. There is intraepithelial (pagetoid) spread in addition to the
subepithelial carcinoma. (H&E, 40x)
Commercial Relationships: Rehan M. Hussain, None; Jared L.
Matthews, None; Sander R. Dubovy, None; Gaofeng Wang, None
Program Number: 6213 Poster Board Number: C0098
Presentation Time: 10:30 AM - 12:15 PM
Genome-wide Analysis of Ocular Adnexal Lymphoproliferative
Disorders Using High Resolution Single Nucleotide
Polymorphism Array
Hiroki Takahashi1, Yoshihiko Usui1, Ryosuke Mitsuhashi1, Naoyuki
Yamakawa1, Aiko Sato-Otsubo2, Yusuke Sato2, Seishi Ogawa2, Ayako
Arai3, Hiroshi Goto1. 1Department of Ophthalmology, Tokyo
Medical University, Tokyo, Japan; 2Cancer Genomics Project, The
University of Tokyo, Tokyo, Japan; 3Department of Hematology,
Tokyo Medical and Dental University, Tokyo, Japan.
Purpose: Ocular adnexal lymphoproliferative disorders include
malignant lymphoma and benign lymphoprolifelative disease. The
aim of this study is to identify the genomic signature of these
disorders, especially ocular adnexal mucosa-associated lymphoid
tissue (MALT) lymphoma, IgG4-related ophthalmic disease (IgG4ROD), and reactive lymphoid hyperplasia (RLH), using highresolution single nucleotide polymorphism array.
Methods: Thirty-nine MALT lymphomas (22 conjunctival MALT
lymphomas and 17 orbital MALT lymphomas), 13 cases of IgG4ROD, and 2 cases of RLH were studied. To determine the genetic
alternations, DNA was extracted from tumor tissues and single
nucleotide polymorphism array experiments were performed
according to the standard protocols for the Affymetrix GeneChip
Human Mapping 250 K NSP arrays. The array data were investigated
using Copy Number Analysis for GeneChips (CNAG) software for
allele-specific copy number analysis. The study was approved by
institutional ethical committees.
Results: In cases of MALT lymphoma, chromosomal aberrations
were detected at chromosomes 3 (36%), 6 (20%), 18 (18%) and 21
(10%). Trisomy 3 was observed in 10 cases (26%) of MALT
lymphoma. Uniparental disomy (UPD) in chromosomes 3 and 6 were
frequently found in MALT lymphoma. Chromosomal aberrations
were found in about 76% of orbital MALT lymphomas, and the
frequency was higher than in conjunctival MALT lymphomas (40%).
No chromosomal aberrations were found in IgG4-ROD and RLH.
Conclusions: In addition to the previously reported chromosomal
aberrations, we detected novel chromosomal aberrations in MALT
lymphoma. MALT lymphoma had different patterns of chromosomal
aberrations. These findings may allow appropriate treatments for
ocular adnexal lymphoproliferative disorders according to the results
of chromosomal aberration pattern.
Commercial Relationships: Hiroki Takahashi, None; Yoshihiko
Usui, None; Ryosuke Mitsuhashi, None; Naoyuki Yamakawa,
None; Aiko Sato-Otsubo, None; Yusuke Sato, None; Seishi
Ogawa, None; Ayako Arai, None; Hiroshi Goto, None
Program Number: 6214 Poster Board Number: C0099
Presentation Time: 10:30 AM - 12:15 PM
Rb1 knockdown selectively induces cone precursor proliferation
and tumor formation dependent on cone-specific signaling
Xiaoliang L. Xu1, Poulos K. Bradford4, Hardeep P. Singh2, David H.
Abramson3, Suresh C. Jhanwar1, David Cobrinik2. 1Department of
Pathology, Memorial Sloan Kettering Cancer Center, New York, NY;
2
Department of Pediatrics, Memorial Sloan Kettering Cancer Center,
New York, NY; 3Ocular Oncology Service, Memorial Sloan
Kettering Cancer Center, New York, NY; 4Albert Einstein College of
Medicine, New York, NY.
Purpose: Retinoblastomas are thought to result from the inactivation
of RB1. We previously found that retinoblastoma has properties of a
cone precursor tumor and depends on cone-related signaling proteins.
Here, we tested whether human cone precursors are uniquely
sensitive to Rb inactivation, and provide direct evidence for cell
origin of retinoblastoma.
Methods: Lentivirus expressing RB1 or control shRNA was injected
into the subretinal space and vitreous of gestational week 20 human
fetal eyes and retinal cell proliferation was evaluated by Ki67
staining after d14 of in vitro culture. We combined CD133 and CD44
immunostaining and cell size to separate cone precursors
(medium+large-CD133highCD44- at 98% purity), rod precursors
(small-CD133highCD44-), and retinal progenitors and glia
(CD133lowCD44+). We knocked down RB1 either in purified or
mixed human fetal retinal cells, either with or without co-knockdown
of cone or Rb-related proteins, and examined proliferation of
different cell types by labeling and co-staining for EdU, Ki67, and
retinal cell type-specific markers. These cone precursors with RB1
knockdown (KD) were injected into subretinal space of nude mice to
check the tumor formation.
Results: In situ RB1 KD in whole human fetal retina caused
significant cone proliferation, especially in the fovea region (Fig. A).
RB1 KD strongly induced proliferation and expression of Ki67 in
purified cone arrestin+ and Crx+ cone precursors (Fig. B) and
apoptosis of Muller glia and progenitors, but did not induce Ki67
expression in other retinal cell types. Co-KD of RB1 with TRbeta2,
MDM2, MycN, SKP2, or p107 blocked the cone cell proliferation.
Co-knockdown of RB1 with p130 promoted cone proliferation and
cell line formation. All of the proliferating cones had failed to label
with EdU labeled prior to RB1 KD, indicating that they are derived
from non-proliferating cells. Xenograft of cone precursors with RB1-
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Genetics
KD caused growth of an ocular mass in nude mice (Fig. C).
Conclusions: These results suggest that Rb is required to suppress
the proliferation of cone precursors but not other retinal types. The
Rb-deficient cone precursor proliferation depended upon cone related
factors TRbeta2, N-Myc, and MDM2. Notably, p130 suppressed
whereas p107 was required for cone precursor proliferation after Rb
deletion. These data provide direct support for a cone precursor
origin of retinoblastoma.
Commercial Relationships: Xiaoliang L. Xu, Gerber Foundation
(F); Poulos K. Bradford, None; Hardeep P. Singh, None; David H.
Abramson, None; Suresh C. Jhanwar, None; David Cobrinik,
None
Support: Development Funds of Pathology in MSKCC, Gerber
Foundation, NIH grant 1R01CA137124
Program Number: 6215 Poster Board Number: C0100
Presentation Time: 10:30 AM - 12:15 PM
Downregulation of MITF Leads to Uveal Melanoma Cell
Apoptosis and Cell Cycle G1 Arrest
Lihua Wang, Xiaoyan Chen, Jiao Wang, Dongsheng Yan. 1School of
Ophthalmology and Optometry, Wenzhou Medical College,
Wenzhou, China.
Purpose: MITF plays a key role in melanocyte development and
tumorigenesis of melanoma. The role of MITF in uveal melanoma,
however, is not fully understood. In the present study, we
investigated the function of MITF in uveal melanoma cells.
Methods: The expression of MITF was determined by Western
blotting in both normal uveal melanocytes and uveal melanoma cell
lines. MITF specific siRNA was used to downregulate MITF
expression by transfection into uveal melanoma cells with
lipofectamine 2000. Cell cycle and proliferation was analyzed by
flow cytometry and MTS assay, respectively. Cell apoptosis was
detected by measurement of caspase 3/7 activity.
Results: MITF was dramatically upregulated in uveal melanoma cell
lines, as compared with normal uveal melanocytes. Transfection of
MITF specific siRNA led to a remarkable decrease of MITF in uveal
melanoma cells. Downregulation of MITF significantly inhibited cell
proliferation, induced cell cycle G1 arrest and apoptosis.
Conclusions: Our results demonstrated that MITF is a potential
therapeutic target in uveal melanoma.
Commercial Relationships: Lihua Wang, None; Xiaoyan Chen,
None; Jiao Wang, None; Dongsheng Yan, None
Support: National Natural Science Foundation of China
81071682&81272286
©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].