Download Aortopathy Gene Testing by Sanger sequencing

Department of Molecular Genetics
Aortopathy Gene Testing by Sanger sequencing
Mutation screening for Marfan syndrome and related disorders has grown to include many genes with
overlapping phenotypes. We have recently added SMAD3, TGFB2, COL3A1 and SKI to our genes for Sanger
sequencing. We now offer some of these genes in a ‘panel’ to be sequenced simultaneously to reduce the
turnaround time of screening each gene consecutively. We are developing an aortopathy gene panel utilising
next generation sequencing (NGS) or massively parallel sequencing (MPS).
Genes tested and associated phenotypes
Gene
FBN1
(Fibrillin-1)
TGFBR1&2
(Transforming growth factor beta
receptor 1 and 2)
COL3A1
(Collagen type III, alpha-1)
TNXB
(tenascin-x) Testing by MLPA only
Associated phenotype
Marfan syndrome
Ectopia lentis, familial
MASS syndrome
Acromicric dysplasia (exons 41 & 42)
Geleophysic dysplasia 2 (exons 41 & 42)
Stiff skin syndrome (exons 37 & 38)
Weill-Marchesani syndrome 2, dominant
Aortic aneurysm, ascending, and dissection
Loeys-Dietz syndrome, type 1A
Loeys-Dietz syndrome, type 2A
Loeys-Dietz syndrome, type 1B
Loeys-Dietz syndrome, type 2B
Ehlers-Danlos syndrome, type III
Ehlers-Danlos syndrome, type IV
Ehlers-Danlos-like syndrome due to tenascin-x
deficiency
MIM
number
154700
129600
604308
102370
614185
184900
608328
609192
608967
610168
610380
130020
130050
606408
Aortic aneurysm, familial thoracic 6
Moyamoya disease 5
Loeys-Dietz syndrome, type 3 (AneurysmsOsteoarthritis syndrome (AOS))
611788
614042
Loeys-Dietz syndrome, type 4
614816
To be developed
Juvenile polyposis/Hereditary Hemorrhagic
Telangiectasia syndrome/Aortopathy
175050
SKI
Shprintzen-Goldberg syndrome
182212
ACTA2
(Actin alpha-2, smooth muscle)
SMAD3
TGFB2
(Transforming growth factor beta 2)
SMAD4
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Pricing and turnaround times (updated May 2013)
Index case (proband)
Sanger sequencing panels (genes are screened and reported together)
Panel
Genes included
Marfan panel
FBN1 sequencing & MLPA
Loeys-Dietz syndrome
panel
TGFBR1
TGFBR2
SMAD3
TGFB2
+/- COL3A1
Fast TAT
Price AUD$
Turnaround
time
$1700
6 weeks
$1200
or
with COL3A1
$1700
6 weeks
extra $300
2-4 weeks
Individual genes (screening is performed consecutively)
Please clearly state the order of the genes to be tested if more than one gene is required
Turnaround time is for each gene
nd
10% discount for the 2 and subsequent gene tested
Gene
Price AUD$
FBN1
COL3A1
TGFBR1 & 2
ACTA2
SMAD3
TGFB2
SKI (exon 1 only)
FBN1 exons 24 to 32 for neonatal Marfan syndrome
FBN1 exons 41 & 42 for Geleophysic and acromicric dysplasia
FBN1 exons 37 & 38 for Stiff skin syndrome
Fast TAT
$1500
$1200
$900
$500
$500
$500
$250
$1000
$250
$250
extra $300
Turnaround
time
6 weeks
6 weeks
6 weeks
6 weeks
6 weeks
6 weeks
4 weeks
3-4 weeks
4 weeks
4 weeks
2-4 weeks
MLPA
(for the detection of deletions and insertions)
Gene
Price AUD$
Turnaround
time
FBN1 (2 kits)
$400
6 weeks
FBN1 & TGFBR2 (2 kits) Analysis of both genes will be performed
$400
6 weeks
TGFBR1 & TGFBR2 (1 kit)
FBN1 & TGFBR1 & TGFBR2 (3 kits) Analysis of all 3 genes will be
$300
6 weeks
$500
6 weeks
COL3A1 & TNXB (1 kit)
$300
6 weeks
together
performed together
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Cascade testing
Once a family-specific mutation has been identified, cascade testing can be used for at risk family members. If
the mutation was not identified in our laboratory a specimen from the proband will be required to test alongside
the family members.
Cascade testing (known mutation)
Price AUD$
Turnaround
time
$250
4 weeks
$200
4 weeks
Predictive testing 2nd sample (sequencing)
$100
4 weeks
MLPA - FBN1
$400
6 weeks
MLPA – TGFBR1 or TGFBR2
$300
6 weeks
Prenatal testing of familial mutation
$600
<2 weeks
Single patient (sequencing)
If the index case was not tested in our laboratory it is advisable to test the
index case alongside the other family members. Please provide a sample
from the index case with the patient to be tested. An additional $50 will
be charged.
Multiple patients (sequencing)
More than one patient tested at the same time for the same familial
mutation
2nd sample received after 1st sample has been tested
Summary of phenotypes for recently added genes
ACTA2: Mutations in ACTA2 associated with TAAD were first reported by Guo et al (2007) in 14 TAAD families.
Other features found in some individuals with mutations included livedo reticularis, iris flocculi and patent ductus
arteriosus. In addition Guo et al (2009) looked at the phenotype of 127 individuals with an ACTA2 mutation from
20 families. The primary vascular disease in these patients was TAAD. Other features included premature onset
coronary artery disease and ischemic strokes. In another cohort of 237 patients with nonfamilial TAAD six
mutations were identified. Other features found in this group include ischemic strokes, Moyamoya disease,
coronary artery disease, bicuspid aortic valve.
Guo, D.-C et al Mutations in smooth muscle alpha-actin (ACTA2) lead to thoracic aortic aneurysms and dissections. Nature Genet. 39:
1488-1493, 2007.
Guo, D.-C. et al Mutations in smooth muscle alpha-actin (ACTA2) cause coronary artery disease, stroke, and Moyamoya disease, along
with thoracic aortic disease. Am. J. Hum. Genet. 84: 617-627, 2009.
SMAD3 : van de Laar et al (2011) found 2 mutations in 99 patients with thoracic aortic aneurysms and
dissections (TAAD) and Marfan-like features but without FBN1, TGFBR1 or TGFBR2 mutations. Regalado et al
(2011) screened 181 patients with familial TAAD and found mutations in four families. The phenotype for
patients with mutations varied but included TAAD alone, TAAD with intracranial aneurysms, abdominal aortic
aneurysms, osteroarthritis, arterial tortuosity and cutaneous and skeletal features of MFS, LDS and AOS. van
de laar (2012) screened SMAD3 for mutations in 393 patients with aneurysms without mutations in FBN1,
TGFBR1 and TGFBR2 and identified mutations in five families. They studied the phenotypic spectrum of
patients with SMAD3 mutations from these families and suggest that joint abnormalities such as osteoarthritis,
osteochondritis dissecans and meniscal anomalies maybe useful discriminating features from other forms of
TAAD.
van de Laar IM, et al Mutations in SMAD3 cause a syndromic form of aortic aneurysms and dissections with early-onset osteoarthritis. Nat
Genet. 2011 Feb;43(2):121-6.
van de Laar IM, et al Phenotypic spectrum of the SMAD3-related aneurysms osteoarthritis syndrome. J Med Genet. 2012 Jan;49(1):47-57.
Regalado ES, Guo DC, et al Exome sequencing identifies SMAD3 mutations as a cause of familial thoracic aortic aneurysm and dissection
with intracranial and other arterial aneurysms. Circ Res.2011 Sep 2;109(6):680-6.
TGFB2: Boileau et al (2012) identified TGFB2 mutations in two unrelated families with autosomal dominant
TAAD using genomewide linkage analysis and whole exome sequencing. Two more mutations were identified in
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a further 276 probands with TAAD. Genome SNP arrays for 898 individuals with thoracic aortic disease did not
detect any deletions or duplications of TGFB2. Aortic dilatation at the level of the sinuses of Valsalva was
present in the majority of the 19 family members with TGFB2 mutations. Other features included skeletal
features, joint laxity, striae and inguinal hernias similar to MFS. Ectopia lentis was absent in these and they did
not have a particular facial appearance or the craniofacial or skin findings of LDS. Joint disease described in
AOS was also not seen.
Lindsay et al (2012) performed SNP array analysis on 8 families with autosomal dominant aortic aneurysms and
MFS-like and LDS-like features including pectus deformity, arachnodactyly, scoliosis, skin striae, hypertelorism,
bifid uvula, bicuspid aortic valve, arterial tortuosity, club feet and thin skin with easy bruising. Ectopia lentis was
not present. Two heterozygous microdeletions including TGFBR2 were detected in two individuals who also had
developmental delay. A further 86 individual with aneurysms and no mutation in FBN1, TGFBR1 and TGFBR2
were screened and six TGFB2 mutations were detected.
Renard et al (2012) initially screened TGFB2 in 40 patients with isolated aortic root dilatation and did not find
any mutations. They then screened a further 146 patients with a wider TAAD phenotype including
cerebrovascular disease, arterial tortuosity, marfanoid skeletal features and mitral valve prolapse and detected
mutations in six patients. Clinical features in the mutation positive patients included thoracic aortic aneurysm,
type A aortic dissection and mitral valve prolapse along with some features of MFS, LDS or AOS. Mutations
were found in individuals with and without a positive family history of TAAD.
Lindsay, M.E. et al. Loss- of function mutations in TGFB2 cause a syndromic presentation of thoracic aortic aneurysm. Nat Genet. 44(8):
922-927 (2012).
Boileau, C. et.al. TGFB2 mutations cause familial thoracic aortic aneurysms and dissections associated with mild systemic features of
Marfan syndrome. Nat.Genet. 44 (8): 916-921 (2012).
Renard, M. et.al. Thoracic aortic-aneurysm and dissection in association with significant mitral valve disease caused by mutations in
TGFB2. Int J Cardiol. (2012).
SMAD4: Andrabi et al (2011) report a family with a SMAD4 mutation in individuals with juvenile polyposis in
addition to mitral valve prolapse, mitral valve regurgitation, and aortic dilatation. SMAD4 mutations have
previously been described in patients with juvenile polyposis and/or hereditary hemorrhagic telangiectasia
syndrome (see OMIM 600993).
Andrabi, S et al SMAD4 mutation segregating in a family with juvenile polyposis, aortopathy, and mitral valve dysfunction. Am. J. Med.
Genet. 155A: 1165-1169, 2011.
Genes and loci known to be associated with TAAD:
TGFBR1:
TGFBR2:
MYH11
ACTA2:
FBN1:
MYLK:
SMAD3:
AAT1 (FAA1) locus:
AAT2 (TAAD1) locus:
1% of familial TAAD
4% of familial TAAD
~1% of familial TAAD (encoding smooth muscle-specific myosin heavy chain)
~10%-14% of familial TAAD
The frequency of mutations in this gene in familial TAAD is unknown
~ 1% of familial TAAD (encoding the myosin light chain kinase (MLCK) protein)
~ 2% of familial TAAD
involved gene unknown
involved gene unknown
Milewicz DM, Regalado E. Thoracic Aortic Aneurysms and Aortic Dissections. 2003 Feb 13 [Updated 2012 Jan 12]. In: Pagon RA, Bird TD, Dolan CR, et al.,
editors. GeneReviews™ [Internet]. Seattle (WA): University of Washington, Seattle; 1993-. Available from: http://www.ncbi.nlm.nih.gov/books/NBK1120/
Next Generation Sequencing panel
We are currently validating an aortopathy NGS panel for diagnostic testing. The genes included are ACTA2,
COL3A1, FBLN4, FBN1, FBN2, MYH11, NOTCH1, SLC2A10, SMAD3, SMAD4, TGFB2, TGFBR1, TGFBR2,
SKI. TNXB and FLN will be added in the next design. We are aiming to have this panel ready for diagnostic
testing by the end of 2013.
Specimen
5 to 10mls EDTA whole blood or extracted DNA from any source with at least 2 unique identifiers.
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Billing
Please indicate who should be invoiced for the testing. The organisation or the individual must have agreed to
pay for the testing. Testing will not commence without billing consent.
Prenatal testing
Prenatal diagnosis will be offered only to families in whom an existing gene mutation has already been
characterised. Where a mutation has been identified by an external source, the laboratory would always seek to
confirm the mutation prior to undertaking prenatal diagnosis. All prenatal testing MUST be booked with the
laboratory as early in the pregnancy as possible and prior to dispatch.
Presymptomatic testing in children and cascade testing
Once a family-specific mutation has been identified, cascade testing can be used for at risk family members. If
the mutation was not identified in our laboratory a specimen from the proband will be required to test alongside
the family members.
It is recommended that two independent samples, collected at different times, be sent for cascade testing.
Presymptomatic testing should be classified and follow the guidelines set out in the NPAAC document
“Classification of Human Genetic Testing - 2007 Edition”. If samples are received and tested at the same time
there will be no additional charge. If the second sample is received and tested after the first sample there will be
an additional cost of $100.
Counselling and Consent
Genetic counselling should be offered and written consent obtained prior to testing, consistent with the NSW
Dept of Health document “DNA Diagnostic Testing for Genetic Disorders”, and the “Consent for DNA Diagnostic
Testing/ Storage” form. Consent should be retained with the patient’s medical record. The lab does NOT require
a copy.
Clinical and Counselling Services
Within Australia details of the clinical and counselling services available in your area can be obtained from your
State Health service or via links from the NSW Genetic Education Program ph [02] 9926 7324; fax [02] 9906
7529; www.genetics.com.au
Transport
Please transport the specimens at room temperature. Referring laboratories will be responsible for arranging
and paying for the transportation of specimens to the laboratory. For countries outside of Australia, blood and
DNA samples without a known infectious risk, do not need quarantine inspection if external packaging is clearly
labeled. i.e. "Contents: Product of human origin, non-hazardous, non-infectious, for diagnostic in-vitro testing
only" and "Exempt human specimen. Diagnostic specimens packed in compliance with IATA packaging
instructions 650". If package is not correctly labeled and incurs a quarantine charge, this fee will be passed onto
the referring laboratory.
AQIS website:
http://www.aqis.gov.au/icon32/asp/ex_casecontent.asp?intNodeId=7974090&intCommodityId=942&Types=non
e&Whi chQuery=Go+to+full+text&intSearch=1&LogSessionID=660320
Address
Postal:
Department of Molecular Genetics, Western Sydney Genetics Program, The Children’s Hospital at Westmead,
Locked Bag 4001, Westmead NSW 2145 AUSTRALIA
Courier:
Department of Molecular Genetics, Western Sydney Genetics Program, The Children’s Hospital at Westmead,
Loading dock 5, Redbank Rd, Northmead NSW 2152 AUSTRALIA
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Contacts
For laboratory enquiries:
A/Prof Bruce Bennetts - Head, Department of Molecular Genetics
Ph [61-2] 9845 3246 Fax[61-2] 9845 3204 Email [email protected]
For clinical enquiries:
Dr Lesley Adès - Head, Marfan Research Group and Clinical Geneticist
Ph [61-2] 9845 3273 Fax[61-2] 9845 3204 Email: [email protected]
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