Download Low frequency of myocilin mutations in Indian primary open

Clin Genet 2004: 65: 333–337
Printed in Denmark. All rights reserved
Copyright # Blackwell Munksgaard 2004
CLINICAL GENETICS
doi: 10.1111/j.1399-0004.2004.00232.x
Short Report
Low frequency of myocilin mutations in
Indian primary open-angle glaucoma
patients
Sripriya S, Uthra S, Sangeetha R, George RJ, Hemamalini A, Paul PG,
Amali J, Vijaya L, Kumaramanickavel G. Low frequency of myocilin
mutations in Indian primary open-angle glaucoma patients.
Clin Genet 2004: 65: 333–337. # Blackwell Munksgaard, 2004
Glaucoma is one of the major causes of blindness in the Indian
population. Mutations in the myocilin (MYOC) gene have been reported
in different populations. However, reports on MYOC mutations in
Indian primary open-angle glaucoma (POAG) patients and juvenile
open-angle glaucoma (JOAG) patients are sparse. We therefore screened
100 unrelated POAG/JOAG patients for MYOC mutations. Patients
with POAG/JOAG were clinically diagnosed. Genomic DNA from such
patients was collected and studied for MYOC mutations by direct
sequencing. Nucleotide variations were compared with unrelated
healthy controls by restriction enzyme digestion. Secondary structure
prediction for the sequence variants was performed by Chou–Fasman
method. A novel mutation in exon 1 (144 G!A) resulting in Gln48His
substitution was observed in 2% of the patients. Four other
polymorphisms were also observed. The novel mutation was seen in four
other affected family members of a JOAG patient. The novel mutation
was found to alter the secondary structure in the glycosaminoglycan
initiation site of the protein. MYOC mutations were found in 2% of the
population studied. MYOC gene may not be playing a significant role in
causing POAG in the Indian population.
S Sripriyaa, S Uthraa,
R Sangeethaa, RJ Georgeb,
A Hemamalinib, PG Paula,
J Amalia, L Vijayab and
G Kumaramanickavela
a
Department of Genetics and Molecular
Biology, Vision Research Foundation
Sankara Nethralaya, and bDepartment of
Glaucoma, Medical Research Foundation
Sankara Nethralaya, Chennai, Tamilnadu,
India
Key words: fibronectin interaction –
glycosaminoglycan initiation site – Indian
population – juvenile open-angle
glaucoma – MYOC – primary open-angle
glaucoma
Corresponding author:
Dr G Kumaramanickavel, Reader &
Head, Department of Genetics and
Molecular Biology, 18, College Road,
Vision Research Foundation, Chennai
600006, Tamilnadu, India.
Tel.: þ91 44 28271616, 28261268;
fax: þ91 44 28254850;
e-mail: [email protected]
Received 22 September 2003, revised
and accepted for publication
5 December 2003
Glaucoma is a heterogeneous progressive opticnerve disorder and is one of the leading causes of
blindness worldwide. Positive family history,
age, increased intraocular pressure, and hypertension are some of the major risk factors of
glaucoma (1). Primary open-angle glaucoma
(POAG) is the major type of glaucoma affecting
2% of world’s population (1). Inheritance in
POAG varies from autosomal dominant to multifactorial, and positive family history represents a
major risk factor of the development of POAG
(2). Six chromosomal loci have been mapped
to POAG (3), and two genes myocilin (MYOC)
and optineurin (OPTN) linked to GLC1A and
E loci are found to be responsible for POAG
and juvenile open-angle glaucoma (JOAG) in
different populations (3–5).
The MYOC gene maps to the chromosomal
region 1q23-24 and was the first gene mapped to
JOAG (6). This gene is expressed in the trabecular meshwork induced by glucocorticoids in a
proportionate manner. The gene spans approximately 20 kb and has 3 exons with the promoter
region having several glucocorticoid response
elements. The protein is expressed, in many of
the ocular and non-ocular tissues, as a 55-kDa
olfactomedin-related secretory glycoprotein with
504 amino acids (7). Stone et al. (8) identified
MYOC mutations in GLC1A locus in POAG
patients and subsequently the same was reported
in other populations (9–11). Frequency ranging
from 2.6 to 4.4% for MYOC mutations has
been reported in Japanese, African-Americans,
Whites, and other populations (9). (Table 1)
333
Sripriya et al.
Table 1. Percentage of mutation in myocilin (MYOC) gene in
primary open-angle glaucoma (POAG) patients reported for
different populations (9, 10, 28–32)
Population
Italy (31)
France (32)
Hong Kong
Chinese (11)
Korea (28)
USA (9)
Iowa
African-American
India
Eastern India (14)
Present study
Canada (9)
Australia (9)
Japan (9)
Morocco (30)
Finland (29)
Percentage of
mutation in
MYOC gene
Total number of
POAG patients
8.0
7.5
4.8
26
237
291
4.4
45
4.3
2.6
727
312
3.5
2.0
3.0
2.8
2.8
1.8
0
56
100
167
390
107
57
136
Most of the mutations observed are missense
mutations in the olfactomedin domain of exon 3
(3). In a report by Rosza et al. (12), mutations
reported in MYOC gene are found to represent
changes in the secondary structure in the regions
of potential, functional regulation of the protein.
Glaucoma is one of the major causes of visual
morbidity in the Indian population, and 1.5
million people are reported to be blind due to
glaucoma in India (13). However, genetic studies
in Indian POAG patients are very few when compared to the West and other populations (14).
There is only one report on the MYOC mutations
in Indians where only 39 sporadic patients and 17
familial cases were screened (14). We have
screened 100 Indian JOAG/POAG patients to
know the frequency of MYOC gene mutations
in this population.
Materials and methods
Clinical material
One hundred unrelated patients diagnosed to
have POAG and JOAG were recruited from the
glaucoma clinic of Sankara Nethralaya Eye Hospital, Chennai, for Glaucoma study (15), after
getting informed consent from them. Patients 40
years of age underwent complete ophthalmic
examination which consisted of measurement of
best corrected visual acuity, slit lamp biomicroscopy, applanation tonometry, gonioscopy,
pachymetry, dilated fundus examination that
include evaluation of the optic disk and macula
with 78D lens with slit lamp and examination of
the retina using indirect ophthalmoscope, Humphrey visual fields, and optic disk documentation.
334
Fifty control subjects above 40 years of age underwent a complete glaucoma evaluation. POAG was
defined as an open angle on gonioscopy, with or
without raised intraocular pressure, typical glaucomatous disk changes with corresponding visual
field defects in the absence of any secondary cause.
JOAG was diagnosed in cases with similar clinical
parameters where the age at onset of disease was
between 10 and 35 years.
Sequence analysis of the MYOC gene
Genomic DNA was extracted from whole blood
samples of patients and controls using phenol
chloroform extraction following proteinase K
digestion (16). Polymerase chain reaction (PCR)
amplification and direct sequencing in ABI Prism
310 Genetic analyzer was performed for all the
100 patients for sequence analysis of the MYOC
gene. The primers used for DNA extraction was
according to that of Lam et al. (10) PCR was
standardized at an annealing temperature of
54 C for all the primers except for primer 1C.
The PCR conditions are as follows: initial denaturation at 94 C for 5 min followed by 30 cycles of
amplification at 94 C for 30 s; 54 and 72 C for
1 min with a final extension step at 72 C for
5 min. The amplified products were electrophoresed in 2% agarose gel for verification and
followed by sequencing. Cycle sequencing was
carried out in automated ABI Prism 310 genetic
analyzer. For some of the sequence alterations,
PCR in 40 controls followed by restriction
enzyme (RE) digestion assay was performed.
Five units of the RE were incubated with 5 ml
of the amplified product with buffer and water
for a total volume of 20 ml at 37 C for 48 h. The
digested products were run on 12% polyacrylamide gel followed by silver staining (17). The
raw data of the sequences run in ABI Prism 310
genetic analyzer was analyzed using SEQUENCE
ANALYSIS software version 1.11.
Secondary structure prediction
The normal sequence and the novel sequence variant of the MYOC gene were analyzed for secondary structure prediction by Chou–Fasman
method (18, 19). The variant was also compared
with the bovine, rat, and mouse MYOC gene for
conserved sequence.
Results
Ninety-four POAG and six JOAG patients
were screened for MYOC mutations. Seventeen
Indian POAG patients and MYOC gene
patients had a strong family history of glaucoma.
Five were autosomal recessive inherited with two
members affected, and twelve were inherited in
autosomal dominant manner. The number of
POAG patients in these 12 families ranges from
two to five. Five sequence alterations were
observed in the study subjects. Two missense
sequence changes, one synonymous codon
change, and two nucleotide variations in the
non-coding regions was observed (Table 2).
The two missense sequence changes observed
include a novel mutation at 144th nucleotide:
[144G!A (Gln48His) and 227G!A (Arg76Lys)
in exon 1]. Gln48His mutation was observed in
two unrelated patients [95% confidence interval
(CI) (0.0047–0.0074)]. One patient from northern
India had JOAG and the other was a POAG
patient from southern India. The POAG patient
with the novel mutation did not have any family
history. The affected and the unaffected family
members of the JOAG patient were also screened
for the mutation. All the four members affected
with the disease had the mutation. The 144G!A
variation resulted in the loss of RE digestion site
for AccI RE. Fifty unrelated control subjects
were screened by RE digestion, and none of
them were seen to have this variation.
First patient
The patient was a 17-year-old male with JOAG
with family history of microcoria and glaucoma.
He was highly myopic (18.0D in the right eye
and 17.0D in the left eye) with miotic pupils
that did not dilate beyond 3 mm. Intraocular
pressure was 33 mmHg in the right eye and
26 mmHg in the left eye on medication. Gonioscopy revealed open angles in both eyes with
prominent iris processes. Examination of the
optic disk with 90D lens showed a 0.6 : 1 cup
disk ratio in the right eye with rim thinning and
0.4 : 1 cup disk ratio in the left eye. Visual fields
on the Humphrey Field Analyzer HFA-24-2
showed a generalized reduction of sensitivity.
Intraocular pressure could not be controlled on
maximum tolerated medical therapy, and he
underwent a trabeculectomy in both eyes following which intraocular pressure was under control.
Second patient
The second case was a 65-year-old lady who was
diagnosed to have POAG. She presented with
intraocular pressures of 21 mmHg in the right
eye and 23 mmHg in the left eye. She had open
angles on gonioscopy, and disk examination in
the right eye showed a 0.6 : 1 cup disk ratio with
an inferior notch; there was no view to the optic
disk in the left eye due to significant cataractous
changes. Visual fields in the right eye were consistently unreliable. Intraocular pressure was
controlled on medication in the right eye, and
she was advised to undergo cataract surgery
combined with trabeculectomy in the left eye.
The 227G!A resulting in Arg 76Lys substitution in exon 1 was the other missense sequence
change observed in 25 of the POAG patients
(95% CI: 0.17–0.33). This sequence change was
the most common nucleotide variation seen
among the study subjects. The other changes in
the non-coding regions include 730 þ 35 A!G in
intron 2 (14 and 95% CI: 0.07–0.17) and 1–83
G!A promoter polymorphism observed in 19
patients (95% CI: 0.11–0.27). A synonymous
codon change resulting in Leu403Leu substitution in exon 3 was seen in one of the POAG
patients.
Secondary structure prediction for the novel mutant
protein
Gln48His mutation was found to remove the
extended sheets in the glycosaminoglycan
(GAG) initiation site (aa 42–45) and was also
found to show a difference in the core–surface
ratio when compared with the wild-type (WT)
protein as shown in Fig. 1.
Discussion
In this study, a novel mutation 144 (G!T) resulting in Gln48His (a positively charged amino acid)
substitution was observed in two unrelated
patients, out of the 100 subjects screened. Fifty
unrelated healthy controls were screened by AccI
RE digestion method, and Gln48His mutation
was observed in none of them. This mutation is
Table 2. Five sequence variants observed in the myocilin (MYOC) gene in 100 Indian primary open-angle glaucoma patients
Location
Nucleotide change
Codon change
Patients’ percentage (n ¼ 100)
Promoter-18C/T
Exon 1
Exon 1
Intron 2
Exon 3
–
144 (G!T)
227 (G!A)
730 þ 35 (A!G)
1209 (C!T)
–
Gln48His
Arg76Lys
–
Leu403Leu
19
2
25
14
1
335
Sripriya et al.
Wild-type myocilin
.
.
.
.
.
.
MRFFCARCCSFGPEMPAVQLLLLACLVWDVGARTAQLRKANDQSGRCQYTFSVASPNESS
helix <---->
<------------------------->
sheet EEEEEEEEEE
EEEEEEEEEEEEEEEE
EEE
EEEEEEEEEE
turns
T
T T
T T
Fig. 1. Results of secondary structure
of the wild-type and Q48H-mutant
protein. Amino acid sequence within
the black box shows region where the
change is observed in the secondary
structure due to Q48H substitution.
Mutant myocilin
.
.
.
.
.
.
MRFFCARCCSFGPEMPAVQLLLLACLVWDVGARTAQLRKANDQSGRCHYTFSVASPNESS
helix <---->
<------------------------->
sheet EEEEEEEEEE
EEEEEEEEEEEEEEEE
EEE
EEEEEE
turns
T
T T
T T
also reported by Mukopadhyay et al. (14) in three
of the 56 eastern Indian POAG patients and 39
amongst them were sporadic patients. The power
of the study is calculated as 27%. The report by
Mukopadhyay et al. (14) on the novel mutation
was published after we submitted our data to the
human genome mutation database and received
the temporary accession number (H972168). The
Gln48His mutation was not reported in any other
population. This suggests that the Gln48His
substitution is unique to the Indian population.
The amino acid glycine at the 48th codon lies in
the conserved sequence region, shown by the
homology of MYOC protein sequence of different species (rat, bovine, mouse, etc.) (14). The
WT MYOC protein has a GAG initiation site
(Asp-Gln-Ser-Gly) at amino acid position 42–45
(7). The predicted secondary structure of the WT
protein has extended sheets in this region and the
Q48H mutation removed these sheets (Fig. 1). It
is interesting to note that GAG is widely
expressed in the trabecular meshwork (TM) tissue
(20) and is involved in the fluid dynamics of
aqueous humor (21) which is reported to be altered
in glaucomatous condition (22). MYOC protein is
also reported to interact with the HepII domain of
fibronectin, the major extra cellular matrix component of the TM tissue (23). It has been suggested by
in vitro studies that normal MYOC protein interacts
with the mutant protein to form insoluble precipitates that obstruct the TM outflow pathway (24).
We observed that the Arg76Lys polymorphism
reported in other populations (10, 11) is one of
the most common polymorphisms (25%) along
with the promoter polymorphism (19%). The
Leu403Leu silent mutation was seen in one of
the POAG patient, which has not been reported
in any other studies. We never found the Pro370Leu mutation in our study, which was reported to
cosegregate with an affected Indian POAG family
by Mukopadhyay et al. (14) Most of the diseasecausing mutations are in the olfactomedin
domain in the exon 3 of the gene (25). MYOC
mutations are reported to have clinical implications in assessing the risk of the development of
336
the disease (26, 27). Some mutations are reported
to restrict to early age at onset while some are
associated with varying age at onset of the disease. Some of the MYOC mutations were found
to have different therapeutic implications (27).
Mutations in the MYOC gene is reported in
POAG patients in different populations; highest
is in Italy (8%) and the lowest is in Finland (0%);
our study showed that it is involved in 2% of
POAG patients from India (9–11). Mutational
screening of MYOC gene in POAG patients of
different populations has shown 45 missense
sequence variations. Mukopadhyay et al. (14)
have predominantly studied the eastern Indian
patients, whereas our study predominantly had
southern Indians though a proportion of patients
was from northern India. Our study did not show
any of the reported POAG-causing mutations
observed in the other populations. We identified
only one possible disease-causing mutation
(Gln48His), and this suggests that MYOC mutations are responsible for only a small percentage
of POAG/JOAG patients in the Indian population, but we suggest that a larger study could
confirm our observation.
Acknowledgements
We gratefully acknowledge and thank the financial support
received from the Chennai Willingdon Corporate Foundation,
India.
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