\set{final}

\def\Author{Rose}
\def\author{rose}
\def\vol{13}
\def\year{2007}
\def\anum{53}
\def\pages{497-503}
\def\txt_title{Myocilin mutations among primary open angle glaucoma patients of Kanyakumari district, South India}
\def\txt_authors{Rajiv Rose, Muthusamy Karthikeyan, Balakrishnan Anandan, Gopalswamy Jayaraman}

\def\rcvd{26 November 2006}
\def\accept{29 January 2007}
\def\publ{2 April 2007}
\def\pdfsize{}
\def\PMID{}


\include{mvstyle.hsm}

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\| Internal defs


\article{


\title{Myocilin mutations among primary open angle glaucoma patients of
Kanyakumari district, South India}

\authors{\mailto{rajivrose@hotmail.com}{Rajiv Rose},
\mailto{muthukarthi@rediffmail.com}{Muthusamy Karthikeyan},
\mailto{anand_gem@yahoo.com}{Balakrishnan Anandan},
\mailto{gjay52@rediffmail.com}{Gopalswamy Jayaraman}}

\institutions{Department of Genetics, Dr. ALM PGIBMS, University of
Madras, Taramani Campus, Chennai, Tamil Nadu, India}

\correspondence{Dr. Gopalswamy Jayaraman, Ph.D., Reader and Coordinator,
Molecular Biology Programme, Department of Genetics, Dr. ALM PGIBMS,
University of Madras, Taramani Campus, Chennai, Tamil Nadu 600113,
India; Phone: +91-44-65361365; FAX: +91-44-24540709; email:
gjay52@rediffmail.com}

\abstract

\abs_purpose{Glaucoma can be defined as optic neuropathy leading to
irreversible blindness if not treated in time. Primary open angle
glaucoma (POAG) is the most common form of glaucoma. The myocilin
(\i{MYOC}) gene has been found to mutate in both sporadic and familial
cases of POAG worldwide. About 90% of these mutations have been seen to
cluster at exon III of the gene. There are documented reports of
mutations in the \i{MYOC} gene among POAG patients from different parts
of India. The southernmost tip of the Indian subcontinent (Kanyakumari
district) has remained isolated from all these studies. The aim of this
study was to indicate or rule out the disease causative role of the
\i{MYOC} gene mutations in these patients by screening the \i{MYOC} gene
for mutations among POAG patients of the Kanyakumari district.}

\abs_methods{One hundred POAG patients from the Kanyakumari District of
South India were recruited for the study. The \i{MYOC} gene was screened
using the PCR-SSCP methodology followed by DNA sequencing. The sequences
were analyzed using BLAST. Secondary structures of the amino acid
sequences with a variation were predicted.}

\abs_results{Two probable disease-causing variations (mutations),
Ser331Thr and Pro370Leu, were each observed in one patient apiece. Two
polymorphisms, (Tyr347Tyr and Thr325Thr) were also observed in the
patients. Ser331Thr is a novel conservative change while Pro370Leu is a
widely reported mutation with an associated severe disease phenotype.}

\abs_conclusions{The presence of the mutations in the patients suggests
the causative role of the \i{MYOC} gene among POAG patients in the
Kanyakumari district of India. The mutation frequency of 2% corresponds
well with the other reports from India and other countries. However, the
mutation rate reported from a population in the eastern part of India
was much higher. Screening of patients from different parts of India is
essential to estimate the overall mutation frequency. More functional
studies on the \i{MYOC} gene are required to elucidate the
pathophysiology of POAG.}

\introduction

\p{Glaucoma is a term used to describe a group of disorders that have
optic neuropathy as a common feature. Optic neuropathy is optic nerve
head cupping or degeneration of the optic nerve. This cupping of the
optic nerve head initially leads to the loss of peripheral vision and,
if not treated in time, results in irreversible blindness [1]. Among the
different forms of glaucoma, the primary open angle glaucoma (POAG) is
the most common type [2,3]. There are two forms of POAG: adult onset and
juvenile onset. These are mainly distinguished based on the age of onset
of the disease. However, juvenile onset POAG usually has a more severe
disease phenotype compared to the adult onset type [2,4].}

\p{India has a high prevalence of glaucoma with POAG being the most
common form of glaucoma [5]. In the WHO report on the prevalence of
glaucoma in India, 1% of the population was stated to be blind of which
12.8% was due to glaucoma [6]. The prevalence of POAG in South Indian
populations has been reported to be 1.62% [7,8]. This prevalence rate is
similar to that observed in Western populations.}

\p{A family history of glaucoma has long been recognized as an important
risk factor for the disease. The common adult onset POAG is inherited as
a complex trait, while the juvenile onset POAG exhibits autosomal
dominant mode of inheritance [9]. The prevalence of POAG among first
degree relatives of affected patients is as high as 16-22% [10,11]. Thus
far, 22 gene loci have been linked to POAG [9,12-26].}

\p{Three genes, \i{MYOC}, \i{OPTN}, and \i{WDR 36}, have been found to
mutate among POAG patients [23,27,28]. The \i{MYOC} gene, located on the
GLC1A locus, was initially known as the trabecular meshwork-inducible
glucocorticoid response (\i{TIGR}) gene. \i{MYOC} sequence variations
associated with POAG were first observed by Stone et al. [27]. Mutations
in the \i{MYOC} gene have now been reported to be present among POAG
patients from almost all parts of the world [1,29-41]. An average
mutation frequency of 1.4-4.6% has been observed among POAG patients
worldwide [29,32,35,42]. The \i{MYOC} gene consists of three exons with
two intervening introns. Exon III is the largest of the three and is
referred to as the olfactomedin-like domain due to its homology to
olfactomedin. More than 90% of the mutations reported so far are
clustered to exon III of the gene [34,43].}

\p{There are three reports currently available on populations from
different parts of India that estimate the mutation frequency among
randomly selected, unrelated POAG patients. Sripriya et al. [40]
reported a mutation frequency of 2% among 100 patients screened from
Chennai (South India). Kanagavalli et al. [39] found a mutation
frequency of 2% among 107 POAG patients screened from Madurai (South
India). While Mukhopadhyay et al. [36] reported mutations among 4 of 56
POAG patients (about 7.1%) screened from Kolkata (East India).}

\p{There are two major reasons to screen a gene for mutations: (a) to
implicate the gene as a cause of a particular disease; and (b) to
identify as many mutations as possible for the purpose of understanding
genotype-phenotype relationships, comparing mutation profiles in
different populations or constructing practical genetic tests for
clinical use [34]. The present study was aimed to implicate or rule out
the involvement of mutations in the MYOC gene in disease causation among
POAG patients of the Kanyakumari district of South India. This region
has never been included in any such genetic or epidemiological study
pertaining to POAG. This study would serve as an initiator for further
POAG research.}

\methods

\subsection{Clinical Samples}

\p{This study recruited 100 patients from hospitals in the Kanyakumari
District. All patients had been given the diagnosis of
adult/juvenile-onset POAG. Eighty-seven of these were adult onset cases,
and 13 were patients with juvenile onset open angle glaucoma.
Twenty-five patients had at least one family member affected by POAG. In
three cases, the family history could not be confirmed. An equal number
of age- and sex-matched controls from the same district were included in
the study. Informed consent was obtained from each individual before
blood was drawn. Each patient underwent a complete ophthalmic
examination, which consisted of measurement of intraocular pressure
(IOP), gonioscopic evaluation of the angle, examination and
documentation of the optic disc, and visual field testing. The criteria
for diagnosing glaucoma, based on previous reports [4], were that the
patient had been (1) treated for glaucoma prior to their involvement in
the study or (2) had at least two of these features: (a) an IOP of
greater than or equal to 22 mmHg, (b) an increased optic cup to disc
ratio (where the normal value is 0.3, and glaucomatous cupping is
greater than or equal to 0.5), and (c) visual field abnormalities with
no other apparent cause for the loss in vision.}

\p{Classification of adult and the juvenile onset POAG was based on age
of disease onset; adult onset was considered to be after 40 years of
age, whereas juvenile onset was before this age [4,9].}

\subsection{Analysis of the \i{MYOC} gene}

\p{Genomic DNA was isolated from the blood samples of patients and
controls using the salting out method of Miller et al. [44]. Eight
primer sets were designed to amplify the gene in fragments of \lt 300 bp
(\tabref{1}) so as to enable analysis of the amplicons using the single
strand conformation polymorphism methodology. PCR amplifications were
carried out in 20 \mu l reaction volumes containing about 100 ng of
genomic DNA, 5 pmol of each primer (forward and reverse), 2 mM of each
dNTP, 0.5 U of Taq polymerase with a standard buffer containing 1.5 mM
MgCl\sub{2}. Amplification was carried out under the following
conditions: initial denaturation 94 \deg C for 5 min, followed by 30
cycles of denaturation 94 \deg C for 30 s, annealing for 30 s (for each
annealing temperature [Ta], please Refer to \tabref{1}), extension -72
\deg C for 30 s, followed by a final extension at 72 \deg C for 2 min.}

\p{The amplicons were mixed with the loading dye (bromophenol blue,
xylene cyanol, formamide, and Na\sub{2}EDTA) and double distilled water
in the following ratio: 2 \mu l amplicon, 2 \mu l dye, and 16 \mu l
double distilled water. The combination was heat denatured for 5 min in
boiling water and electrophoresed on a composite (acrylamide, bis
acrylamide, and agarose) gel [45] for 13 to 14 h. The percentage of the
gels used and the electrophoresis voltage varied for amplicons of the
different primers sets. Gels were silver stained as per the protocol of
Bassam et al. [46]. The amplicons with a mobility shift were
reamplified, column purified, and sequenced using dye termination
chemistry and read using the 96 capillary 3730x1 DNA analyzer (Applied
Biosystems, Foster City, CA). Sequences were analyzed using BLAST to
identify the variation(s).}

\p{DNA sequences with a variation were translated in silico to obtain
the corresponding amino acid sequences. The amino acid sequences were
subjected to the secondary structure prediction using the
Garnier-Osguthorpe-Robson (GOR) prediction method [47,48], a means of
predicting the secondary structure of proteins. This method forecasts
the secondary structure of a sequence by calculating the probability for
each of the four structure classes (helix, sheet, turn, and loop) based
on the central residue and its neighbors from the precalculated
matrices. The highest is then selected for each residue.}

\results

\p{Four different types of sequence variations were obtained. Two of
these were nonsynonymous mutations i.e., the variations were both in the
nucleotide sequence, and the corresponding amino acid sequence (missense
mutation), namely Ser331Thr and Pro370Leu, showed up in at least one
patient (\figref{1} and \figref{2}). These were probable disease-causing
variations. The other two were synonymous variations, meaning the
changes were in the nucleotide sequence alone and not in the
corresponding amino acid sequence: (1) Tyr347Tyr in four patients and
(2) Thr325Thr in one patient. All sequence variations were observed in
the heterozygous state and only in the patients' samples.}

\p{The Ser331Thr mutation (T\gt A transversion at nucleotide 991) was
observed in one patient, who also had the Thr325Thr synonymous mutation
(G\gt A transition at nucleotide 975). This patient was 68 years old at
the time of sample collection. At the time of blood drawing the
patient's IOP was 20.6 mmHg OD and 20.6 mmHg OS. The patient was was
being treated with 0.5% levobunolol for POAG. The cup to disc ratio
revealed a totally excavated disc in the left eye and was 0.9 in the
right eye. The left eye was totally blind with only light perception,
and the right eye had about 25\deg\ of vision. The patient was
hypertensive (180/100 mmHg). The patient denied a family history of the
disease.}

\p{The Pro370Leu (C\gt T transition at nucleotide 1109) was the other
mutation observed in one patient only. The patient was 57 years old at
the time of sample collection and she had been affected by glaucoma at
about 16 years of age. During the last stages of her vision, she had
highly elevated pressures (readings not available), open angles, and no
other systemic or ocular abnormalities that could have resulted in the
loss of vision, indicating POAG. This is a typical example of a rapid
progressive JOAG with an early age of onset. It was later learned she
had a family history of glaucoma. Her brother developed JOAG before the
age of 30 and six of her uncles (distant relatives) developed either
POAG or primary angle closure glaucoma (PACG). Her brother with POAG,
three of her uncles (first cousins once removed), and one second cousin
who had POAG/PACG were also included in the present study. However, none
of them had any variation in the MYOC gene.}

\p{The other synonymous variation or polymorphism was the Tyr347Tyr
(1041 T\gt C) change. This was seen in four patients.}

\p{Random sequencing of five patients and five normal control samples of
each primer set confirmed the negative results of SSCP analysis.}

\discussion

\p{This is the first such study from this part of India. The patients
from the Kanyakumari district had two mutations: (1) Pro370Leu (1109
C\gt T) and (2) Ser331Thr (991 T\gt A). The Ser331Thr is a novel
mutation with no report of it available at present as far as our
literature search showed. The other mutation has been reported in other
populations worldwide.}

\p{The Ser331Thr mutation observed in this study is a conservative
change of serine to threonine. However its absence in the controls and
the effect of this change on the predicted secondary structure of the
protein (\figref{3}) suggests that it might have a causal effect.
Alhough there is no direct evidence of this being a causal factor of
glaucoma, the benign nature of this variant cannot be assumed. The fact
that this change is not mentioned in the literature and that it was
absent in the normal controls indicates this is a mutation or a probable
disease-causing variation.}

\p{An earlier report mentions that though the nonconservative changes
were presumably more injurious to the functioning of the myocilin
protein than conservative changes, the conservative changes have also
been classified as probable disease-causing mutations if they were
absent in the controls [34].}

\p{The Pro370Leu alteration was observed in one patient, who had a
severe disease phenotype with an early age of onset of the disease
(diagnosed at 16 years), rapid progression of the disease, and a
positive family history of the disease. The severe nature of the disease
phenotype associated with this variant correlates well with earlier
reports [30,33,49]. The proband reported by Damji et al. [50] had a
similar clinical history as that of the aforedescribed patient: early
age of onset, much before sample collection, lack of initial
ophthalmological examination records, and a family history of the
disease.}

\p{This mutation has also been reported in an Indian patient from
Kolkata (eastern part of India) [36]. The patient had pressures of 24
mmHg OD and 32 mmHg OS and cup to disc ratios of 0.8 OD 0.7 OS. Vision
tests revealed presence of an arcuate scotoma in the superior and
inferior halves with nasal steps OD and a scotomatous defect in the
superonasal quadrant OS.}

\p{The occurrence of this mutation in patients of varying ethnicity
(Indian, English, French, North American, Japanese, and German
populations) suggests this is a prevalent mutant allele of MYOC. The
severe nature of the disease in patients with this mutation indicates
that the loss of proline at this position may acutely affect the normal
function of the protein [32]. However, further studies are needed to
correlate disease severity with this mutation.}

\p{The change C\gt T transition at nucleotide 1109, results in a change
from proline (a polar amino acid) to a hydrophobic leucine molecule.
This transition occurs in the CpG dinucleotide. The importance of CpGs
in the disease causation was suggested by Mukhopodhyay, et al. [36].}

\p{The translationally silent polymorphism in codon 347 has a relatively
high incidence in subjects from the Kanyakumari district (about 4%).
These observations indicate this is a frequent polymorphism found among
POAG patients of this part of India.}

\p{Both the mutations presented here have been predicted to affect the
myocilin protein's secondary structure. Secondary structure prediction
of the Ser331Thr mutant amino acid sequence by GOR revealed a removal of
turn "T" at the amino acid 332 followed by an addition of a \beta\ sheet
"E" at amino acid 332 and 333. A substitution of \beta\ sheet "E" to
turn "T" at amino acid 325 was predicted and \beta\ sheet "E" was
expected to coil "C" at 337. The Pro370Leu results in a change from a
polar amino acid to a hydrophobic amino acid. This mutation also results
in changes in the predicted secondary structure. Moreover, it has been
elucidated that proline has a side chain that inhibits (\alpha\ helix)
formation and fits poorly in the \alpha\ helix conformation, while
leucine is one of the good \alpha\ helix formers [51]. Since the
function of the \i{TIGR/MYOC} gene has not yet been fully elucidated,
the effects of single amino acid changes cannot be precisely predicted
and can only be hypothesized [52].}

\p{The findings suggest that the \i{MYOC} gene is involved in the
causation of POAG in this part of India as well. The mutation rate was
2% among POAG patients of the Kanyakumari district This is not a high
rate, but the frequency coincides with that reported from other parts of
India and the world.}

\p{A larger data set including more patients and functional studies on
the \i{MYOC} gene are required to elucidate the pathophysiology of
POAG.}

\acknowledgements

\p{We wish to sincerely thank Dr. Bejan Singh, Dr. Rooshita Bejan Singh,
and Dr. Hector. This work was supported by Council of Scientific and
Industrial Research in the form of a Senior Research Fellowship. We
thank the patients, their family members, and the control subjects for
participating in this study.}

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\endreferences

}

\beginfigures

\figfile{1}{
\figtitle{1}{Heterozygous Ser331Thr mutation and Thr325Thr polymorphism in the
\i{MYOC} gene}

\p{Chromatogram sequence derived from patient Ngl 12 with the T\gt A
transversion and G\gt A transition (indicated by arrows) compared to the
normal control.}

\ctr{\gifimage{1}{400}{502}{39}}

}

\figfile{2}{
\figtitle{2}{Heterozygous Pro370Leu mutation in the \i{MYOC} gene}

\p{Comparison of chromatogram sequences derived from patient R6 and
normal control. Arrow marks the C\gt T transition compared to the normal
control.}

\ctr{\gifimage{2}{400}{499}{34}}

}

\figfile{3}{
\figtitle{3}{Secondary structure prediction of the mutant sequence (Ser331Thr) using
the GOR method}

\p{Comparison between the predicted secondary structures of control
(\panel{A}) amino acid sequences of myocilin and the amino acid
sequences of myocilin with the Ser331Thr variant (\panel{B}; only the
region where there was a change in the predicted secondary structure has
been shown).}

\ctr{\gifimage{3}{700}{259}{29}}

}

\begintables

\tabfile{1}{
\tabtitle{1}{PCR amplification conditions}

\p{The following are given for each primer set: primer name, sequence,
binding site, annealing temperature, and expected amplicon size for each
primer set. The asterisk indicates the annealing temperature (Ta).}

\ctr{\gifimage{1}{800}{672}{112}}

}