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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. References 1. Wilson R & Martone J. The Glaucomas 2nd edn. Louis: Mosby St, 1996: 753–768. 2. Wolfs RCW, Klaver CW, Ramrattan RS et al. Genetic risk of primary open angle glaucoma. Arch Opthalmol 1998: 116: 1640–1645. 3. Sarfarazi M. Recent advances in molecular genetics of glaucomas. 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