Abstract: PURPOSE: To assess the effects of previously uncharacterized PITX2 missense mutations found in patients with Axenfeld-Rieger syndrome and to determine the functional roles of the C-terminal region of PITX2. METHODS: Recombinant PITX2 proteins were analyzed with the use of cellular immunofluorescence, electrophoretic mobility shift, reporter transactivation, and protein half-life assays in human trabecular meshwork cells. RESULTS: Two homeobox mutations, R43W and R90C, resulted in severely reduced DNA-binding and transcriptional activation despite normal nuclear localization. L105V, located C-terminal to the homeodomain, resulted in normal localization, reporter gene transactivation, and protein half-life, but with an altered mobility shift pattern of protein-DNA complexes. N108T, also located C-terminal to the homeodomain, resulted in an altered mobility shift pattern and with slightly increased reporter transactivation and shortened protein half-life. The PITX2 C-terminal region contains at least three domains, each with distinct modulating effects on reporter transactivation. CONCLUSIONS: PITX2 homeobox mutations predictably resulted in decreased function of the protein. However, the two C-terminal mutations exhibited only subtle defects on PITX2 transactivation and protein-DNA binding, suggesting that ocular development is sensitive to even slight alterations of PITX2 function. The C-terminal mutations L105V and N108T lie in a domain that inhibits PITX2 transcriptional activation. These two mutations produce electrophoretic mobility shift assay patterns representing altered protein-DNA interactions that may be important for accurate target gene selection. Additionally, N108T resulted in a less stable PITX2 mutant protein with elevated activity that may result in stochastic dysregulation during critical stages of development. Together, the results clearly indicate that stringent control of PITX2 is required for normal ocular development and function.
Abstract: Mutations in the homeobox transcription factor PITX2 result in Axenfeld Rieger syndrome which is associated with anterior segment dysgenesis and an increased risk of glaucoma. To understand the pathogenesis of the defects resulting from PITX2 mutations, it is essential to know the normal functions of PITX2 and its interaction with the network of proteins in the eye. Yeast two-hybrid screening was performed using a cDNA library from human trabecular meshwork primary cell line to detect novel PITX2 interacting proteins and study their role in ARS pathogenesis. After screening of ~1X106 clones, one putative interacting protein was identified named PRKC apoptosis WT1 regulator (PAWR). This interaction was further confirmed by retransformation assay in yeast cells as well as coimmunoprecipitation in ocular cells and nickel pulldown assay in vitro. PAWR is reportedly a pro-apoptotic protein capable of selectively inducing apoptosis primarily in cancer cells. Our analysis indicates that the homeodomain and the adjacent inhibitory domain in PITX2 interact with the C terminal leucine zipper domain of PAWR. Endogenous PAWR and PITX2 were found to be located in the nucleus of ocular cells and to colocalize in the mesenchyme of the iridocorneal angle of the developing mouse eye, consistent with a role in the development of the anterior segment of the eye. PAWR was also found to inhibit PITX2 transcriptional activity in ocular cells. These data suggest PAWR is a novel PITX2 interacting protein that regulates PITX2 activity in ocular cells. This information sheds new light in understanding ARS and associated glaucoma pathogenesis.
Abstract: PURPOSE: Defects in cytochrome P450 1B1 (CYP1B1) cause primary congenital glaucoma. However, defects in the gene have also been reported in primary open-angle glaucoma (POAG). Since POAG is primarily a complex disease, we examined the potential of coding single nucleotide polymorphisms (cSNPs) in the gene for association with the disease. METHODS: Five coding SNPs - c.514 C>G (Arg48Gly), c.727 G>T (Ala119Ser), c.1666 C>G (Leu432Val), c.1719 C>T (Asp449Asp), and c.1730 A>G (Asn453Ser) - were genotyped in 264 unrelated POAG patients and 95 controls. In addition, 542 normal individuals selected from various ethnic groups representing the Indian population were also genotyped for these cSNPs. The patterns of linkage disequilibrium between the SNPs and haplotype variations for comparison between POAG patients and controls as well as different ethnic groups of the Indian population were determined using Haploview. Allelic variants of Leu432Val were cloned by site-directed mutagenesis of normal CYP1B1 cDNA, which were used for transfection of retinal pigment epithelium (RPE) cells. The generation of reactive oxygen species (ROS) was quantified by measuring fluorescence emission by degradation of CM-H2DCFDA using a fluoremeter. RESULTS: The c.1666G allele of the Leu432Val in CYP1B1 showed a statistically significant higher representation among POAG patients compared to controls (p=0.0001; Odds ratio=6.027; 95% CI: 3.863-9.401) suggesting it to be a potential risk allele toward disease predisposition. Analysis of genotype frequencies of the polymorphism between the two groups demonstrated GG as a potential risk genotype (p=0.0001; Odds ratio=15.505; 95% CI: 5.529-43.474) for the disease. CYP1B1 Val432 was estimated to generate higher ROS in RPE cells compared to its allelic variant (Leu432; p=0.0245 for 15 min and p=0.0197 for 30 min). Comparison of haplotype diversities revealed CGGTA as the risk haplotype for the disease (p=0.0001, by Fisher's exact test). CONCLUSIONS: We report CYP1B1 c.1666G (Val432) as a susceptible allele for POAG and CGGTA as the risk haplotype for the disease. Higher ROS generation by Val432 in CYP1B1 might lead to apoptotic change that leads to glaucoma. Remarkable variation of the cSNPs observed among ethnic groups of India could provide insight for future epidemiological studies on POAG in these population groups.
Abstract: Analyses of frequency profiles of markers on disease or drug-response related genes in diverse populations are important for the dissection of common diseases. We report the results of analyses of data on 405 SNPs from 75 such genes and a 5.2 Mb chromosome, 22 genomic region in 1871 individuals from diverse 55 endogamous Indian populations. These include 32 large (>10 million individuals) and 23 isolated populations, representing a large fraction of the people of India. We observe high levels of genetic divergence between groups of populations that cluster largely on the basis of ethnicity and language. Indian populations not only overlap with the diversity of HapMap populations, but also contain population groups that are genetically distinct. These data and results are useful for addressing stratification and study design issues in complex traits especially for heterogeneous populations.
Abstract: BACKGROUND: We investigated the molecular basis of primary open-angle glaucoma (POAG) using Opticin (OPTC) as a candidate gene on the basis of its expression in the trabecular meshwork cells involved in the disease pathogenesis. Two hundred POAG patients and 100 controls were enrolled in this study. The coding sequence of OPTC was amplified by PCR from genomic DNA of POAG patients, followed by SSCP, DHPLC and DNA sequencing. Subsequent bioinformatic analysis, site-directed mutagenesis, quantitative RT-PCR and western blot experiments were performed to address the functional significance of a 'silent' change in the OPTC coding region while screening for mutations in POAG patients. RESULTS: We detected two missense (p.Glu66Gly & p.Ile89Thr) and one silent change (p.Phe162Phe; c.602 C>T) that was present in 3 different patients but in none of the 100 controls screened. The mutant (c.602T) mRNA was predicted to have remarkably different secondary structure compared to the wild-type transcript by in silico approaches. Subsequent wet-lab experiments showed lower expression of the gene both at the mRNA and protein levels. CONCLUSION: Our study suggests OPTC as a candidate gene for POAG. Further, it highlights the importance of investigating the 'silent' variations for functional implication that might not be apparent from only in silico analysis.
Abstract: OBJECTIVE: To identify and evaluate MYOC variant alleles among patients with primary open-angle glaucoma (POAG) and age-matched control subjects in an Indian population. METHODS: Three hundred fifteen patients with POAG and 100 unrelated control subjects from the same ethnic background were enrolled in the study. The coding sequence of MYOC was amplified by polymerase chain reaction using genomic DNA, followed by sequencing of the polymerase chain reaction products. Four single nucleotide polymorphisms were genotyped in different Indian subpopulations comprising 1466 individuals using SEQUENOM's homogeneous MassEXTEND assay. RESULTS: One novel mutation (Gly399Asp), 6 reported mutations (Gln48His, Thr256Met, Thr353Ile, Gln368Stop, Pro370Leu, and Ala427Thr), and 6 single nucleotide polymorphisms were identified in MYOC. Ala427Thr was identified in a patient with POAG and Parkinson disease. Four single nucleotide polymorphisms genotyped in control subjects were highly heterozygous and displayed a similar pattern of linkage disequilibrium among all linguistic groups. CONCLUSIONS: MYOC mutations account for 2.2% of POAG cases. The Gln368Stop mutation (common among persons of the white race) found in 2 families does not seem to be of white race origin. Identification of a MYOC mutation (Ala427Thr) in a patient with POAG and Parkinson disease is interesting with respect to reported interaction of myocilin with synucleins. CLINICAL RELEVANCE: Studying the genetics of POAG is helpful for preclinical identification and for better disease management.
Abstract: Glaucoma is a heterogeneous group of optic neuropathies, with a
complex genetic basis. Among the established candidate genes known to be
involved in the disease, myocilin has been reported to cause a small percentage
of adult onset and a major percentage of juvenile onset cases of glaucoma.
Mutations in different regions of the gene have been found to be associated with
a wide spectrum of glaucoma phenotypes. The gene has also been implicated in
primary congenital glaucoma as well as in digenic cases of the disease. The article
intends to explore the functional aspects of the protein in normal trabecular
meshwork (TM) and molecular basis of TM cell dysfunction as a result of mutation
in the protein as revealed from the current studies. We also report occurrence in
an Indian POAG family a mutation (Q368X), common among Caucasians, and
the studies in progress on myocilin-related genes that could serve as candidates
for glaucoma.
Abstract: PURPOSE: CYP1B1, a member of the cytochrome P450 superfamily of enzymes, has been implicated in primary congenital glaucoma (PCG). Recent studies suggest a role of CYP1B1 in primary open-angle glaucoma (POAG) as a modifier locus. The purpose of the study was to further investigate the potential role of CYP1B1 in POAG patients. METHODS: Two hundred unrelated Indian POAG patients and 100 unrelated ethnically matched controls were enrolled in this study. The coding sequence of CYP1B1 was amplified by polymerase chain reaction (PCR) from genomic DNA, followed by direct DNA sequencing to identify the allelic variants. RESULTS: Six mutations were identified in nine patients and none of the controls examined. One novel mutation (R523T) was detected in the homozygous condition while three reported (W57C, E229K, and R368H) and two novel mutations (S515L and D530G) were found in the heterozygous state. The homozygous mutation of a conserved residue, detected in a familial juvenile onset POAG (JOAG) patient (lacking MYOC or OPTN mutations), cosegregated with the disease locus in an autosomal recessive mode of transmission. All the novel mutations (R523T, S515L and D530G) were detected in a region of CYP1B1 that did not harbor any of the 34 point mutations implicated in PCG. In addition, six previously reported (p.R48G, p.A119S, p.V432L, p.D449D, p.N453S, and 372-12C>T in intron 1) and four novel (p.V395V, p.P400P, p.V518A, and c.2016C>G in the 3'-UTR) single nucleotide polymorphism (SNPs) were also observed in POAG patients and controls. CONCLUSIONS: Our observation suggests that on rare occasions CYP1B1 may be primarily responsible for JOAG by possible monogenic association, and this observation emphasizes the importance of screening for mutation in this gene of JOAG patients that are determined not to harbor mutations in previously characterized candidate genes and loci for POAG.
Abstract: PURPOSE: Myocilin gene defects have been originally implicated in primary open angle glaucoma (POAG). Based on multiple reports for the occurrence of Gln48His mutation (c.144G>T; HGMD accession number CM023962) among Indian POAG patients, we wanted to estimate the prevalence of this mutation in primary open angle and primary congenital glaucoma (PCG) in India and assess its role in the causation of the disease. METHODS: Two hundred cases each of POAG and PCG were screened for the Gln48His mutation by RFLP (AccI) analysis of the PCR amplicons followed by confirmation of the c.144G>T change by direct sequencing. RESULTS: The Gln48His mutation was detected in 9 different glaucoma patients (four POAG and five PCG). While all four POAG cases were heterozygous, among PCG cases, four were heterozygous and one exhibited homozygous genotype for the mutation. One each of POAG and PCG patients was detected to be heterozygous for CYP1B1 mutation (c.1656C>T, Pro437Leu) and (c.1449G>A, Arg368His), respectively. None of the 300 ethnically matched normal controls contained either the MYOC or CYP1B1 mutation(s). CONCLUSIONS: The myocilin mutation, Gln48His, represents an allelic condition involving a spectrum of glaucoma phenotypes in Indian populations, and could be a potential risk factor towards disease predisposition among patients of Indian origin. The study also highlights the role of MYOC as a candidate in different glaucoma subtypes that needs to be investigated further.
Abstract: Indian population, comprising of more than a billion people, consists of 4693 communities with several thousands of endogamous groups, 325 functioning languages and 25 scripts. To address the questions related to ethnic diversity, migrations, founder populations, predisposition to complex disorders or pharmacogenomics, one needs to understand the diversity and relatedness at the genetic level in such a diverse population. In this backdrop, six constituent laboratories of the Council of Scientific and Industrial Research (CSIR), with funding from the Government of India, initiated a network program on predictive medicine using repeats and single nucleotide polymorphisms. The Indian Genome Variation (IGV) consortium aims to provide data on validated SNPs and repeats, both novel and reported, along with gene duplications, in over a thousand genes, in 15,000 individuals drawn from Indian subpopulations. These genes have been selected on the basis of their relevance as functional and positional candidates in many common diseases including genes relevant to pharmacogenomics. This is the first large-scale comprehensive study of the structure of the Indian population with wide-reaching implications. A comprehensive platform for Indian Genome Variation (IGV) data management, analysis and creation of IGVdb portal has also been developed. The samples are being collected following ethical guidelines of Indian Council of Medical Research (ICMR) and Department of Biotechnology (DBT), India. This paper reveals the structure of the IGV project highlighting its various aspects like genesis, objectives, strategies for selection of genes, identification of the Indian subpopulations, collection of samples and discovery and validation of genetic markers, data analysis and monitoring as well as the project's data release policy.
Abstract: PURPOSE: To evaluate the role of the optineurin gene (OPTN) in Indian primary open angle glaucoma (POAG) patients from different parts of the country. METHODS: Two hundred patients with POAG and 200 ethnically matched normal controls were recruited from various parts of India for the study. The entire coding region of OPTN along with the intron-exon boundaries were screened by PCR and single strand conformation polymorphism (SSCP) followed by direct sequencing. A rapid screening method was developed for some of the observed variants by denaturing high performance liquid chromatography (dHPLC). Four variants were also confirmed by digesting the amplicon with appropriate restriction enzymes. RESULTS: Seven nucleotide changes were observed in OPTN of which one was a putative mutation in exon 16 (Arg545Gln) that was observed in six POAG patients and not in the controls (p<0.05). The remaining variants comprised four single nucleotide polymorphisms (SNPs) in the coding region (Thr34Thr, Met98Lys, Arg149Arg, and Asn303Lys) and two in intron 6 (879-10G>A and 879-5C>T). But frequencies of the minor allele were not significantly different among the patients and controls. The Met98Lys variant that was identified to be a potential risk factor for NTG and POAG in some Asian populations and also for modulating IOP in Caucasian populations, did not exhibit any significant association to the disease phenotype. CONCLUSIONS: Despite a putative mutation (Arg545Gln) in some patients, the present study does not suggest a significant involvement of OPTN in POAG patients of Indian origin.
Abstract: Glaucoma is the second largest blinding disorder, after cataract, affecting about 67 million people worldwide. In India about 1.5 million people are blind due to glaucoma. Primary open angle glaucoma is the major sub-type of glaucoma affecting all ages and is genetically complex. Myocilin and optineurin are two different genes that have been implicated for primary open angle glaucoma. This review is focused on the studies being conducted in India on primary open angle glaucoma to identify the molecular defects and new directions undertaken using bioinformatic approaches towards a better understanding of the disease.
Abstract: Glaucoma represents a heterogeneous group of optic neuropathies, with different genetic bases. It can affect all ages generally with a rise in intra-ocular pressure. Three major types of glaucoma have been reported: primary open angle glaucoma (POAG), primary acute closed angle glaucoma (PACG) and primary congenital glaucoma (PCG), as well as a few others associated with developmental abnormalities. In recent years impressive progress has been made in the molecular genetic studies of POAG and PCG. These include the discovery of three genes--Myocilin, Optineurin and CYP1B1--defects in which results in Mendelian transmission of glaucoma. Identification of single nucleotide polymorphisms in multiple other genes that are associated with glaucoma and alteration of drug sensitivity are enriching our knowledge regarding the complex nature of the disease. This review attempts to present the recent progress made in the molecular genetics of glaucoma.
Abstract: PURPOSE: Glaucoma is the second leading cause of blindness worldwide after cataract. Defects in the myocilin gene (MYOC) have been shown to be associated with Primary Open Angle Glaucoma (POAG), the most common form of the disease, especially in its juvenile form. Most of the reported mutations in MYOC are in POAG patients of Caucasian origin. A few studies have been reported on Asian patients (such as Chinese, Japanese, and Koreans) but none from the Indian subcontinent. The purpose of this study was to investigate the molecular basis of POAG among Indians, using MYOC as the candidate gene, and broaden our understanding on the pathogenesis caused by MYOC. METHODS: Fifty-six unrelated POAG patients, comprising 39 sporadic cases and 17 patients having familial history for POAG were enrolled in this study. The coding sequence of the gene was amplified by polymerase chain reaction (PCR) using genomic DNA from 30 POAG patients, followed by sequencing of the PCR products. Nucleotide changes were detected by identifying double peaks in the chromatogram due to heterozygosity and pairwise BLAST analysis of the sequence output data against the normal copy of the MYOC cDNA. Alteration of restriction sites due to nucleotide changes was identified. Twenty-six patients (not sequenced) and controls were screened for nucleotide changes by allele specific restriction digestion of the PCR products followed by separation of the digested DNA fragments by polyacrylamide gel electrophoresis. RESULTS: From a pool of 56 unrelated POAG patients two mutations were identified. A putative novel mutation (144 G->T; Gln48His) of a conserved amino acid was detected in the exon 1 of MYOC from three unrelated patients but none in the 51 control samples examined. The other mutation (1109 C->T; Pro370Leu), located in exon 3 and detected in a family affected with POAG, cosegregated with the disease and was not present in control samples. Two single nucleotide polymorphisms (SNPs) were identified; one in the promoter region (-83 G->A) and the other in the coding sequence (227 G->A; Arg76Lys). These two SNPs were found to be highly heterozygous both in the control (0.480) and the patient (0.477) populations, and were observed to be in linkage disequilibrium. CONCLUSIONS: The presence of a novel non-conservative change in codon 48 of MYOC in 3 POAG patients, but none in the healthy controls, suggests a causal association of the mutation with the disease, either singly or in combination with other genetic loci. The other point mutation (Pro370Leu) detected in the members of an affected POAG family represents a hotspot of mutation in the gene. Two identified SNPs (-83 G->A and 227 G->A) are not associated with the disease phenotype but could be used as highly informative markers in POAG affected families to determine any causal association of MYOC with the disease, and for identification of presymptomatic carriers in the family, where applicable. A comparison of our data with other studies revealed that these two polymorphisms are in complete linkage disequilibrium among Asians, but not among other ethnic groups studied so far.
Abstract: PURPOSE: Glaucoma is a complex neurodegenerative disorder of the eye. Primary Open Angle Glaucoma (POAG) is the most common type, accounting for over half of the total cases. Recently, a significant difference in the distribution of the codon 72 polymorphism of the tumor suppressor gene p53 between control subjects and POAG patients of Chinese origin (p=0.00782) was demonstrated. The proline residue at codon 72 of the p53 gene was significantly over represented in the POAG patients relative to healthy controls. The purpose of this study was to investigate whether the reported association between the p53 polymorphism and POAG is a common phenomenon irrespective of geographical location or ethnicity of the population. METHODS: Sixty seven unrelated POAG patients, ranging from 10-65 years of age (mean+/-SD of 41.16+/-18.52 years), and 112 control subjects having a similar age range of 18-63 years (mean+/-SD of 36.64+/-14.65 years) were enrolled in this study. A region of the p53 gene encompassing two polymorphic sites, a 16 bp duplication in intron 3 and a BstU I RFLP in exon 4, were amplified by polymerase chain reaction from Indian POAG patients and normal healthy controls. A single base change (G to C) in codon 72 alters the amino acid residue from arginine to proline and removes the polymorphic BstU I site mentioned above. The amplified DNA fragments were digested with the restriction enzyme and the digestion patterns of the fragments were used to identify the alleles for both the polymorphic sites. RESULTS: No significant association between p53 alleles and Indian POAG patients were observed by analyzing either codon 72 polymorphism (p=0.5627) or the intronic 16 bp duplication polymorphism (p=0.059). Haplotype analysis, reported to be a better predictor of association of the p53 gene with different types of cancer, was also performed and no association of any haplotype was detected with POAG (p=0.1831). CONCLUSIONS: Association between the p53 gene encoding for proline at codon 72 and POAG presumably exists in some ethnic populations but cannot be used as a predictor for the role of the gene as a common regulator of cell death of retinal ganglions leading to POAG.
