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Sarcoglycanopathy is the most frequent form of autosomal recessive limb-girdle muscular dystrophies caused by mutations in SGCB gene encoding beta-sarcoglycan proteins. In this study, we describe a shared, common haplotype co-segregating in 14 sarcoglycanopathy cases from 13 unrelated families from south Indian region with the likely pathogenic homozygous mutation c.544T>G (p.Thr182Pro) in SGCB. Haplotype was reconstructed based on 5 polymorphic markers surrounding the c.544T>G mutation in the cases and related family members as well as 150 unrelated controls from general Indian populations using PLINK1.9. We identified haplotype H1= G, A, T, G, G, T at a significantly higher frequency in cases compared to related controls and general Indian population. Upon segregation analysis within the family pedigrees, H1 is observed to co-segregate with c.544T>G in a homozygous state in all the pedigrees of cases except one indicating a probable event of founder effect. Furthermore, Identical-by-descent and inbreeding coefficient analysis revealed relatedness among 33 new pairs of seemingly unrelated individuals from sarcoglycanopathy cohort and a higher proportion of homozygous markers, thereby indicating towards common ancestry. Since all these patients are from the south Indian region, we suggest this region to be a primary target of mutation screening in patients diagnosed with sarcoglycanopathy.

Morphocumin coordinated RuII–p-cymene complex acts as a type-I photosensitizer to inhibit growth of CSC enriched 3D-spheroids of oral squamous carcinoma downregulating stemness genes (cMYC, SOX2, OCT4) with no systemic toxicity to zebrafish embryos in the dark.

Eight new ruthenium(II) complexes of N,N-chelating pyrazolylbenzimidazole ligands of the general formula [RuII(p-cym)(L)X]+ [where the ligand L is 2-(1H-pyrazol-1-yl)-1H-benzo[d]imidazole (L1) substituted at the 4 position of the pyrazole ring by Cl (L2), Br (L3), or I (L4) and X = Cl- and I-] were synthesized and characterized using various analytical techniques. Complexes 1 and 3 were also characterized by single-crystal X-ray crystallography, and they crystallized as a monoclinic crystal system in space groups P21/n and P21/c, respectively. The complexes display good solution stability at physiological pH 7.4. The iodido-coordinated pyrazolylbenzimidazole ruthenium(II) p-cymene complexes (2, 4, 6, and 8) are more resistant toward hydrolysis and have less tendency to form monoaquated complexes in comparison to their chlorido analogues (1, 3, 5, and 7). The halido-substituted 2-(1H-pyrazol-1-yl)-1H-benzo[d]imidazole ligands, designed as organic-directing molecules, inhibit vascular endothelial growth factor receptor 2 (VEGFR2) phosphorylation. In addition, the ruthenium(II) complexes display a potential to bind to DNA bases. The cytotoxicity profile of the complexes (IC50 ca. 9-12 μM for 4-8) against the triple-negative breast cancer cells (MDA-MB-231) show that most of the complexes are efficient. The lipophilicity and cellular accumulation data of the complexes show a good correlation with the cytotoxicity profile of 1-8. The representative complexes 3 and 7 demonstrate the capability of arresting the cell cycle in the G2/M phase and induce apoptosis. The inhibition of VEGFR2 phosphorylation with the representative ligands L2 and L4 and the corresponding metal complexes 3 and 7 in vitro shows that the organic-directing ligands and their complexes inhibit VEGFR2 phosphorylation. Besides, L2, L4, 3, and 7 inhibit the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and proto-oncogene tyrosine-protein kinase (Src), capable of acting downstream of VEGFR2 as well as independently. Compounds L2, L4, 3, and 7 have a lesser effect on ERK1/2 and more prominently affect Src phosphorylation. We extended the study for L2 and 3 in the Tg(fli1:gfp) zebrafish model and found that L2 is more effective in vivo compared to 3 in inhibiting angiogenesis.

Background: Stem cell-like properties in cancer cells are found to be responsible for its aggressive behaviour. However, this has not been studied with respect to the bimodal NOTCH-pathway-activity status, found in oral cancer. Methods:Oral-SLCCs were enriched in 3D-spheroids. Constitutively-active and inactive status of NOTCH-pathway was achieved by genetic or pharmacological approaches. RNA sequencing and real-time PCR was performed for gene expression studies. in vitro cytotoxicity assessments were performed by AlamarBlue assay and in vivoeffects were studied by xenograft growth in zebrafish embryo. The t tests were performed to estimate statistical significance of the study. Results: Here, we have demonstrated the stochastic plasticity on NOTCH-activity axis; maintaining both NOTCH-active and inactive states of oral stem-like cancer cells (Oral-SLCCs). While Oral-SLCCs with inactive NOTCH-pathway status showed higher proliferation and aggressive tumor growth, the Cisplatin refraction was associated with active-status of NOTCH-pathway; suggesting the crucial role of plasticity on NOTCH-axis. The differentially expressed genes between NOTCH-pathway active and inactive clones clearly suggested the upregulation of JAK-STAT signaling in subset of Oral-SLCCs with lower NOTCH-pathway activity status. Confirming the function; the 3D-spheroids generated by oral-SLCCs with lower NOTCH-activity-status displayed significantly higher sensitivity to JAK-selective drugs, Ruxolitinib or Tofacitinib and siRNA mediated downregulation of tested partners STAT 3 and 4. Therefore, we adopted the strategy of synthetic lethality, where Oral-SLCCs were reprogrammed to maintain the inactive status of NOTCH-pathway by exposure to γ-secretase inhibitors, LY411575 or RO4929097 followed by targeting with JAK-inhibitors, Ruxolitinib or Tofacitinib. This resulted in a very significant inhibition in viability of 3D-spheroids as well as xenograft formation in Zebrafish embryos; whereas inhibition of either of these pathway alone were largely ineffective. Conclusion: We have demonstrated the stochastic cellular plasticity on NOTCH-activity axis. Study revealed for the first time that NOTCH-HES and JAK-STAT pathways may act as synthetic lethal pair, and as novel targets against diverse states of stemness in oral cancer. Therefore, we have provided the rational for sequential combination of NOTCH and JAK inhibitors as possible therapeutic strategy against aggressive oral cancer.

Purpose PACG is one of the leading causes of blindness where lens thickness is a major risk factor for narrow-angle individuals. To our knowledge, no literature has been reported on candidate gene for lens thickness as a quantitative trait (QT). Here, we performed a genome-wide association analysis on lens thickness in the narrow-angle individuals. Materials and Methods We conducted a genome-wide association study (GWAS) in the narrow angle individuals to investigate comprehensive genetic insights on lens thickness. Results In QT-GWAS, we identified 145 genome-wide suggestive significant loci in the discovery cohort. Subsequently, we observed 13 SNPs that showed statistical significance around the region of PTRRM. Regional association analysis for top significant genotyped variants identified PTPRM as the most likely candidate for increased LT. Integrative bioinformatic analyses confirmed that the associated genomic region has potential regulatory roles for modulating transcription as enhancers. In the replication cohort, the sentinel genotype SNP was further associated significantly (P-value =0.000448) with high LT individuals. In both cohorts, the T allele of rs1941137 in the PTPRM gene indicates as a risk allele for the increased LT. Conclusion In this study, we discovered evidence of a genomic association between chromosomal areas around the PTPRM and increased lens thickness, resulting in a narrow angle. The regulatory components corresponding to PTPRM variations might have a role in the thicker lens. We report that the genomic region near PTPRM, a gene of potential interest, is associated with increased lens thickness.

Correction for ‘A photoactive lysosome targeting RuII complex downregulates stemness genes in oral squamous cell carcinoma’ by Souryadip Roy et al., Inorg. Chem. Front., 2022, 9, 5840–5852, https://doi.org/10.1039/D2QI01079H.

BackgroundSarcoglycanopathies (SG) is the most frequent form of autosomal recessive limb-girdle muscular dystrophies (LGMD) leading to progressive muscle wasting and weakness, predominantly characterized by limb-girdle weakness. LGMDR4 is caused by mutations in SGCB encoding for the beta-sarcoglycan proteins. In this study, we describe a shared, common haplotype cosegregating in 14 SG cases from 13 unrelated families with the likely pathogenic homozygous mutation c.544A>C (p.Thr182Pro) in SGCB.MethodsThe genotypes of five selected markers (rs10009426, rs6824707, rs2271046, rs35414474 and rs17611952) surrounding the c.544A>C (p.Thr182Pro) were extracted from the variant call format (VCF) generated from whole-exome sequencing (WES) of 14 cases and 14 related family members as controls. The linkage data file was constructed and linkage disequilibrium (LD) plots were generated using HaploView to visualize patterns of LD. Further, haplotype reconstructions based on the 6 markers were conducted using PLINK1.9. using the expectation-maximization (EM) algorithm, an iterative method to find maximum likelihood. Subsequently, the R programming language was used to determine and compare plots of the haplotype frequencies and percentages for both groups to infer the risk haplotypes.ResultsFour strong LD blocks were identified in control group: rs10009426 to rs6824707 (0.27□kb), rs6824707 to rs2271046 (41.6 kb), rs10009426 to rs2271046 (41.8 kb) and rs35414474 to rs17611952 (0.17 kb) which were absent in the case group. Similarly, a total of nine haplotypes were estimated in cases and controls of which haplotype H1= G, A, T, G, G, T showed significant statistical difference in the frequency between cases and controls. H1 is also observed to cosegregate with c.544A>C (p.Thr182Pro) in the pedigrees of all the cases.ConclusionThe identification of c.544A>C (p.Thr182Pro) mutation in 14 cases from India indicates a probable event of founder effect. Further, the H1 haplotype, cosegregating with this mutation, convincingly sheds light on the recent developments in population genetics allowing insights into demographic and population history. This haplotype can also be used as a genetic marker to screen individuals with genetic susceptibility as carriers and provide genetically informed risk stratification and management in the prevention of SG.

The clinico-genetic architecture of sarcoglycanopathies in Indian patients is reported only as short series. In the present study, we aimed to investigate the clinical picture, genetic basis, and disease progression of patients genetically confirmed to have sarcoglycanopathy. Next-generation sequencing was performed in 68 probands with suspected sarcoglycanopathy. A total of 35 different variants were detected in the sarcoglycan genes in 68 probands (M = 37; age range, 5-50 years). Consanguinity was present in 44 families. Thirty-two variants are predicted to be pathogenic/likely pathogenic, among which 25 (78.13%) are reported, and 7 (21.87%) are novel. The clinical diagnosis was confirmed in a total of 64 (94.12%) probands with biallelic variations [SGCA(n=18); SGCB(n=34); SGCG(n=7); SGCD(n=5)]. The most common mutation was c.544A C (p.Thr182Pro) in SGCB, and detected in 20 patients (29.42%). The majority of pathogenic mutations are homozygous (n = 30; 93.75%). Variants in 4 cases are of uncertain significance. Thirty-three patients lost ambulation at a mean age of 15.12 ± 9.47 years, after 7.76 ± 5.95 years into the illness. Only 2 patients had cardiac symptoms, and one had respiratory muscle involvement. The results from this study suggest that mutations in SGCB are most common, followed by SGCA, SGCG, and SGCD. The novel variations identified in this study expand the mutational spectrum of sarcoglycanopathies. To the best of our knowledge, this is the first study from India to describe a large cohort of genetically confirmed patients with sarcoglycanopathy and report its disease progression.

Glaucoma is a heterogeneous group of optic neuropathies and is one of the leading causes of irreversible blindness worldwide. Primary angle closure glaucoma (PACG) is a major subtype, prevalent mostly in east and south Asia, where occludable anterior chamber angle is considered as a primary risk factor, which in turn could be responsible for high intraocular pressure (IOP) and subsequent neurodegeneration of retinal ganglion cells. Clinically, IOP is considered as a major risk factor for glaucoma and viewed as an important endophenotype to promote the disease severity. To investigate the comprehensive genomic insights, we conducted a genomewide association study (GWAS) on IOP in individuals with occludable angle (

Primary angle closure glaucoma (PACG) is one of the major causes of blindness worldwide. The underlying genetic aetiology is complex in nature and molecular mechanism remains elusive. Here, we identify genomic alterations using haplotype-based genome-wide association study in 148 PACG and 92 anatomically predisposed non-glaucomatous individuals. Logistic regression was performed on each common haplotype (within blocks of 3-8 SNPs) across the genotype and a total of 59 SNPs were found below genome wide suggestive threshold (

The PITX2 'homeobox' and FOXC1 and FOXC2 'forkhead box' transcription factors are critical for eye development and cause human ocular diseases when mutated. We have identified biochemical and genetic links between these transcription factors and a transcriptional regulator protein PRKC apoptosis Wilms' tumor 1 regulator (PAWR) that we propose to functionally connect all these proteins in a common pathway critically involved in eye development. We discovered all binary physical interactions between FOXC1, PITX2, FOXC2 and PAWR. Importantly, PAWR modulates the abilities of PITX2, FOXC1 and FOXC2 to activate their genetic targets. Together with either FOXC1 or FOXC2, PAWR increases PITX2 activity. PAWR reduces PITX2 activity in the absence of FOXC1 or FOXC2. At the same time, PAWR also exerts different regulatory effects on different FOXC target sites. Furthermore, morpholino knockdown of pitx2, foxc1 and pawr in zebrafish indicate that PAWR, FOXC1 and PITX2 genetically interact, and are in the same developmental pathway. These data for the first time tie PITX2, FOXC1, FOXC2 and PAWR into a common regulatory pathway. We have therefore identified a functional link between three transcription factors, modulated by PAWR, which we propose underlies the similar ocular phenotypes and glaucoma pathology caused by mutations of these genes.

Mutations in the homeobox transcription factor PITX2 result in Axenfeld-Rieger syndrome (ARS), 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 a human trabecular meshwork primary cell line to detect novel PITX2-interacting proteins and study their role in ARS pathogenesis. After screening of approximately 1 x 10(6) 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 co-immunoprecipitation in ocular cells and nickel pulldown assay in vitro. PAWR is reportedly a proapoptotic 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 co-localize 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.

Glaucoma is the second largest cause of blindness worldwide, affecting almost 60 million people (Quigley and Broman 2006). Defects in MYOC and CYP1B1 have been implicated in primary open angle glaucoma (POAG) and primary congenital glaucoma (PCG), respectively. Variants in both the genes have been detected in both the diseases indicating a higher complexity in the pathogenesis of the disease. In this context, we present here the case of a POAG patient who is found to be a compound heterozygote for CYP1B1 mutations and a homozygote for MYOC variant. None of the three nonsynonymous changes were found in 170-unrelated controls with matched age and ethnicity. Analysis of the family members reveal that proband’s younger sister, not affected, was homozygous for the MYOC variant but lacked one variant in CYP1B1. This is in contrast to other reports that showed implication of CYP1B1 in POAG. This case is unique, since both parents of the proband do not show any symptom of POAG, despite of having one defective allele for both MYOC and CYP1B1.

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.

Purpose Capturing organ-specific phenomes in genetic diseases is an uphill task for the eye as it comprises tissue types derived from all three germinal layers. We attempted to deconstruct genetic eye diseases (GEDs) into primary phenotypic features, to understand the complex genome-phenome relationship in GEDs. Methods Using phenotype, molecular basis, and gene description features in OMIM as a primary resource, we analyzed gene-phenotype information. All ocular and systemic phenotypes were categorized and ranked based on occurrence. Clustering was performed on shared ocular features to identify genetic interactions and the largest cluster of each phenotype was used for functional analyses. Results We collected 527 GEDs associated with 440 unique protein-coding genes. We indexed 787 ocular and 3094 systemic features, for an average of 2.17 ocular and 8.14 systemic features, respectively, per disease unit. The most common ocular features included nystagmus, hypertelorism, and myopia, while neurological and skeletal are the most common systemic groups associated with GEDs. Functional analyses revealed pathways relevant to GEDs (e.g., extracellular matrix organization in ONH3 [glaucoma]) and protein metabolism in EOM35 (nystagmus) phenotype clusters. Conclusions Our work imparts a structure in dissecting GEDs into unique phenotypes to study the relationship between genes and diseases involving the eye.

Purpose Mutations in the homeobox transcription factor paired-like homeodomain transcription factor 2 (PITX2) cause Axenfeld–Reiger syndrome (ARS), which is associated with anterior segment dysgenesis (ASD) and glaucoma. To understand ARS pathogenesis, it is essential to know the normal functions of PITX2 and the proteins with which PITX2 interacts in the eye. Therefore, we used a unique cDNA library that we created from human trabecular meshwork (TM) primary cells to discover PITX2-interacting proteins (PIPs). Methods A human TM cDNA library was created from primary cells in the ProQuest Two-Hybrid prey vector: pEXP-AD502. Human PITX2A and PITX2C isoforms were used independently as “bait” to identify novel PIPs. A total of 1.25×106 clones were screened by yeast two-hybrid (Y2H) analyses. PIPs obtained from each Y2H experiment were confirmed by yeast retransformation and mammalian co-immunoprecipitation assays. Results EGF-containing fibulin-like extracellular matrix protein 2 (EFEMP2) was identified by both PITX2A and PITX2C isoforms as a novel PIP from Y2H analyses. EFEMP2 is 443 amino acids long with six epidermal growth factor (EGF)-like modules and one fibulin-like module. The PITX2-interaction domain in EFEMP2 lies between the second EGF-like module and the COOH-terminal fibulin-like module. Co-immunoprecipitation assays in COS-7 cells confirmed the interaction between PITX2 and EFEMP2. Conclusions We discovered EFEMP2 as a novel PITX2-interacting protein. Further, our cDNA library made from human TM primary cells is a unique and effective resource to identify novel interacting proteins for glaucoma and ASD candidates. This resource could be used both for discovery and validation of interactomes identified from in silico analysis.

Glaucoma refers to a heterogeneous group of optic neuropathies with a complex genetic basis. Glaucoma is also the ‘silent thief of sight’ since half of affected individuals are unaware of having glaucoma due to lack of symptoms. Juvenile glaucoma and congenital glaucoma (CG) are two major subtypes of glaucoma. As the names suggest, both CG and juvenile glaucoma have an early age of onset and more severe phenotype than the adult-onset form. Significant progress has been made in past few years in molecular genetic studies of juvenile glaucoma and CG. Myocilin (MYOC) and Cytochrome P4501B1 (CYP1B1) have been discovered, initially as causal candidate genes for juvenile glaucoma and CG, respectively. Later, however, the involvement of CYP1B1 in juvenile glaucoma and MYOC in CG placed juvenile glaucoma and CG as part of a broader disease spectrum. While mutations in a single gene can be causal to juvenile glaucoma or CG, simultaneous mutations of several genes can also be causal to these forms of glaucoma. In this article, we present the recent progress made in molecular genetic studies of juvenile glaucoma and CG and describe the underlying complexities between these forms of glaucoma.

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.

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.

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.

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.

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.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.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 372-12CT in intron 1) and four novel (p.V395V, p.P400P, p.V518A, and c.2016CG in the 3'-UTR) single nucleotide polymorphism (SNPs) were also observed in POAG patients and controls.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.

To evaluate the role of the optineurin gene (OPTN) in Indian primary open angle glaucoma (POAG) patients from different parts of the country.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.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 (p0.05). The remaining variants comprised four single nucleotide polymorphisms (SNPs) in the coding region (Thr34Thr, Met98Lys, Arg149Arg, and Asn303Lys) and two in intron 6 (879-10GA and 879-5CT). 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.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.

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.

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.

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.

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.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.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.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.

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.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.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).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.

Genetic and biochemical mechanisms linking onset or progression of Alzheimer Disease and prion diseases have been lacking and/or controversial, and their etiologies are often considered independent. Here we document a novel, conserved and specific genetic interaction between the proteins that underlie these diseases, amyloid-β precursor protein and prion protein, APP and PRP, respectively. Knockdown of APP and/or PRNP homologs in the zebrafish (appa, appb, prp1, and prp2) produces a dose-dependent phenotype characterized by systemic morphological defects, reduced cell adhesion and CNS cell death. This genetic interaction is surprisingly exclusive in that prp1 genetically interacts with zebrafish appa, but not with appb, and the zebrafish paralog prp2 fails to interact with appa. Intriguingly, appa & appb are largely redundant in early zebrafish development yet their abilities to rescue CNS cell death are differentially contingent on prp1 abundance. Delivery of human APP or mouse Prnp mRNAs rescue the phenotypes observed in app-prp-depleted zebrafish, highlighting the conserved nature of this interaction. Immunoprecipitation revealed that human APP and PrP(C) proteins can have a physical interaction. Our study reports a unique in vivo interdependence between APP and PRP loss-of-function, detailing a biochemical interaction that considerably expands the hypothesized roles of PRP in Alzheimer Disease.

CYP1B1 has been implicated in primary congenital glaucoma with autosomal recessive mode of inheritance. Mutations in CYP1B1 have also been reported in primary open angle glaucoma (POAG) cases and suggested to act as a modifier of the disease along with Myocilin (MYOC). Earlier reports suggest that over-expression of myocilin leads to POAG pathogenesis. Taken together, we propose a functional interaction between CYP1B1 and myocilin where 17β estradiol acts as a mediator. Therefore, we hypothesize that 17β estradiol can induce MYOC expression through the putative estrogen responsive elements (EREs) located in its promoter and CYP1B1 could manipulate MYOC expression by metabolizing 17β estradiol to 4-hydroxy estradiol, thus preventing it from binding to MYOC promoter. Hence any mutation in CYP1B1 that reduces its 17β estradiol metabolizing activity might lead to MYOC upregulation, which in turn might play a role in glaucoma pathogenesis. It was observed that 17β estradiol is present in Human Trabecular Meshwork cells (HTM) and Retinal Pigment Epithelial cells (RPE) by immunoflouresence and ELISA. Also, the expression of enzymes related to estrogen biosynthesis pathway was observed in both cell lines by RT-PCR. Subsequent evaluation of the EREs in the MYOC promoter by luciferase assay, with dose and time dependent treatment of 17β estradiol, showed that the EREs are indeed active. This observation was further validated by direct binding of estrogen receptors (ER) on EREs in MYOC promoter and subsequent upregulation in MYOC level in HTM cells on 17β estradiol treatment. Interestingly, CYP1B1 mutants with less than 10% enzymatic activity were found to increase the level of endogenous myocilin in HTM cells. Thus the experimental observations are consistent with our proposed hypothesis that mutant CYP1B1, lacking the 17β estradiol metabolizing activity, can cause MYOC upregulation, which might have a potential implication in glaucoma pathogenesis.