Intergenic variants, rs1200114 and rs1200108 are genetically associated along with a decreased ATP1B1 expression in Fuchs Endothelial Corneal Dystrophy.

Fuchs endothelial corneal dystrophy (FECD) is an age-related, bilateral corneal condition, characterized by apoptosis of the terminally differentiated endothelial cells. A genome-wide association study (GWAS) conducted in the European population in 2017, identified a new single nucleotide polymorphism (SNP), rs1200114 in the intergenic region between long intergenic non-protein coding RNA 970 (LINC00970) and ATPase Na+/K+ transporting subunit beta 1 (ATP1B1). The major focus of the current study is to understand the genetic association of this intergenic variant, rs1200114 with FECD in the Indian population. Sanger sequencing followed by statistical analysis indicated a significant difference in the allelic frequency between controls and cases (P = 0.01) with the minor allele 'G' of rs1200114 imparting a 1.64 fold increased risk for the disease. Luciferase reporter assay revealed no significant difference in the luciferase activity between allele 'A' and 'G' of rs1200114. However, quantitative RT-PCR assay revealed lower expression of ATP1B1 in FECD subjects compared with controls (P = 0.007). Therefore, to find whether another nearby SNP imparts regulatory effect, tag SNP association analysis was carried out; which revealed a significant association of another SNP, rs1200108, present in the intergenic region between LINC00970 and ATP1B1 with FECD (P = 0.009). The protective allele 'A' of rs1200108 displayed reduced reporter activity as opposed to the risk allele 'G' (P = 0.014). Furthermore, haplotype 'A-A' of rs1200108 - rs1200114 was present at a higher frequency in control subjects, suggesting it as a protective haplotype. Altogether, this study inferred the genetic association of rs1200114 and rs1200108 along with the decreased expression of ATP1B1 related to FECD pathogenesis in the Indian population.

Quantitative analysis of circulating levels of vimentin, clusterin and fibulin-5 in patients with pseudoexfoliation syndrome and glaucoma.

Pseudoexfoliation (PEX) is a multifactorial age-related disease involving deposition of extracellular fibrillar material on anterior ocular tissues. If left untreated, the early stage of deposition termed as pseudoexfoliation syndrome (PEXS) may lead to the advanced stage of pseudoexfoliation glaucoma (PEXG) characterised by increased intraocular pressure, damage to the optic nerve and subsequent irreversible blindness. The etiology of PEX is complex and identification of novel factors associated with the disease is needed. This study aimed to identify the involvement of vimentin in pseudoexfoliation pathology and assess the levels of vimentin, clusterin and fibulin-5 in the circulating fluids of PEX patients compared to controls. Eighty-seven participants (35 controls, 35 PEXS and 17 PEXG) were enrolled for this case-control study. The expression of vimentin in lens capsules of patients and age-sex matched controls was assayed by qRT-PCR, western blotting and immunohistochemistry. Aqueous humor (AH) vimentin levels and plasma levels of vimentin, clusterin and fibulin-5 were assayed through ELISA. Increased vimentin was observed in the lens capsule of patients compared to controls at mRNA and protein levels. Compared to control (11.5 ± 1.4 ng/ml [±SEM]), the AH vimentin concentrations were significantly higher (ANOVA p = 0.01) in PEXS (16.4 ± 1.8 ng/ml) and PEXG (20.1 ± 2.5 ng/ml). Compared to controls (372.2 ± 15.1 ng/ml), plasma vimentin levels were significantly higher (ANOVA, p < 0.001) in PEXS (449.9 ± 15.7 ng/ml) and PEXG (535.5 ± 25.0 ng/ml). The plasma and aqueous humor levels of vimentin showed a positive correlation of 0.31. The plasma levels of clusterin were 298.9 ± 19.0 µg/ml, 367.8 ± 25.6 µg/ml and 272.9 ± 16.8 µg/ml in controls, PEXS and PEXG, respectively and were significantly higher in PEXS (p = 0.03) compared to control. Plasma fibulin-5 levels were 149 ± 32.2 pg/ml, 187.6 ± 32.3 pg/ml and 203.8 ± 27.3 pg/ml in controls, PEXS and PEXG, respectively and there was no significant difference in its levels between the groups (ANOVA p = 0.49). In conclusion, vimentin is upregulated in PEX affected eyes. Increased vimentin levels in plasma and AH differentiate PEXS and PEXG from controls.

Epigenetic silencing of heat shock protein 70 through DNA hypermethylation in pseudoexfoliation syndrome and glaucoma.

This study is intended to investigate the epigenetic regulation of the most conserved molecular chaperone, HSP70 and its potential role in the pathophysiology of pseudoexfoliation syndrome (PEXS) and glaucoma (PEXG), a protein aggregopathy, contributing significantly to world blindness. Expression levels of HSP70 were significantly decreased in the lens capsule (LC) of PEXS but not in PEXG compared with that in control. Bisulfite sequencing of the LC of the study subjects revealed that the CpG islands (CGIs) located in the exonic region but not in the promoter region of HSP70 displayed hypermethylation only in PEXS individuals. There was a corresponding increase in DNA methyltransferase 3A (DNMT3A) expression in only PEXS individuals suggesting de novo methylation in this stage of the disease condition. On the other hand, peripheral blood of both PEXS and PEXG cases showed hypermethylation in the exonic region when compared with non-PEX controls displaying tissue-specific effects. Further, functional analyses of CGI spanning the exon revealed a decreased gene expression in the presence of methylated in comparison with unmethylated reporter gene vectors. Treatment of human lens epithelial B-3 (HLE B-3) cells with DNMT inhibitor restored the expression of HSP70 following depletion in methylation level at exonic CpG sites. In conclusion, a decreased HSP70 expression correlates with hypermethylation of a CGI of HSP70 in PEXS individuals. The present findings enhance our current understanding of the mechanism underlying HSP70 repression, contributing to the pathogenesis of PEX.

Pseudoexfoliation and Alzheimer's associated CLU risk variant, rs2279590, lies within an enhancer element and regulates CLU, EPHX2 and PTK2B gene expression.

Genetic variants at PTK2B-CLU locus pose as high-risk factors for many age-related disorders. However, the role of these variants in disease progression is less characterized. In this study, we aimed to investigate the functional significance of a clusterin intronic SNP, rs2279590, that has been associated with pseudoexfoliation, Alzheimer's disease (AD) and diabetes. We have previously shown that the alleles at rs2279590 differentially regulate clusterin (CLU) gene expression in lens capsule tissues. This polymorphism resides in an active regulatory region marked by H3K27Ac and DNase I hypersensitive site and is an eQTL for CLU expression. Here, we report the presence of an enhancer element in surrounding region of rs2279590. Deletion of a 115 bp intronic region flanking the rs2279590 variant through CRISPR-Cas9 genome editing in HEK293 cells demonstrated a decreased clusterin gene expression. Electrophoretic mobility shift and chromatin immunoprecipitation assays show that rs2279590 with allele 'A' constitutes a transcription factor binding site for heat shock factor-1 (HSF1) but not with allele 'G'. By binding to allele 'A', HSF1 abrogates the enhancer effect of the locus as validated by reporter assays. Interestingly, rs2279590 locus has a widespread enhancer effect on two nearby genes, protein tyrosine kinase 2 beta (PTK2B) and epoxide hydrolase-2 (EPHX2); both of which have been previously associated with AD as risk factors. To summarize, our study unveils a mechanistic role of the common variant rs2279590 that can affect a variety of aging disorders by regulating the expression of a specific set of genes.

REVIEW: Current understanding of the pathogenesis of Fuchs' endothelial corneal dystrophy.

Fuchs' endothelial corneal dystrophy (FECD) is the most prominent reason for corneal-endothelial transplantations across the globe. The disease pathophysiology manifests through a combination of various genetic and non-heritable factors. This review provides a comprehensive list of known genetic players that cause FECD, and discusses the prominent pathological features that participate in disease progression, such as channel dysfunction, abnormal extracellular matrix deposition, RNA toxicity, oxidative stress, and apoptosis. Although current practices to correct visual acuity involve surgical intervention, this review also discusses the scope of various non-surgical therapeutics to remedy FECD.

Altered unfolded protein response and proteasome impairment in pseudoexfoliation pathogenesis.

Pseudoexfoliation (PEX), an ocular disorder involving deposition of proteinaceous fibrils on the surface of anterior eye tissues, is a major contributing factor to worldwide glaucoma. Excessive production and accumulation of fibrillar materials in PEX could be an indication of proteostasis imbalance. This study aims at investigating the differential expression of various genes involved in unfolded protein response and ubiquitin proteasome pathway in pseudoexfoliation (PEX) patients compared to non-PEX controls using lens capsule tissue as the study material. The custom RT2 Profiler PCR array was used to identify a set of stress-related candidate genes that were differentially expressed in PEX. The expression of the highly deregulated genes was validated by qRT-PCR and subsequently their protein expression was checked through immunoblotting and immunostaining. Proteasome-Glo based assay and TUNEL assay were employed to detect specific proteasomal activity and apoptotic activity, respectively in the study subjects. Increased ER stress markers, Synoviolin1, Eukaryotic initiation factor 2-alpha kinase 3, DnaJ (Hsp40) homolog, subfamily B, member 11, Caspase 12, Heat shock 70 kDa protein 5, Heat shock 60 kDa protein 1 and Calnexin were observed in the lens capsule of PEX individuals compared to age-matched controls. On the other hand, increased ubiquitin B mRNA expression followed by significant downregulation of proteasome subunits; 26 S proteasome non-ATPase regulatory subunit 1, and proteasome subunit alpha-type 5 was found in pseudoexfoliation syndrome (PEXS) individuals. Decrease in chymotrypsin-like proteasome activity and increased apoptosis were also observed in PEX subjects. The present findings provide evidence for alterations in endoplasmic reticulum-related stress response and ubiquitin proteasome function in lens capsule of PEX individuals. Altogether, our study has identified deregulated expression of candidate genes in ER-UPR pathway and implicates proteasome impairment as a causative factor in PEX pathogenesis.

Artemisinin and curcumin inhibit Drosophila brain tumor, prolong life span, and restore locomotor activity.

Deletion of tumor suppressor gene, lethal(2)giant larvae [l(2)gl], leads to brain tumor in Drosophila melanogaster at larval stage of development and severe brain dysplasia in mice. We have studied the effect of two potential antitumor drugs artemisinin and curcumin in the perspective of inhibiting l(2)gl brain tumor. Efficacies of these drugs are characterized morphologically by measuring brain sizes of untreated and treated larvae on the basis of tumor inhibition and anatomically by looking at the cellular patterning via antibody staining of the third instar Drosophila larval brains. Behavioral experiments were done in form of locomotion to correlate tumor inhibition with the revival of brain function and longevity assays to assess general health span. It was observed that both drugs show antitumor properties individually and in combination when larvae were treated with these drugs. We also found evidence for reactive oxygen species-mediated action of these drugs. Both the drugs when treated individually or together show better median life span and locomotory response. Although the efficacies of various treatments varied, overall, the positive effects of artemisinin and curcumin demonstrate a potential applicability of these drugs against brain tumor in higher organisms. It also paves a way for a simpler model system for screening such natural products for antitumor property.

Current understanding of genetics and epigenetics in pseudoexfoliation syndrome and glaucoma.

Pseudoexfoliation is a complex, progressive, and systemic age-related disorder. The early stage of deposition of extracellular fibrillar material on ocular and extraocular tissues is termed as pseudoexfoliation syndrome (PEXS). The severe advanced stage is known as pseudoexfoliation glaucoma (PEXG), which involves increased intraocular pressure and optic nerve damage. Through genome-wide association and candidate gene studies, PEX has been associated with numerous genetic risk variants in various gene loci. However, the genetic basis of the disease fails to explain certain features of PEX pathology, such as the progressive nature of the disease, asymmetric ocular manifestation, age-related onset, and only a subset of PEXS individuals developing PEXG. Increasing evidence shows an interplay of genetic and epigenetic factors in the pathology of complex, multifactorial diseases. In this review, we have discussed the genetic basis of the disease and the emerging contribution of epigenetic regulations in PEX pathogenesis, focusing on DNA methylation and non-coding RNAs. Aberrant methylation patterns, histone modifications, and post-transcriptional regulation by microRNAs lead to aberrant gene expression changes. We have reviewed these aberrant epigenetic changes in PEX pathology and their effect on molecular pathways associated with PEX. We have further discussed some possible genetic/epigenetic-based diagnoses and therapeutics for PEX. Although studies to understand the role of epigenetic regulations in PEX are just emerging, epigenetic modifications contribute significantly to PEX pathogenesis and may pave the way for better and targeted therapeutics.

Genetic variants and haplotypes in fibulin-5 (FBLN5) are associated with pseudoexfoliation glaucoma but not with pseudoexfoliation syndrome.

Pseudoexfoliation (PEX) is a multifactorial age-related disease involving deposition of extracellular proteinaceous aggregates on anterior ocular tissues. The present study aims to identify functional variants in fibulin-5 (FBLN5) as risk factors for the development of PEX. Thirteen tag single-nucleotide polymorphisms (SNPs) in FBLN5 were genotyped using TaqMan SNP genotyping technology to identify association between SNPs of FBLN5 and PEX in an Indian cohort comprising 200 control and 273 PEX patients (169 PEXS and 104 PEXG). Functional analysis of risk variants was done through luciferase reporter assays and electrophoretic mobility shift assay (EMSA) using human lens epithelial cells. Genetic association and risk haplotype analysis showed a significant association of rs17732466:G>A (NC_000014.9:g.91913280G>A) and rs72705342:C>T (NC_000014.9:g.91890855C>T) within FBLN5 as risk factors with the advanced severe stage of the disease, pseudoexfoliation glaucoma (PEXG). Reporter assays showed allele-specific regulatory effect of rs72705342:C>T on gene expression, wherein, construct containing the risk allele showed a significant decrease in the reporter activity compared with the one with protective allele. EMSA further validated higher binding affinity of the risk variant to nuclear protein. In silico analysis predicted binding sites for two transcription factors, GR-α and TFII-I with risk allele at rs72705342:C>T, which were lost in the presence of protective allele. The EMSA showed probable binding of both these proteins to rs72705342. In conclusion, the present study identified the novel association of two genetic variants in FBLN5 with PEXG but not with PEXS, distinguishing between the early and the later forms of PEX. Further, rs72705342:C>T was found to be a functional variant.

Dickkopf-1 and ROCK2 upregulation and associated protein aggregation in pseudoexfoliation syndrome and glaucoma.

AIMS: The etiology of pseudoexfoliation (PEX), a stress-induced fibrillopathy and a leading cause of secondary glaucoma worldwide, remains limited. This study aims to understand the role of the Wnt antagonist Dickkopf-related protein 1 (DKK1) in PEX pathophysiology and assess its candidature as a biomarker for PEX. MAIN METHODS: Expression levels of DKK1 and Wnt signaling genes were assayed in the anterior ocular tissues of study subjects by qRT-PCR, Western blotting, and immunohistochemistry. Protein aggregation was studied through Proteostat staining. Role of DKK1 in protein aggregation and regulation of target Wnt signaling genes was elucidated through overexpression and knockdown studies in Human Lens Epithelial cells (HLEB3). Levels of DKK1 in circulating fluids were assayed through ELISA. KEY FINDINGS: DKK1 upregulation was observed in lens capsule and conjunctiva tissues of PEX individuals compared to controls correlating with an upregulation of the Wnt signaling target, ROCK2. Proteostat staining showed increased protein aggregates in lens epithelial cells of PEX patients. HLE B-3 cells overexpressed with DKK1 showed increased protein aggregates along with upregulation of ROCK2, and knockdown of DKK1 in HLE B-3 cells demonstrated downregulation of ROCK2. Further, ROCK2 inhibition by Y-27632 in DKK1 overexpressed cells showed that DKK1 regulated protein aggregation via ROCK2. Also, increased levels of DKK1 were observed in patients' plasma and aqueous humor compared to controls. SIGNIFICANCE: This study shows that DKK1 and ROCK2 might play a role in protein aggregation in PEX. Further, elevated levels of DKK1 in aqueous humor serve as a fair classifier of pseudoexfoliation glaucoma.

Role of clusterin gene 3'-UTR polymorphisms and promoter hypomethylation in the pathogenesis of pseudoexfoliation syndrome and pseudoexfoliation glaucoma.

Pseudoexfoliation (PEX) is a multifactorial age-related disease characterized by the deposition of extracellular fibrillar aggregates in the anterior ocular tissues. This study aims to identify the genetic and epigenetic contribution of clusterin (CLU) in PEX pathology. CLU is a molecular chaperone upregulated in PEX and genetically associated with the disease. Sequencing of a 2.9 kb region encompassing the previously associated rs2279590 in 250 control and 313 PEX [(207 pseudoexfoliation syndrome (PEXS) and 106 pseudoexfoliation glaucoma (PEXG)] individuals identified three single nucleotide polymorphisms (SNPs), rs9331942, rs9331949 and rs9331950, in the 3'-UTR of CLU of which rs9331942 and rs9331949 were found to be significantly associated with PEXS and PEXG as risk factors. Following in silico analysis, in vitro luciferase reporter assays in human embryonic kidney cells revealed that risk alleles at rs9331942 and rs9331949 bind to miR-223 and miR-1283, respectively, suggesting differential regulation of clusterin in the presence of risk alleles at the SNPs. Further, through bisulfite sequencing, we also identified that CLU promoter is hypomethylated in DNA from blood and lens capsules of PEX patients compared to controls that correlated with decreased expression of DNA methyltransferase 1 (DNMT1). Promoter demethylation of CLU using DNMT inhibitor, 5'-aza-dC, in human lens epithelial cells increased CLU expression. Chromatin immunoprecipitation assays showed that the demethylated CLU promoter provides increased access to the transcription factor, Sp1, which might lead to enhanced expression of CLU. In conclusion, this study highlights the different molecular mechanisms of clusterin regulation in pseudoexfoliation pathology.

Wide-spread enhancer effect of SNP rs2279590 on regulating epoxide hydrolase-2 and protein tyrosine kinase 2-beta gene expression.

Polymorphisms in the PTK2B-CLU locus have been associated with various neurodegenerative disorders including pseudoexfoliation glaucoma, Alzheimer's and Parkinson's. Many of these genomic variants are within enhancer elements and modulate genes associated with the disease pathogenesis. However, mechanisms by which they control the gene expression is unknown. Previously, we have shown that clusterin enhancer element surrounding rs2279590 intronic variant, a risk factor in the pathogenesis of pseudoexfoliation glaucoma modulates gene expression of clusterin (CLU), protein tyrosine kinase 2 beta (PTK2B) and epoxide hydrolase 2 (EPHX2). Here, we explored the mechanism by which rs2279590 enhancer regulates their gene expression through chromosome conformation capture assays. 3C assays revealed a strong enhancer-promoter chromatin interaction between rs2279590 enhancer and promoters of genes CLU, PTK2B and EPHX2 in the HEK293 wild type cells. Moreover, genomic knockout of rs2279590 element significantly decreases the chromatin-chromatin cross-linking frequency suggesting gene regulation at transcriptional level through formation of chromatin loop. In addition, molecular assays showed a significantly decreased expression of EPHX2 but not PTK2B at both mRNA and protein level in the lens capsule of pseudoexfoliation affected patients in comparison to control subjects implying a role of EPHX2 in the pathogenesis of pseudoexfoliation.

Fuchs Endothelial Corneal Dystrophy associated risk variant, rs3768617 in LAMC1 shows allele specific binding of GFI1B.

AIMS: To investigate the genetic and functional association of an intronic variant of LAMC1, rs3768617 with Fuchs endothelial corneal dystrophy (FECD) in the Indian population. METHODS: Blood samples were collected from age and sex matched 356 controls and 120 FECD patients after a detailed assessment via specular microscopy. Genomic DNA was extracted and genotyping was done by fluorescence based capillary electrophoresis. The genetic association of rs3768617 polymorphisms was computed by the chi-square (χ2) test. Bioinformatics studies were performed to find the allele specific binding of different transcription factors in the region of rs3768617 and functional evaluation assessed by luciferase assay followed by Electrophoretic Mobility Shift Assay (EMSA) and Chromatin Immunoprecipitation assay (ChIP). Immunofluorescence assay was carried out to check for any differential expression of GFI1B between control and FECD endothelium samples. RESULTS: SNP rs3768617 {chr1:183123365 (GRCh38.p13)} was found to be genetically associated with FECD in Indian population (p = 2.646 × 10-8). Luciferase assay suggested that the rs3768617 locus has a regulatory role. In silico analysis showed that the transcription factor, GFI1B binds to the risk allele 'G' of rs3768617, but not to the protective allele 'A' which was also experimentally validated by EMSA. High enrichment of DNA flanking the surrounding region of rs3768617 was also found in presence of GFI1B specific antibody in ChIP assay. There was a 0.63 fold decrease in GFI1B expression in FECD affected corneal endothelium compared to control endothelium. CONCLUSIONS: The genetic association of rs3768617 in LAMC1 with FECD pathogenesis is mediated by GFI1B, thus finding the functional role of LAMC1 in FECD pathogenesis.

Is pseudoexfoliation glaucoma a neurodegenerative disorder?

Pseudoexfoliation (PEX) is a systemic age-related progressive disorder with ocular manifestations. The earlier stage of the disease, pseudoexfoliation syndrome (PEXS) involves deposition of white fibrillar aggregates on anterior and posterior eye tissues. It is also the cause of most common form of secondary glaucoma known as pseudoexfoliation glaucoma (PEXG). Studies in the past decade highlight the role of many genetic and environmental factors as the underlying cause of PEX pathogenesis. Latest research findings by various researchers and us present the view of PEX as a type of neurodegenerative disorder. Epidemiological studies have shown association of PEX with different forms of neurodegenerative diseases like Alzheimer's, agerelated macular degeneration and open angle glaucoma. Also, sharing of common genetic risk factors, abnormal protein aggregation and most importantly, progressive degeneration of neurons with age are some of the identifiable features seen in both PEX and other neurodegenerative diseases. In this review, we have compared the pathological symptoms and factors involved in the disease manifestation of PEXG with various forms of neurodegenerative disorders and categorized PEXG as a progressive neurodegenerative disorder.

A Forward Genetic Approach to Mapping a P-Element Second Site Mutation Identifies DCP2 as a Novel Tumor Suppressor in Drosophila melanogaster.

The use of transposons to create mutations has been the cornerstone of Drosophila genetics in the past few decades. Second-site mutations caused by transpositions are often devoid of transposons and thereby affect subsequent analyses. In a P-element mutagenesis screen, a second site mutation was identified on chromosome 3, wherein the homozygous mutants exhibit classic hallmarks of tumor suppressor mutants, including brain tumor and lethality; hence the mutant line was initially named as lethal (3) tumorous brain [l(3)tb]. Classical genetic approaches relying on meiotic recombination and subsequent complementation with chromosomal deletions and gene mutations mapped the mutation to CG6169, the mRNA decapping protein 2 (DCP2), on the left arm of the third chromosome (3L). Thus the mutation was renamed as DCP2l(3)tb Fine mapping of the mutation further identified the presence of a Gypsy-LTR like sequence in the 5'UTR coding region of DCP2, along with the expansion of the adjacent upstream intergenic AT-rich sequence. The mutant phenotypes are rescued by the introduction of a functional copy of DCP2 in the mutant background, thereby establishing the causal role of the mutation and providing a genetic validation of the allelism. With the increasing repertoire of genes being associated with tumor biology, this is the first instance of mRNA decapping protein being implicated in Drosophila tumorigenesis. Our findings, therefore, imply a plausible role for the mRNA degradation pathway in tumorigenesis and identify DCP2 as a potential candidate for future explorations of cell cycle regulatory mechanisms.

De novo variants in an extracellular matrix protein coding gene, fibulin-5 (FBLN5) are associated with pseudoexfoliation.

Fibulin-5 (FBLN5), an extracellular scaffold protein, plays a crucial role in the activation of Lysyl oxidase like-1 (LOXL1), a tropoelastin crosslinking enzyme, and subsequent deposition of elastin in the extracellular matrix. Following study identifies polymorphisms within FBLN5 gene as risk factors and its aberrant expression in the pathogenesis of an ocular disorder, pseudoexfoliation (PEX). Exons and exon-intron boundaries within FBLN5 gene were scanned through fluorescence-based capillary electrophoresis for polymorphisms as risk factors for PEX pathogenesis in recruited study subjects with Indian ethnicity. mRNA and protein expression of FBLN5 was checked in lens capsule of study subjects through qRT-PCR and western blotting, respectively. In vitro functional analysis of risk variants was done through luciferase reporter assays. Thirty study subjects from control and PEX affected groups were scanned for potential risk variants. Putative polymorphisms identified by scanning were further evaluated for genetic association in a larger sample size comprising of 338 control and 375 PEX affected subjects. Two noncoding polymorphisms, hg38 chr14:g.91947643G>A (rs7149187:G>A) and hg38 chr14:g.91870431T>C (rs929608:T>C) within FBLN5 gene are found to be significantly associated with PEX as risk factors with a p-value of 0.005 and 0.004, respectively. Molecular assays showed a decreased expression of FBLN5 at both mRNA and protein level in lens capsule of pseudoexfoliation syndrome (PEXS) affected subjects than control. This study unravels two novel risk variants within FBLN5 gene in the pathogenesis of PEX. Further, a decreased expression of FBLN5 in PEXS affected lens capsules implicates a pathogenic link between extracellular matrix maintenance and onset of PEX.

rs4246215 is targeted by hsa-miR1236 to regulate FEN1 expression but is not associated with Fuchs' endothelial corneal dystrophy.

Fuchs' Endothelial Corneal Dystrophy (FECD) is a genetically complex disorder that affects individuals above 40 years of age; molecular pathogenesis of its associated genes is poorly understood. This study aims at assessing the association of flap endonuclease 1 (FEN1) polymorphisms, c.-69G>A (rs174538) and c.4150G>T (rs4246215) with FECD. Comet assay analysis reaffirmed that endogenous DNA damage was greater in FECD individuals. However, genetic analysis in 79 FECD patients and 234 unrelated control individuals prove that both the FEN1 polymorphisms, c.-69G>A (rs174538) and c.4150G>T (rs4246215), failed to show any genetic association with the FECD disease phenotype. In silico analysis and luciferase reporter assay identified 'G' allele of the 3'UTR located FEN1 polymorphism c.4150G>T as the target for binding of hsa-miR-1236-3p. This study indicates that although FEN1 polymorphisms, c.-69G>A (rs174538) and c.4150G>T (rs4246215) are not genetically associated with FECD, its transcript regulation reported in other diseases such as lung cancer which are genetically associated by rs4246215 could be mediated through miRNA, hsa-miR-1236-3p.

Genetic association of TCF4 intronic polymorphisms, CTG18.1 and rs17089887, with Fuchs' endothelial corneal dystrophy in an Indian population.

PURPOSE: To assess the genetic association of transcription factor 4 (TCF4) intronic polymorphisms and CTG18.1 allele in individuals with Fuchs' endothelial corneal dystrophy (FECD) individuals from a sample Indian population. METHODS: Forty-four FECD patients and 108 unrelated age-matched controls were recruited with informed consent for this study. Three, single nucleotide polymorphisms (SNPs) spanning the third intronic region of TCF4 (rs613872, rs17089887, and rs17089925) and an unstable trinucleotide repeat CTG18.1 allele were genotyped by direct sequencing using Sanger's method. The association of polymorphisms was analyzed using χ(2) test and logistic regression. RESULTS: SNP rs17089887 (P = 0.013) and CTG18.1 (P = 2 × 10(-4)) alleles were found to be significantly associated with FECD in the sample Indian population. However, the other two SNPs, rs613872 and rs17089925, were not likewise associated. Thirty-four percent of FECD subjects and 5% of control individuals harbor more than 50 trinucleotide repeats, which was considered as the disease threshold. CONCLUSIONS: TCF4 poses a major contributor to FECD manifestation globally, with a significant association of rs17089887 and CTG18.1 allele in the Indian population.