A Study on the Candidate Gene Association and Interaction with Measures of UV Exposure in Pseudoexfoliation Patients from India.

PURPOSE: Environmental and genetic factors are associated with development of Pseudoexfoliation syndrome (XFS). Here we intended to elucidate the association of candidate genes in relevance to UV exposure in these patients. METHODS: This is a case-control study of 309 subjects (N = 219 controls and 90 XFS cases) from India. PCR based direct sequencing was performed for candidate genes (LOXL1, POMP and TMEM136) followed by genotype and haplotype analysis. The promoter methylation status was assessed by Methylation specific PCR based direct sequencing of genomic DNA for all samples. The methylation status was compared with that of primary fibroblasts cultures established from patient's Tenon's tissue samples in subset of these patients. RESULTS: SNPs rs3825942, rs41435250, rs8818 (LOXL1) and rs3737528 (POMP) showed significant association with XFS. LOXL1 gene haplotype GAGC (rs1048661- rs3825942- rs41435250-rs8818) was associated with lower risk for XFS with a p value 4.1961 × 10-6 (OR =0; 95%CI, 0.000-0.003). POMP gene haplotypes for intronic SNPs (rs1340815- rs3737528- rs913797) TCC and TTC were associated with increased risk for the disease (OR > 1.0). Significant correlation for SNPs rs3825942 of LOXL1 (ρ= -0.132) and rs3737528 of POMP (ρ = 0.12) was observed with measure of lifetime UV exposure (CUVAF value). Reduced LOXL1 gene expression was observed in cultured tenon fibroblasts from the patients that correlated with differential methylation of the Sp-1 binding sites at -253, -243bp upstream to the transcription start site of LOXL1 promoter region. CONCLUSION: Our results suggest a possible interaction for LOXL1 gene haplotype (GAGC) with the measure of ocular UV exposure in pseudoexfoliation syndrome.

Evaluating the impact of ocular UV exposure for the development for pseudoexfoliation syndrome in a South Indian population.

CLINICAL RELEVANCE: Pathophysiology of pseudoexfoliation syndrome (XFS) can be influenced by environmental factors such as solar exposure/occupational factors and genetic factors. BACKGROUND: The study aims to assess the association of lifetime ocular UV exposure and its impact on the risk of development of XFS. METHODS: All eligible subjects underwent a comprehensive ocular examination. XFS was defined as precipitates on the pupillary border, cornea, and angle of anterior chamber or lens in at least one eye without any clinical signs of glaucoma. A standardised questionnaire was administered to assess the lifetime UV exposure. Conjunctival ultraviolet autofluorescence (CUVAF) photography was taken to detect the conjunctival changes with exposure to UV radiation. Ascorbic acid concentration in the aqueous humour was measured. RESULTS: A total of 404 subjects of which 274 (controls) and 130 (XFS cases) were studied. There were 204 males (50.5%) and 200 females (49.5%).Lifetime UV exposure (OR: 1.14, 95% CI: 1.02-1.30, p: 0.032), CUVAF damage (OR: 1.03, 95% CI: 1.01-1.06, p: 0.008) and outdoor worker (OR: 1.87, 95% CI: 1.18-3.00, p: 0.008) were positively associated with XFS. Usage of spectacles (OR: 0.63, 95% CI: 0.39-0.95, p: 0.030) and ascorbic acid concentration in aqueous (OR: 0.47, 95% CI: 0.23-0.99, p: 0.038) were found to be protective against XFS. CONCLUSION: Besides genetic factors, environmental factors such as lifetime ocular UV exposure and outdoor work are significantly associated with the risk of XFS. CUVAF can be used as a non-invasive tool to detect preclinical sun damage in outdoor workers.

Increased Desmosine in the lens capsules is associated with augmented elastin turnover in Pseudoexfoliation syndrome.

Pseudoexfoliation syndrome (PXF) is an idiopathic disease with a high prevalence rate. The elastosis disorder is contributed by genetic and non-genetic factors. Elastin dysregulation associated with the disease mechanism is incompletely understood. This study evaluated the molecules of the elastogenesis machinery in PXF. Lens capsule and aqueous humor (aqH) samples (age/sex-matched) were collected from the eyes with PXF alone and PXF with glaucoma (PXF-G) undergoing Extra Capsular Cataract Extraction (ECCE) surgery. The Elastin turnover was assessed by estimating Desmosine levels in the lens capsules by HPLC analysis. Expression of elastogenesis genes [EMILIN1, CLU, FBN1, FN1, FBLN5, FBLN4 and LOXL1] were evaluated in the lens capsule by qPCR while the proteins were assessed in aqH by western blot analysis. The Desmosine content in the lens capsules were 3-fold and 6-fold elevated in PXF (P = 0.02) and PXF-G (P = 0.01) respectively compared to the cataract-alone, indicating increased elastin degradation. A significant increase in the transcript levels of the CLU, FBLN4, EMILIN1, FBLN5, FN1, FBN1, LOXL1 along with significant changes in protein expression of CLU, FBLN5, FBN1 and LOXL1 signified up-regulation of the elastogenesis machinery. The study provides direct evidence of augmented elastin degradation and turnover in the lens capsule of PXF marked by increased Desmosine content and the expression of proteins involved in mature elastin formation.

Elastin modulation and modification by homocysteine: a key factor in the pathogenesis of Pseudoexfoliation syndrome?

BACKGROUND: Pseudoexfoliation syndrome (PXF) is an idiopathic, elastogenesis-associated systemic disease characterised by amyloid-like material aggregates in the eye. Elevated plasma and aqueous humour (aqH) homocysteine (Hcy) is reportedly associated with PXF. This study is aimed to probe Hcy-mediated alterations in elastin expression. METHODOLOGY: Lens level of Hcy (total Hcy (tHcy)), mRNA expression of Eln, CBS and MTR in lens capsule, protein expression of elastin in aqH were estimated by enzyme immunoassay, quantitative PCR and western blot, respectively in PXF, PXF with glaucoma (PXF-G) cases, in comparison with cataract-alone disease controls. Human lens epithelial cells (hLECs) were exposed to Hcy and homocysteine thiolactone (HCTL) to evaluate elastin expression in vitro. Furthermore, elastin recombinant protein was incubated with Hcy and HCTL to assess secondary and tertiary structural modifications based on circular dichroism spectroscopy, spectrophotometric and SEM studies. RESULTS: The lens tHcy was significantly high in PXF (p=0.02) and PXF-G (p=0.009). Eln expression was elevated in PXF and PXF-G (p=0.0007). Elastin level in aqH was elevated in PXF (p=0.01) and PXF-G (p=0.002). Hcy (200 µM) and HCTL (1 µM) promoted elastin expression at mRNA level by 36-fold (p=0.02) and 10-fold (p=0.05), respectively, and at protein level by nearly two-fold in cultured hLECs. Secondary structure changes in elastin protein caused by Hcy were evident from 34.11% drop in α-helix and 6.17% gain in ß-sheet. Fluorescence, spectral assays and SEM analyses showed aggregation and amyloid formation of elastin with homocysteinylation. CONCLUSION: The study reveals that lens accumulation of Hcy associated with hyperhomocysteinaemia is characteristic of PXF that augments elastin expression. Hcy causes structural changes promoting elastin aggregation, thereby contributing to defective elastin in PXF and PXF-G.

Correlation of Aqueous Humor Lysyl Oxidase Activity with TGF-ß Levels and LOXL1 Genotype in Pseudoexfoliation.

PURPOSE: Pseudoexfoliation (PXF) is a microfibrillopathy involving disordered elastogenesis. Abnormal extracellular matrix (ECM) production underlies the pathophysiology of PXF. The enzyme Lysyl oxidase (LOX) and its isoforms are known to cross-link the elastin and collagen. Though the etiopathogensis of PXF is not well understood, studies report on the genetic risk involving LOXL1 gene. This study aims to screen LOXL1 coding variants rs1048661 and rs3825942 in the South Indian population and the implication of the single nucleotide polymorphism (SNP) with LOX activity. The levels of transforming growth factor ß (TGF-ß) in aqueous humor and its correlation with the LOX activity were also examined. METHODS: Blood, plasma, and aqueous aspirates were prospectively collected from PXF cases with and without glaucoma and cataract cases as controls. DNA was extracted from 48 PXF cases without glaucoma, 12 PXF cases with glaucoma, and 40 age-matched cataract-alone controls without PXF/glaucoma for analyzing LOX SNPs. LOX activity was measured in aqueous humor and plasma of 30 PXF cases without glaucoma, 24 age-matched cataract-alone controls without PXF/glaucoma, and 14 PXF cases with glaucoma. Protein levels of LOX, LOXL1, LOXL2, and total TGF-ß were estimated in plasma and aqueous humor by ELISA. RESULTS: The specific activity of LOX in aqueous humor was found to be significantly lowered in PXF cases compared with cataract-alone controls (p = 0.014). This decrease in LOX activity in PXF cases was associated with high-risk GG haplotype. However, this was not statistically significant and a larger sample size is warranted. TGF-ß1 and TGF-ß2 negatively correlated with LOX activity in aqueous humor (p = 0.028; p = 0.046, respectively). CONCLUSIONS: The LOXL1 SNPs, rs1048661 and rs3825942, are associated with PXF in the South Indian population correlating with lowered LOX activity in the aqueous humor. The increased level of total TGF-ß in the aqueous humor of PXF cases is possibly associated with LOX regulation which needs further investigation.