Association of Rare CYP39A1 Variants With Exfoliation Syndrome Involving the Anterior Chamber of the Eye.

Importance: Exfoliation syndrome is a systemic disorder characterized by progressive accumulation of abnormal fibrillar protein aggregates manifesting clinically in the anterior chamber of the eye. This disorder is the most commonly known cause of glaucoma and a major cause of irreversible blindness. Objective: To determine if exfoliation syndrome is associated with rare, protein-changing variants predicted to impair protein function. Design, Setting, and Participants: A 2-stage, case-control, whole-exome sequencing association study with a discovery cohort and 2 independently ascertained validation cohorts. Study participants from 14 countries were enrolled between February 1999 and December 2019. The date of last clinical follow-up was December 2019. Affected individuals had exfoliation material on anterior segment structures of at least 1 eye as visualized by slit lamp examination. Unaffected individuals had no signs of exfoliation syndrome. Exposures: Rare, coding-sequence genetic variants predicted to be damaging by bioinformatic algorithms trained to recognize alterations that impair protein function. Main Outcomes and Measures: The primary outcome was the presence of exfoliation syndrome. Exome-wide significance for detected variants was defined as P < 2.5 × 10-6. The secondary outcomes included biochemical enzymatic assays and gene expression analyses. Results: The discovery cohort included 4028 participants with exfoliation syndrome (median age, 78 years [interquartile range, 73-83 years]; 2377 [59.0%] women) and 5638 participants without exfoliation syndrome (median age, 72 years [interquartile range, 65-78 years]; 3159 [56.0%] women). In the discovery cohort, persons with exfoliation syndrome, compared with those without exfoliation syndrome, were significantly more likely to carry damaging CYP39A1 variants (1.3% vs 0.30%, respectively; odds ratio, 3.55 [95% CI, 2.07-6.10]; P = 6.1 × 10-7). This outcome was validated in 2 independent cohorts. The first validation cohort included 2337 individuals with exfoliation syndrome (median age, 74 years; 1132 women; n = 1934 with demographic data) and 2813 individuals without exfoliation syndrome (median age, 72 years; 1287 women; n = 2421 with demographic data). The second validation cohort included 1663 individuals with exfoliation syndrome (median age, 75 years; 587 women; n = 1064 with demographic data) and 3962 individuals without exfoliation syndrome (median age, 74 years; 951 women; n = 1555 with demographic data). Of the individuals from both validation cohorts, 5.2% with exfoliation syndrome carried CYP39A1 damaging alleles vs 3.1% without exfoliation syndrome (odds ratio, 1.82 [95% CI, 1.47-2.26]; P < .001). Biochemical assays classified 34 of 42 damaging CYP39A1 alleles as functionally deficient (median reduction in enzymatic activity compared with wild-type CYP39A1, 94.4% [interquartile range, 78.7%-98.2%] for the 34 deficient variants). CYP39A1 transcript expression was 47% lower (95% CI, 30%-64% lower; P < .001) in ciliary body tissues from individuals with exfoliation syndrome compared with individuals without exfoliation syndrome. Conclusions and Relevance: In this whole-exome sequencing case-control study, presence of exfoliation syndrome was significantly associated with carriage of functionally deficient CYP39A1 sequence variants. Further research is needed to understand the clinical implications of these findings.

The Ocular Surface in Medically Controlled Glaucoma: An In Vivo Confocal Study.

PURPOSE: To study clinical and in vivo laser scanning confocal microscopy (LSCM) ocular surface findings in stable, medically controlled primary open-angle glaucoma (MCPOAG) patients. METHODS: We recruited 100 consecutive patients with MCPOAG and 50 healthy controls. Patients had to have been treated with the same medical regimen without variation for the 18 months before enrollment and were excluded if there was a history of dry eye prior to glaucoma diagnosis. Each participant underwent ocular surface clinical and LSCM examination. RESULTS: In MCPOAG patients, subbasal nerve length and tortuosity and dendritic cell density were increased compared to controls (P < 0.01), but there were no clinical abnormalities. Patients treated with preserved drugs (n = 80) had reduced tear film breakup time (P < 0.05, ANOVA), and those preserved with benzalkonium chloride (n = 72) had reduced Schirmer test values (P < 0.001). Patients (n = 50) treated with two or more drugs had increased lissamine green conjunctival staining (P < 0.001, LSD post hoc test). Patients (n = 29) treated with three or more eye drops daily had decreased Schirmer test values. Laser scanning confocal microscopy showed subbasal changes related to preservatives, type and number of drugs, and number of eye drops. CONCLUSIONS: In stable MCPOAG patients without dry eye history, the ocular surface changes due to antiglaucoma medications are mostly subclinical. Active ingredients, preservatives, number of concomitant drugs, and number of eye drops instilled per day are all elements that can induce ocular surface changes. The clinical relevance of these changes remains to be determined.

Genetic variants and cellular stressors associated with exfoliation syndrome modulate promoter activity of a lncRNA within the LOXL1 locus.

Exfoliation syndrome (XFS) is a common, age-related, systemic fibrillinopathy. It greatly increases risk of exfoliation glaucoma (XFG), a major worldwide cause of irreversible blindness. Coding variants in the lysyl oxidase-like 1 (LOXL1) gene are strongly associated with XFS in all studied populations, but a functional role for these variants has not been established. To identify additional candidate functional variants, we sequenced the entire LOXL1 genomic locus (∼40 kb) in 50 indigenous, black South African XFS cases and 50 matched controls. The variants with the strongest evidence of association were located in a well-defined 7-kb region bounded by the 3'-end of exon 1 and the adjacent region of intron 1 of LOXL1. We replicated this finding in US Caucasian (91 cases/1031 controls), German (771 cases/1365 controls) and Japanese (1484 cases/1188 controls) populations. The region of peak association lies upstream of LOXL1-AS1, a long non-coding RNA (lncRNA) encoded on the opposite strand of LOXL1. We show that this region contains a promoter and, importantly, that the strongly associated XFS risk alleles in the South African population are functional variants that significantly modulate the activity of this promoter. LOXL1-AS1 expression is also significantly altered in response to oxidative stress in human lens epithelial cells and in response to cyclic mechanical stress in human Schlemm's canal endothelial cells. Taken together, these findings support a functional role for the LOXL1-AS1 lncRNA in cellular stress response and suggest that dysregulation of its expression by genetic risk variants plays a key role in XFS pathogenesis.

The genetics of POAG in black South Africans: a candidate gene association study.

Multiple loci have been associated with either primary open angle glaucoma (POAG) or heritable ocular quantitative traits associated with this condition. This study examined the association of these loci with POAG, with central corneal thickness (CCT), vertical cup-to-disc ratio (VCDR) and with diabetes mellitus in a group of black South Africans (215 POAG cases and 214 controls). The population was homogeneous and distinct from other African and European populations. Single SNPs in the MYOC, COL8A2, COL1A1 and ZNF469 gene regions showed marginal associations with POAG. No association with POAG was identified with tagging SNPs in TMCO1, CAV1/CAV2, CYP1B1, COL1A2, COL5A1, CDKN2B/CDKN2BAS-1, SIX1/SIX6 or the chromosome 2p16 regions and there were no associations with CCT or VCDR. However, SNP rs12522383 in WDR36 was associated with diabetes mellitus (p = 0.00008). This first POAG genetic association study in black South Africans has therefore identified associations that require additional investigation in this and other populations to determine their significance. This highlights the need for larger studies in this population if we are to achieve the goal of facilitating early POAG detection and ultimately preventing irreversible blindness from this condition.

Lack of association between lysyl oxidase-like 1 polymorphisms and primary open angle glaucoma: a meta-analysis.

AIM: To study the associations between lysyl oxidase-like 1 (LOXL1) polymorphisms and primary open angle glaucoma (POAG) remain inconsistent. In this study, we have performed a meta-analysis to investigate the association of LOXL1 polymorphisms with POAG risk. METHODS: Published literature from PubMed and other databases were retrieved. All studies evaluating the association between LOXL1 polymorphisms (rs2165241, rs1048661, rs3825942) and POAG risk were included. Pooled odds ratio (OR) and 95% confidence interval (CI) were calculated using random- or fixed-effects model. RESULTS: Twelve studies were identified as eligible articles, with thirteen (2098 cases and 16 473 controls), thirteen (1795 cases and 2916 controls) and sixteen population cohorts (2456 cases and 2846 controls) for the association of rs2165241, rs1048661 and rs3825942 with POAG risk respectively. Overall analyses showed no association between each LOXL1 polymorphism and POAG risk, and the negative associations were remained when the subjects were stratified as Caucasian and Asian. The heterozygote of rs2165241 was associated with reduced POAG risk in hospital-based populations (TC vs CC: OR, 0.79, 95%CI: 0.63-0.99), and rs1048661 was associated with increased POAG risk in hospital-based populations in a dominant model (TT vs CC+CT: OR, 1.23, 95%CI: 1.01-1.50); however, these associations were not found in population-based subjects. CONCLUSION: This meta-analysis suggests that LOXL1 polymorphisms are not associated with POAG risk. Given the limited sample size, the associations of LOXL1 polymorphisms with POAG risk in hospital-based populations await further investigation.

The role of lysyl oxidase-like 1 DNA copy number variants in exfoliation glaucoma.

PURPOSE: To investigate whether DNA copy number variants (CNVs) in the lysyl oxidase-like 1 (LOXL1) gene are associated with exfoliation glaucoma (XFG) in black South Africans. METHODS: Black South African subjects with XFG and age-matched unaffected controls were recruited from the St. John Eye Hospital in Soweto (Johannesburg, South Africa) and East London Hospital Complex (Eastern Cape, South Africa) using standard clinical examination techniques. A customized array comparative genomic hybridization (aCGH) from Roche NimbleGen was designed to cover a 1.5 million base genomic region centered on the LOXL1 gene on chromosome 15. Twenty selected XFG cases were examined using this custom aCGH to identify common CNVs in the LOXL1 gene. The potential DNA copy number variants identified from aCGH were further validated using TaqMan probe-based CNV real-time PCR in a data set containing 91 XFG cases and 52 controls. The frequencies of CNVs in the LOXL1 region were compared between the XFG cases and the controls using Fisher's exact test. RESULTS: Several DNA CNV variants were identified in the LOXL1 genomic region using aCGH in the selected XFG cases. However, we were unable to validate these candidate CNVs using real-time PCR-based TaqMan CNV assays. There was no significant difference in the frequency of the DNA copy number variants in the LOXL1 region between the XFG cases and the controls. CONCLUSIONS: This represents the first DNA CNV study of LOXL1 in the black South African population with XFG. Our study did not identify any significant DNA copy number alterations in the genomic region containing the LOXL1 gene. This suggests that other as yet unknown causal variants of LOXL1 or variants in other genes in linkage disequilibrium with the LOXL1 locus contribute to the genetic risk of XFG in black South Africans.

Myocilin mutations in black South Africans with POAG.

PURPOSE: Myocilin (MYOC) mutations are associated with primary open-angle glaucoma (POAG) in multiple populations. Here we examined the role of MYOC mutations in a black South African population with primary open-angle glaucoma (POAG). METHODS: Unrelated black South African subjects with POAG and unaffected controls were recruited from the St. John Eye Hospital (Soweto, Johannesburg, South Africa) and East London Hospital Complex (Eastern Cape, South Africa). A complete eye examination including visual field assessment was performed in all subjects. Blood samples were obtained for DNA extraction. The complete coding region of MYOC was sequenced using the PCR-based Sanger method. Identified mutations were compared to known MYOC mutations. RESULTS: One hundred-thirteen POAG cases and 131 controls were recruited for analysis. A total of 19 variants were observed. Probable glaucoma-causing mutations were observed in 4.4% of POAG cases. A previously reported glaucoma-causing mutation, Tyr453MetfsX11, was observed in three cases and one control. Two other sequence variants, Gly374Val and Lys500Arg, occurred only in cases. Other sequence variants, including 6 novel variants, occurred in at least one control. CONCLUSIONS: A small minority of black South Africans with POAG carry MYOC mutations. The Gly374Val mutation might represent a novel glaucoma-causing mutation. The Tyr453MetFSX11 mutation appears to be a glaucoma-causing mutation with incomplete penetrance.

Major LOXL1 risk allele is reversed in exfoliation glaucoma in a black South African population.

PURPOSE: To investigate whether variants in the lysyl oxidase-like 1 (LOXL1) gene are associated with exfoliation glaucoma (XFG) and primary open-angle glaucoma (POAG) in an ancestral population from South Africa. METHODS: Black South African subjects with XFG, POAG, and age matched unaffected controls were recruited from the St. John Eye Hospital in Soweto, Johannesburg, South Africa, using standard clinical examination techniques. Fifty individuals were collected for each of the three groups: XFG, POAG, and normal controls. The complete coding region of LOXL1 was sequenced using the PCR-based Sanger method. The allele frequencies of the identified sequence variants were compared between XFG or POAG and controls using Fisher's exact test. RESULTS: A large number of coding variants were identified, including rs1048661 (R141L), rs3825942 (G153D), S159A, S161L, rs41435250 (A320A), rs13329473 (F489F), and T567A. The allele frequencies of both rs3825942 and rs1048661 differed significantly between the XFG and control subjects from South Africa (p=5.2 x 10(-13) and 1.7 x 10(-5), respectively). The G allele for rs1048661 (encoding arginine) was the risk allele which is similar to other populations. The A allele of rs3825942 (encoding aspartic acid) was the risk allele, in sharp contrast to the G allele (encoding glycine) reported in multiple other populations. There was no significant difference in the allele frequencies of coding variants in LOXL1 between POAG and control subjects. CONCLUSIONS: This represents the first genetic association study of LOXL1 in an ancestral African population with XFG. We have confirmed the association between variants of LOXL1 and XFG. To date, the G allele of the major susceptibility variant rs3825942 has consistently been shown in multiple populations to increase the risk of XFG. Surprisingly, we have found a strong association with the opposite allele in the South African population. This suggests that other as yet unknown causal variants of LOXL1 contribute to the genetic risk of XFG.