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

Abstract

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.

Figure 1.. Eye of a Patient With Exfoliation Syndrome

The typical white exfoliation material deposits on the surface of the lens are visible (blue arrows) with the aid of a slit lamp at 16 × magnification under white light. Exfoliation material may not be visible on unaided examination.

Figure 2.. Sequencing and Analytical Approaches to Identify Genes Associated With Exfoliation Syndrome

^aEach participant with exfoliation syndrome was matched to at least 1 participant without exfoliation syndrome (for every individual with exfoliation syndrome, ≥1 individual without exfoliation syndrome could be recruited as a matching control). Matching was by geographical site of recruitment and self-reported ancestry. ^bMost disease-causing genetic variants are maintained at low frequencies by purifying evolutionary selection. ^cPolymorphism Phenotyping version 2 (Polyphen-2) is a widely used computer algorithm to identify genetic variants that damage protein function. Such variants are likely to cause disease. Alleles predicted to be benign were excluded because their inclusion could have masked disease associations caused by damaging allelic variants. ^dMany medical conditions are caused by haploinsufficiency, which is a state where 1 copy of a gene has been damaged by alterations, leaving the remaining normally functioning gene copy insufficient to sustain normal function. In many of these conditions, different alterations within a given gene causes the same damaging consequences to the encoded protein product. In this study, the burden test was designed to compare the number (or burden) of damaging genetic alterations found in each gene among persons with exfoliation syndrome vs those without exfoliation syndrome. ^eValidation of original discovery stage findings in 2 independently enrolled validation cohorts increases the confidence that the original observations were not false discoveries. ^fGenes identified to be significantly associated with disease during the discovery and validation stages were characterized further using post hoc experimental assays to garner additional insights into disease pathogenesis.

Figure 3.. CYP39A1 Association With Presence of Exfoliation Syndrome

Forest plot describing the association between the burden of damaging CYP39A1 allelic variants and exfoliation syndrome among 20 441 participants from 14 countries. The height of the data marker is proportional to the size of the sample. The width of the diamond represents the 95% CI. P values were calculated using the Cochran-Mantel-Haenszel meta-analysis method. There was no meta-analysis conducted for self-reported Black African ancestry because this would simply duplicate the analysis from South Africa as reported elsewhere in the Figure. Further information on selection of the study participants based on self-reported ancestry appears in the eAppendix in Supplement 1. ^aAffected individuals had exfoliation syndrome and unaffected individuals did not have it. ^bIncluded participants from Japan and Singapore who self-identified as having Asian ancestry. ^cIncluded participants from Austria, Canada, Georgia, Germany, Greece, Poland, Romania, Russia, Spain, Turkey, and the US.

Figure 4.. Experimental Determination of Functional Enzymatic Activity for CYP39A1 Variants

Box and whisker plots of functional enzymatic activity of 1 wild-type CYP39A1 variant, 3 common variants, 42 rare variants predicted to be damaging (by Polymorphism Phenotyping version 2 [Polyphen-2]; software that predicts the possible effect of amino acid substitutions on human protein function), and 5 variants predicted by Polyphen-2 to be benign. Three data points (representing 3 independent biological replicates) were plotted for all tested CYP39A1 genetic variants. The crossbars represent the median level of activity, the boxes represent the interquartile range, and the whiskers portray the range of enzymatic activity for each variant. Individual biological replicates are shown as individual data points. Amino acid substitutions are caused by coding-sequence genetic variation. The case-control distribution of all variants predicted by Polyphen-2 to be damaging appears in eTable 10 in Supplement 1. The enzymatic activity of individual variants was expressed as a proportion relative to the wild-type CYP39A1 enzymatic activity. The functional enzymatic activity of each tested CYP39A1 variant was determined by the relative 24(S)-7α,24-dihydroxycholesterol product abundance it produced with respect to wild-type CYP39A1. The product abundance was calculated by integrating the area under the 24(S)-7α,24-dihydroxycholesterol product peak (eFigure 10 in Supplement 1), averaged across 3 independent samples for each tested variant. The empirical distribution of the wild-type CYP39A1 enzymatic activity was defined using 14 independent biological replicates. The design and validation of the experimental system appear in eFigures 9 and 10 in Supplement 1.

Figure 5.. Expression of CYP39A1 in Eye Tissues From Participants With Self-Reported European Ancestry

Box plots showing relative messenger RNA expression levels of cytochrome P450 enzymes involved in cholesterol metabolism. The crossbars represent median relative messenger RNA expression levels, the boxes represent the interquartile range, the whiskers extend to the most extreme observed values with 1.5 × interquartile range of the nearer quartile, and the dots represent observed values outside that range. The ocular tissues were derived from patients with late-stage exfoliation syndrome (n = 23 [10 males and 13 females]; mean age, 79.1 years [SD, 8.8 years]) and those without exfoliation syndrome (n = 23 [12 males and 11 females]; mean age, 72.7 years [SD, 8.1 years]) using quantitative real-time polymerase chain reaction. The affected tissues and the unaffected control tissues were matched by age and sex if possible. Expression levels of CYP39A1 were significantly reduced in all ocular tissues from patients with exfoliation syndrome compared with controls, with the most significant differences seen in the ciliary body and retina. In contrast, CYP7A1, CYP7B1, CYP27A1, and CYP51A1 were reduced only in specific ocular tissues derived from patients with exfoliation syndrome, whereas CYP46A1 levels were not changed. Expression levels were normalized relative to the GAPDH housekeeping gene. P values were calculated based on the unpaired 2-tailed t test. A cholesterol metabolism schematic appears in eFigure 16 in Supplement 1.