Rare variants in methionine synthase reductase are associated with iridociliary cysts

Abstract

Purpose : Our purpose is to determine a genetic cause for iris cysts, which have been reported to cause glaucoma by an angle closure mechanism.

Methods : Individuals from a 3-generation dominant pedigree with multiple iridociliary cysts were genotyped with HumanCoreExome 530K beadchip. Linkage analysis was performed using a parametric dominant model with MERLIN. Two regions of suggestive linkage with multipoint LOD score of 2.71 (chr 1q25.3, chr5p13.5) were identified. Whole exome sequencing of pooled affected and unaffected samples was done on the Illumina HighSeq100 platform, and variants were aligned, filtered, and annotated. Mutation segregation was confirmed. Mutation screening in twenty-six isolate cyst cases was done by Sanger sequencing. Ocular tissue expression of this candidate gene and functional studies were performed.

Results : A single candidate mutation was identified in the methionine synthase reductase gene (MTRR c.A1468>G, p.T490A), a regulator of homocysteine metabolism. The T490A mutation segregated in all affected family members, except for one individual without the chromosome 5 haplotype. Among unrelated cases, this mutation was identified in one individual (1.9% allele frequency [1/26]) who had elevated plasma homocysteine of 26 μM (normal 5-15 μM). This allele frequency of 1.9% was overrepresented in cyst cases compared to ethnically similar controls (0.5% in the ExAC database). MTRR was expressed in all human ocular tissues, including iris and ciliary body, by quantitative RT-PCR and western blot analysis. Cycloheximide pulse-chase experiments in HEK 293T cells showed that the MTRR p.T490A on the M22/L175 variant background was less stable than wild-type. Biochemical analysis revealed that the mutant protein has impaired flavin mononucleotide (FMN, 44±12%) and flavin adenine dinucleotide (82±17%) content, and decreased electron transport from FMN to downstream electron acceptors including decreased reduction of cytochrome c [k_cat 0.16±0.02 vs. 1.73±0.12 sec^-1].

Conclusions : We discovered a novel mutation in MTRR associated with multiple iridociliary body cysts. This gene is expressed in both the iris and ciliary body. The MTRR p.T490A mutation decreases protein stability and alters electron transport properties leading to loss of protein function. The biological mechanism of such deleterious MTRR variants in the pathogenesis of iridociliary cyst formation remains to be determined.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.