Abstract 53: Missense Pathogenic Variants in ANO1 Predispose to Moyamoya Disease

Abstract

Introduction: Moyamoya disease (MMD) is a cerebrovascular disease often causing pediatric onset strokes and characterized by progressive bilateral occlusion of the distal internal carotid arteries and formation of a compensatory network of collateral vessels. Several genes harbor variants that increase the risk for MMD, but the majority of cases of European descent does not have an identified genetic cause. Hypothesis: MMD is associated with significant genetic heterogeneity, i.e., many genes in the human genome can be altered to predispose to this condition. Methods: To identify novel genes for MMD, exome sequencing was performed on DNA from 145 individuals from 80 families with one or more affected members. Bioinformatics filtering included a CADD (GRCh37-v1.4) score > 20 and a minor allele frequency < 0.0001 in gnomAD (v2.1.1 controls). For a subset of ANO1 mutant alleles, we characterized the channel activities via patch recording and assessed protein localization in heterologous cell cultures. Results: We identified 6 rare variants in ANO1 ( TMEM16A ), which encodes an evolutionarily conserved calcium-activated chloride channel. One rare heterozygous variant, p.Met658Val (CADD: 22.9, absent in gnomAD), was found in two very distantly related MMD families and segregated with disease in multiple affected members; p.Glu459Lys and p.Arg890Gln were identified in two unrelated affected probands. Through Matchmaker Exchange (MyGene2) collaboration, we identified 3 additional rare variants: homozygous p.Glu170Lys in a consanguineous MMD family, heterozygous p.Thr740Ile in a MMD case and p.Ala333Ser in a case with ischemic stroke. Functional analyses determined that p.Glu170Lys affects channel gating and calcium sensitivity, producing much smaller chloride current, but the channel itself is more sensitive to calcium, which means it is more likely to open. Ano1 knock out (KO) in mice is lethal by 1 week of age with pathology in multiple organ systems. Smooth muscle cells-specific KO is not lethal, and phenotyping of these mice is ongoing. Conclusions: ANO1 is a novel gene predisposing to MMD and future studies will focus on defining the role of ANO1 protein to connect the altered gene to the occlusive lesions observed in MMD patients.