Rare variants in ANO1, encoding a calcium-activated chloride channel, predispose to moyamoya disease

Abstract

Moyamoya disease, a cerebrovascular disease leading to strokes in children and young adults, is characterized by progressive occlusion of the distal internal carotid arteries and the formation of collateral vessels. Altered genes play a prominent role in the aetiology of moyamoya disease, but a causative gene is not identified in the majority of cases. Exome sequencing data from 151 individuals from 84 unsolved families were analysed to identify further genes for moyamoya disease, then candidate genes assessed in additional cases (150 probands). Two families had the same rare variant in ANO1, which encodes a calcium-activated chloride channel, anoctamin-1. Haplotype analyses found the families were related, and ANO1 p.Met658Val segregated with moyamoya disease in the family with an LOD score of 3.3. Six additional ANO1 rare variants were identified in moyamoya disease families. The ANO1 rare variants were assessed using patch-clamp recordings, and the majority of variants, including ANO1 p.Met658Val, displayed increased sensitivity to intracellular Ca2+. Patients harbouring these gain-of-function ANO1 variants had classic features of moyamoya disease, but also had aneurysm, stenosis and/or occlusion in the posterior circulation. Our studies support that ANO1 gain-of-function pathogenic variants predispose to moyamoya disease and are associated with unique involvement of the posterior circulation.

Keywords: genetic heterogeneity; genotype-phenotype; pathogenesis; smooth muscle cells; stroke genetics.

Figure 1

Pedigrees of the families and protein map of the variants identified. (A) Pedigrees of families. Frame indicates the haplotype shared by Families MM137 and MM001. (B) Schematic representation of anoctamin-1 protein (986 amino acids), which features cytoplasmic, transmembrane and extracellular domains. Above the protein, ANO1 variants identified in this study are shown at their location on the schematic. Below the protein are rare missense and nonsense variants identified in the general population (gnomAD v2.1.1 with minor allele frequency <0.0001). Height indicates the number of individuals. CA = cancer; d. = age of death; DS = Down syndrome; dx = age of diagnosis; MI = myocardial infarct.

Figure 2

Characterization of Ca²⁺ activated Cl⁻ current in wild-type and mutant anoctamin-1. (A) Representative traces of whole-cell currents of anoctamin-1 wild-type (WT) and mutants recorded with a voltage-family protocol from −80 to 80 mV with increments of 10 mV, followed by depolarization of membrane potential to 80 mV, with pipette solution containing 50 nM Ca²⁺. R77Q = p.Arg77Gln; E170K = p.Glu170Lys; S196T = p.Ser196Thr; E459K = p.Glu459Lys; M658V = p.Met658Val; T740I = p.Thr740Ile; R890Q = p.Arg890Gln. (B) Averaged current-voltage (I-V) relationships of WT and mutant anoctamin-1. (C) Current magnitudes measured at indicated time points at +80 mV of recordings as in A. (D) Ratio of current magnitudes measured at indicated time points at −80 mV to those at +80 mV of recordings as in A, to indicate the rectification level of calcium-activated chloride channel (CaCC) current. R890Q was excluded from statistics due to small current amplitude. (E) Normalized conductance (G/G_{+80 mV}) of anoctamin-1 WT and mutants at different voltages. The conductance ratio was calculated by normalization of the current magnitudes at +80 mV immediately following the voltage family protocol (intersected with the vertical dash line as in A). (F) Representative traces of inside-out recordings of anoctamin-1 WT and mutants with membrane potential held at +80 mV, in response to solutions containing a series of Ca²⁺ concentrations. (G) Ca²⁺-response curves of anoctamin-1 WT and mutants, fitted of recordings as in F with a Hill equation. (H) The logarithm of half-activation points (logEC₅₀) of WT and mutant anoctamin-1, calculated from fitting as in G.

Figure 3

Imaging of affected individuals. (A) MM001, IV-3. Magnetic resonance angiography (MRA) demonstrating bilateral internal carotid occlusion (arrows) with moyamoya vascular changes. (B) MM001, IV-3. MRA of the posterior circulation demonstrating occlusive changes of the basilar artery (blue arrow), with narrowing of bilateral posterior cerebral arteries (red arrows). (C) MM001, IV-3. Diffusion weighted MRI of the brain demonstrating a right midbrain infarct (arrow). (D) MM001, III-2. MRA demonstrating a steno-occlusion of the left internal carotid artery (ICA) with numerous pial collaterals visualized (arrow). The right internal carotid appears normal. (E) MM035, II-1. MRA demonstrating a bilateral occlusion of the supraclinoid segment of the internal carotid arteries with extensive pial collaterals (arrows). (F) MM035, II-1. MRA demonstrating bilateral stenosis of the posterior cerebral arteries (arrows). (G and H) M117, II-1. Right side middle cerebral artery (MCA) occlusion without involvement of the ICA, but with mild basal moyamoya collaterals and extensive collaterals from the posterior cerebral artery and anterior cerebral artery (ACA). (I) M117, II-1. Left side occlusion of the ACA and MCA, narrowing of the terminal part of the ICA, residual perfusion from the choroid artery, and leptomeningeal collaterals.