Genotype-phenotype correlation in CLCN4-related developmental and epileptic encephalopathy

Abstract

CLCN4-related disorder is a rare X-linked neurodevelopmental condition with a pathogenic mechanism yet to be elucidated. CLCN4 encodes the vesicular 2Cl -/H + exchanger ClC-4, and CLCN4 pathogenic variants frequently result in altered ClC-4 transport activity. The precise cellular and molecular function of ClC-4 remains unknown; however, together with ClC-3, ClC-4 is thought to have a role in the ion homeostasis of endosomes and intracellular tra cking. We reviewed our research database for patients with CLCN4 variants and epilepsy, and performed thorough phenotyping. We examined the functional properties of the variants in mammalian cells using patch-clamp electrophysiology, protein biochemistry, and confocal uorescence microscopy. Three male patients with developmental and epileptic encephalopathy were identi ed, with differing phenotypes. Patients #1 and #2 had normal growth parameters and normalappearing brains on MRI, while patient #3 had microcephaly, microsomia, complete agenesis of the corpus callosum and cerebellar and brainstem hypoplasia. The p.Gly342Arg variant of patient #1 signi cantly impaired ClC-4's heterodimerization capability with ClC-3 and suppressed anion currents. The p.Ile549Leu variant of patient #2 and p.Asp89Asn variant of patient #3 both shift the voltage dependency of transport activation by 20 mV to more hyperpolarizing potentials, relative to the wild-type, with p.Asp89Asn favouring higher transport activity. We concluded that p.Gly342Arg carried by patient #1 and the p.Ile549Leu expressed by patient #2 impair ClC-4 transport function, leading to epilepsy and developmental impairment without clear brain malformation. In contrast, p.Asp89Asn variant results in a gain-of-transport function in ClC-4, causing severe brain malformation with associated developmental impairment, epilepsy, microcephaly, and microsomia.