目的:CLCN4变异最近被确定为X连锁神经发育障碍的遗传原因。这项研究旨在拓宽CLCN4相关疾病的表型谱,并将其与CLCN4变体的功能后果相关联。
方法:我们描述了13例CLCN4相关神经发育障碍患者。我们使用异源表达分析了未报道的变体的功能后果,生物化学,共聚焦荧光显微镜,膜片钳电生理学,和小基因剪接分析。
结果:我们确定了五个新颖的(p。R41W,p.L348V,p.G480R,p.R603W,c.1576+5G>A)和三个已知(p。T203I,p.V275M,13例中国患者中p.A555V)致病性CLCN4变异。p.V275M变体以高频率被发现并且在四个无关的个体中可见。所有患者均患有全球发育迟缓(GDD)/智力残疾(ID)。八个人都有癫痫发作,其中62.5%发展为难治性癫痫。五名没有癫痫发作的个体显示中度至重度GDD/ID。在大多数患者中,发育延迟先于癫痫发作。变体p.R41W,p.L348V,和p.R603W损害ClC-4的阴离子/交换功能。p.R41W部分损害ClC-3/ClC-4缔合。p.G480R降低ClC-4表达水平并损害与ClC-3的异源二聚化。c.1576+5G>A变体导致外显子10的22bp缺失。
结论:我们进一步定义和拓宽了CLCN4相关神经发育病症的临床和突变谱。p.V275M变异可能是中国患者潜在的热点CLCN4变异。五个新的变体导致ClC-4的功能丧失。运输功能障碍,蛋白质不稳定,细胞内运输缺陷,或ClC-4寡聚化失败可能导致导致CLCN4相关神经发育障碍的病理生理事件。
OBJECTIVE: CLCN4 variations have recently been identified as a genetic cause of X-linked neurodevelopmental disorders. This study aims to broaden the phenotypic spectrum of CLCN4-related condition and correlate it with functional consequences of CLCN4 variants.
METHODS: We described 13 individuals with CLCN4-related neurodevelopmental disorder. We analyzed the functional consequence of the unreported variants using heterologous expression, biochemistry, confocal fluorescent microscopy, patch-clamp electrophysiology, and minigene splicing assay.
RESULTS: We identified five novel (p.R41W, p.L348V, p.G480R, p.R603W, c.1576 + 5G > A) and three known (p.T203I, p.V275M, p.A555V) pathogenic CLCN4 variants in 13 Chinese patients. The p.V275M variant is found at high frequency and seen in four unrelated individuals. All had global developmental delay (GDD)/intellectual disability (ID). Seizures were present in eight individuals, and 62.5% of them developed refractory epilepsy. Five individuals without seizures showed moderate to severe GDD/ID. Developmental delay precedes seizure onset in most patients. The variants p.R41W, p.L348V, and p.R603W compromise the anion/exchange function of ClC-4. p.R41W partially impairs ClC-3/ClC-4 association. p.G480R reduces ClC-4 expression levels and impairs the heterodimerization with ClC-3. The c.1576 + 5G > A variant causes 22 bp deletion of exon 10.
CONCLUSIONS: We further define and broaden the clinical and mutational spectrum of CLCN4-related neurodevelopmental conditions. The p.V275M variant may be a potential hotspot CLCN4 variant in Chinese patients. The five novel variants cause loss of function of ClC-4. Transport dysfunction, protein instability, intracellular trafficking defect, or failure of ClC-4 to oligomerize may contribute to the pathophysiological events leading to CLCN4-related neurodevelopmental disorder.