{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,3]],"date-time":"2026-06-03T16:53:14Z","timestamp":1780505594920,"version":"3.54.1"},"reference-count":33,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2023,9,15]],"date-time":"2023-09-15T00:00:00Z","timestamp":1694736000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Neurol."],"abstract":"<jats:sec><jats:title>Objective<\/jats:title><jats:p>The dysfunction of the <jats:italic>CLCN4<\/jats:italic> gene can lead to X-linked intellectual disability and Raynaud\u2013Claes syndrome (MRXSRC), characterized by severe cognitive impairment and mental disorders. This study aimed to investigate the genetic defects and clinical features of Chinese children with <jats:italic>CLCN4<\/jats:italic> variants and explore the effect of mutant ClC-4 on the protein expression level and subcellular localization through <jats:italic>in vitro<\/jats:italic> experiments.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>A total of 401 children with intellectual disabilities were screened for genetic variability using whole-exome sequencing (WES). Clinical data, including age, sex, perinatal conditions, and environmental exposure, were collected. Cognitive, verbal, motor, and social behavioral abilities were evaluated. Candidate variants were verified using Sanger sequencing, and their pathogenicity and conservation were analyzed using <jats:italic>in silico<\/jats:italic> prediction tools. Protein expression and localization of mutant ClC-4 were measured using Western blotting (WB) and immunofluorescence microscopy. The impact of a splice site variant was assessed with a minigene assay.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>Exome analysis identified five rare <jats:italic>CLCN4<\/jats:italic> variants in six unrelated patients with intellectual disabilities, including two recurrent heterozygous <jats:italic>de novo<\/jats:italic> missense variants (p.D89N and p.A555V) in three female patients, and two hemizygous missense variants (p.N141S and p.R694Q) and a splicing variant (c.1390-12T &amp;gt; G) that are maternally inherited in three male patients. The p.N141S variant and the splicing variant c.1390-12(T &amp;gt; G were novel, while p.R694Q was identified in two asymptomatic heterozygous female patients. The six children with <jats:italic>CLCN4<\/jats:italic> variants exhibited a neurodevelopmental spectrum disease characterized by intellectual disability (ID), delayed speech, autism spectrum disorders (ASD), microcephaly, hypertonia, and abnormal imaging findings. The minigene splicing result indicated that the c.1390-12T &amp;gt; G did not affect the splicing of <jats:italic>CLCN4<\/jats:italic> mRNA. <jats:italic>In vitro<\/jats:italic> experiments showed that the mutant protein level and localization of mutant protein are similar to the wild type.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusion<\/jats:title><jats:p>The study identified six probands with <jats:italic>CLCN4<\/jats:italic> gene variants associated with X-linked ID. It expanded the gene and phenotype spectrum of <jats:italic>CLCN4<\/jats:italic> variants. The bioinformatic analysis supported the pathogenicity of <jats:italic>CLCN4<\/jats:italic> variants. However, these <jats:italic>CLCN4<\/jats:italic> gene variants did not affect the ClC-4 expression levels and protein location, consistent with previous studies. Further investigations are necessary to investigate the pathogenetic mechanism.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fneur.2023.1096969","type":"journal-article","created":{"date-parts":[[2023,9,18]],"date-time":"2023-09-18T06:33:42Z","timestamp":1695018822000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":6,"title":["Novel variants in the CLCN4 gene associated with syndromic X-linked intellectual 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