Patient-derived neural organoids reveal developmental impairments associated with a novel GJB1 mutation in X-linked Charcot-Marie-Tooth disease

Charcot-Marie-Tooth disease (CMT) is one of the most prevalent inherited peripheral neuropathies. CMT type X1 (CMTX1), caused by mutations in GJB1 gene, represents the most common X-linked subtype with central nervous system (CNS) involvement. Here, we report the identification and functional characterization of a novel GJB1 variant (c.554C > T, p.Thr185Ile) in a CMTX1-affected family and its pathogenic impact using patient-derived induced pluripotent stem cells (iPSCs) and three-dimensional (3D) neural organoid models. The GJB1 gene encodes connexin 32 (Cx32), a gap junction protein. Immunofluorescent analysis revealed aberrant intracellular reduction and aggregation of the mutant Cx32 protein, suggesting impaired gap junction function. iPSC-derived neural organoids carrying the GJB1 mutation exhibited significant delay in neural differentiation and disrupted neural rosette organization. These findings underscore the critical role of Cx32 in neural development and provide a physiologically relevant platform for underlying CMTX1 pathological mechanisms on central nervous system. The established GJB1 -variant organoid model holds promise for investigating genotype-phenotype correlations and facilitating the development of targeted therapeutic strategies for CMTX1. • A novel GJB1 c.554C > T variant was identified in a CMTX1 family. • GJB1 mutation causes mis-location and reduced levels of the Cx32 protein. • Patient iPSC-derived neural organoids showed disrupted neural organization. • RNA-Sequencing revealed impaired cell adhesion and neurodevelopment in mutant cells.