The Charcot-Marie-Tooth Neuropathy (CMTX3) Complex Structural Variation Causes Differential SOX3 Spatiotemporal Expression

Charcot-Marie-Tooth (CMT) neuropathy is a clinically and genetically heterogeneous group of diseases characterised by the length dependent axonal degeneration of peripheral nerves. We previously mapped a rare form of X-linked CMT, CMTX3, to a 5.7-Mb interval on chromosome Xq26.3-q27.1 and excluded the coding region of all known genes in the linkage interval for mutations. Whole genome sequencing subsequently identified a 78-kb region of chromosome 8q24.3, that had been duplicated and inserted into the CMTX3 locus between the genes HAPSTR2 and SOX3. The 78-kb insertion, which contains a partial transcript of ARHGAP39, fully segregated in families with CMTX3 and was absent in neurologically normal controls. To retain the CMTX3 insertion and investigate its consequences in appropriate neuronal tissue, we generated induced pluripotent stem cells (iPSC) from CMTX3 fibroblasts. Using bulk RNA sequencing of patient-derived spinal motor neurons, ARHGAP39 was deemed non-pathogenic by excluding both the formation of novel fusion transcripts and dosage effects from the partial duplication. Subsequent NanoString expression analyses of candidate genes within the CMTX3 locus, across different stages of neuronal differentiation, identified spatiotemporal dysregulation of SOX3. NanoString showed reduced SOX3 expression in patient iPSC. RNA sequencing detected SOX3 downregulation in CMTX3 neuroepithelial progenitor cells, which was further confirmed by quantitative proteomics. Given the early onset and relatively rapid progression of CMTX3, these data prioritise SOX3 as a leading candidate gene, consistent with its role as one of the earliest transcription factors expressed in the developing nervous system and a key regulator of neuronal fate.