Investigating mechanisms of the CMTX3 insertion using induced pluripotent stem cell (iPSC) derived motor neurons

Abstract

Charcot-Marie-Tooth neuropathy (CMT) is an incurable genetic condition characterised by the length dependent degeneration of motor and sensory nerves. CMTX3 is a rare, X-linked form of CMT, caused by a ~78 kb interchromosomal insertion of chromosome 8q24.3 into Xq27.1. This structural variation (SV) does not disrupt the sequence of known genes, and the pathomechanism remains unknown. It has been hypothesised that the CMTX3 SV may cause disease by dysregulating the expression of nearby genes. Given the tissue-specific nature of transcriptional regulation, it is essential that disease-relevant tissue is used to investigate this hypothesis. Therefore, patient-derived induced pluripotent stem cells (iPSC) were used to obtain neuronal tissue harbouring the complex SV. iPSC were differentiated into caudal neuroepithelial progenitors (NEP), motor neuron progenitors (MNP), and spinal motor neurons (sMN). Transcriptomic analyses showed dysregulation of SOX3 in CMTX3 patient iPSC and NEP, but not in the later developmental stages. This provided evidence that the CMTX3 SV can alter the spatiotemporal regulation of nearby genes. This result has identified SOX3 as a high-priority candidate gene for CMTX3 neuropathy. Promoter Capture Micro-C, a powerful technique to assess regulatory chromatin interactions, was used to investigate the potential mechanisms driving the observed gene dysregulation in CMTX3 NEP. It was theorised that the CMTX3 SV may form ectopic chromatin interactions with genes localising to the CMTX3 linkage region. Although the results of this preliminary experiment were inconclusive, the study flagged potential interactions of interest that can be addressed in future studies. The results of this thesis represent significant progress towards elucidating the pathomechanism of CMTX3 neuropathy and has generated an invaluable pre-clinical model that will continue to inform disease biology and guide future development of treatment therapies.