Repeat expansion panel analysis for undiagnosed inherited peripheral neuropathies: methodological validation and clinical characterization of a NOTCH2NLC-related axonal CMT family

Background and objectives Charcot–Marie–Tooth disease (CMT), as the most common type of inherited peripheral neuropathy (IPN), is a heterogeneous group of diseases characterized by degeneration of peripheral motor and sensory nerves, while 35–70% of CMT patients remain genetically undiagnosed. Recent studies implicate pathogenic repeat expansions (e.g., NOTCH2NLC , RFC1 , LRP12 ) in IPNs. We evaluated whether targeted repeat expansion screening improves molecular diagnosis in PMP22 CNV-negative and WES-negative IPN patients. Methods We screened 22 genetically undiagnosed Chinese IPN index patients (negative for PMP22 CNV and WES) for repeat expansions across a 51-gene panel using targeted long-read sequencing (PacBio Sequel II) for 47 genes, and RP-PCR and AL-PCR used to validate 4 loci with low capture efficiency owing to their high-GC content that impairs PCR amplification. For a NOTCH2NLC -positive family, promoter CpG methylation was assessed by next-generation sequencing-based bisulfite sequencing PCR. Results We identified a pathogenic GGC repeat expansion in NOTCH2NLC (90–119 repeats) segregating in four affected individuals from a four-generation Han Chinese axonal CMT family, with a cohort diagnostic yield of 1/22 (4.5%). Affected individuals manifested adolescent-to-middle-age onset, slowly progressive axonal motor-sensory neuropathy with oculofaciopharyngeal involvement and marked intrafamilial clinical heterogeneity. Serial EMG suggested early myogenic features evolving to neurogenic changes. Thigh MRI demonstrated distal-predominant fatty replacement and atrophy. Serial brain MRI revealed slowly progressive periventricular white matter hyperintensity without corticomedullary junction DWI abnormalities. Methylation analysis showed that no differences in methylation levels were observed at most CpG sites (43/47) in the NOTCH2NLC promoter region among the six family members, with only four CpGs showing inter-individual differences. Conclusion A stepwise repeat expansion panel offers a diagnostic avenue for IPN patients negative for PMP22 CNV and WES. However, NOTCH2NLC repeat expansion size and promoter methylation did not explain the pronounced intrafamilial heterogeneity.