The aim of this study was to identify novel or rare genetic variants in Charcot–Marie–Tooth (CMT) patients in whom common CMT genes had previously been excluded, and to assess their pathogenicity through morphological, molecular, and in-silico analyses.

Twelve patients (median age 28; range 21–50) with clinically and electrophysiological confirmed CMT were included in this study. Clinical parameters, CMTNSv2 scores, and sural nerve biopsies were analyzed. WES was performed, followed by Sanger sequencing confirmation, in-silico prediction, and functional studies.

All biopsies demonstrated demyelinating pathology with large fiber loss, thinly myelinated fibers, irregular myelin folding, and variable axonal degeneration. WES identified variants in INF2, GJB1, EGR2, SORD, ITPR3, and FBLN5 genes. INF2 included a benign heterozygous missense (c.3703C>T, p. Pro1235Ser) and a novel intronic deletion (c.104715395delG) with no effect on protein expression. A heterozygous c.379A>C (p.Ile127Leu) variant in GJB1, consistent with CMTX1, was identified; XCI analysis showed a random inactivation pattern in the patient’s nerve tissue. A heterozygous duplication in EGR2 (c.364_369dupCCTCCT, p. Pro172-Pro173dup) was detected; in-silico analysis showed no significant impact on protein structure. Other rare variants included a pathogenic homozygous SORD deletion (c.757del) and heterozygous ITPR3 and FBLN5 variants of uncertain significance.

These findings emphasize the value of integrating genetic, morphological, and molecular analyses for accurate interpretation of novel and rare CMT variants and highlight the need for further functional studies to clarify their phenotypic impact and improve genotype–phenotype correlations.