Despite the rapid growth in the number of genes associated with Charcot-Marie-Tooth disease in the last decade, many patients with axonal forms lack a genetic diagnosis.

A pedigree was constructed, based on family members' clinical data. Next-generation sequencing of nuclear and mitochondrial DNA (mtDNA) was performed for three affected family members. Muscle biopsies from four family members were used for analysis of muscle histology and ultrastructure, mtDNA sequencing, and RNA quantification. Biopsies of the lateral femoral cutaneous nerve were collected from two affected family members for histologic and ultrastructural studies.

Clinical examination and electrodiagnostic testing showed a motor and sensory polyneuropathy with predominantly axonal features. Pedigree analysis revealed inheritance only through the maternal line, a pattern consistent with a mitochondrial DNA disorder. Whole exome sequencing and mtDNA sequencing identified a mutation in the mtRNA-Val gene, m.1661A>G, which is predicted to disrupt a Watson-Crick base pair in the T-loop stem of this tRNA. This mutation was present at nearly 100% heteroplasmy in both blood and skeletal muscle. Muscle biopsies showed chronic denervation/reinnervation changes, with subtle abnormalities suggestive of mitochondrial pathology, while biochemical analysis of electron transport chain (ETC) enzyme activities showed reduction in multiple components of the ETC. Northern blots from skeletal muscle total RNA showed reduction in the quantity of mtRNA-Val, but not mtRNA-Leu, in subjects compared to unrelated age- and sex-matched controls. Nerve biopsies from two affected family members demonstrated ultrastructural mitochondrial abnormalities (hyperplasia, hypertrophy and crystalline arrays) consistent with a mitochondrial neuropathy.

Our findings identify a previously unreported cause of Charcot-Marie-Tooth (CMT) disease, a mutation in the mitochondrial tRNA-Valine, in a large Venezuelan family. This work expands the list of CMT-associated genes from protein-coding genes to a mitochondrial tRNA.