Identification and functional characterization of novel GDAP1 variants in Chinese patients with Charcot-Marie-Tooth disease

Multiplex ligation-dependent probe amplification (MLPA) and whole-exome sequencing (WES) were performed in 30 unrelated CMT patients. Minigene assay was used to verify the effect of a novel splicing variant (c.694+1G>A) on pre-mRNA. Primary fibroblast cell lines were established from skin biopsies to characterize the biological effects of the novel variants p.L26R and p.S169fs. The mitochondrial structure was observed by an electron microscope. The expression level of protein was analyzed by Western Blotting. Mitochondrial dynamics and mitochondrial membrane potential (MMP, Δψm) were analyzed via immunofluorescence study. Mitochondrial ATP levels were analyzed via bioluminescence assay. The rate of oxygen consumption was measured with a Seahorse Bioscience XF-96 extracellular flux analyzer.

To identify and characterize the pathogenicity of novel variants in Chinese patients with Charcot-Marie-Tooth disease.

We identified 10 pathogenic variants in three known CMT related genes, including three novel variants (p.L26R, p.S169fs, c.694+1G>A) and one known pathogenic variant (p.R120W) in GDAP1. Further, we described the clinical features of patients carrying pathogenic variants in GDAP1 and found that almost all Chinese CMT patients with GDAP1 variants present axonal type. The effect of c.694+1G>A on pre-mRNA was verified via minigene splice assay. Cellular biological effects showed ultrastructure damage of mitochondrial, reduced protein levels, different patterns of mitochondrial dynamics, decreased mitochondrial membrane potential (Δψm), ATP content, and defects in respiratory capacity in the patient carrying p.L26R and p.S169fs in GDAP1. Interpretation: Our results broaden the genetic spectrum of GDAP1 and provided functional evidence for mitochondrial pathways in the pathogenesis of GDAP1 variants.