Mutation Analyses Of Charcot-marie-tooth Disease And Functional Study Of GDAP1

Charcot-Marie-Tooth(CMT)disease refers to a group of sensorimotor inherited neuropathies with an estimated frequency of 4 in 10000 individuals.Up to now,more than 80 genes have been demonstrated to be associated with CMT.Owing to CMT's clinical and genetic heterogeneity,it is expensive and time-consuming to screen all the possible causative genes using conventional Sanger sequencing.Consequently,high-throughput targeted next-generation sequencing(NGS)combined multiplex ligation-dependent probe amplification(MLPA)was employed in genetic diagnosis of CMT.Additionally,Sanger sequencing in the 5'-UTR(untranslated region)of GJB1 was supplemented due to the region was not covered in NGS.MLPA was firstly performed in all subjects and NGS was carried out among those patients without PMP22 duplication or deletion.Clinical data and molecular characterization were analyzed and summarized.Functional studies were carried out if variants were assigned as ‘uncertain significance' according to the American College of Medical Genetics and Genomics(ACMG).Section I: Mutation analysis and genotype-phenotype correlation in CMT patientsObjective: To evaluate the efficiency of MLPA combined targeted NGS in patients with CMT and perform a comprehensive analysis of clinical phenotypes and genetic distributions of a cohort of patients with Charcot-Marie-Tooth disease(CMT)in southeast China.Methods: One hundred and fifty-two unrelated CMT patients were enrolled from southeast China.We performed multiplex ligation-dependent probe amplification(MLPA)testing in all patients and next-generation sequencing(NGS)among those patients without PMP22 rearrangements.ACMG standards and guideline was used to interpret the sequence variants identified by targeted NGS.Results: We obtained a genetic diagnosis in 109(72.7%)patients.PMP22 duplications were identified in 40 patients and deletions in 12 patients.In addition,we found 22 novel variants and 36 known mutations in 57 patients.PMP22,GJB1 and MFN2 are the most common causative genes in CMT1(demyelinated form),intermediate CMT,and CMT2(axonal form),respectively.In this study,we identified a higher proportion of intermediate CMT,a relatively high frequency of NDRG1 mutations and clinical features of later onset age in CMT1 A patientsConclusions: Our results broaden the genetic and clinical spectrum of CMT patients,which can help optimize the genetic and clinical diagnosis.Section II: Functional characterization of novel variants in GDAP1Objective: To characterize the biochemical and biological effects of novel variants in GDAP1(p.L26 R,p.S169 fs and c.490+1G>A)identified in CMT patients using targeted NGS.Methods: Mitochondrial structure tissue was observed by electron microscope.Primary fibroblast cell lines were established from skin biopsies with punches.The effects of variants in GDAP1 to mitochondrial dynamics and expression level of protein were analyzed by Western Blotting and immunofluorescence study.The effects of variants in GDAP1 to mitochondrial ATP levels were analyzed using bioluminescence assay.Splicing reporter minigene assay was used to evaluate the effect on splicing of the unclassified genetic variant.Results: Electron microscope study confirmed damage of mitochondrial structure in muscular tissue of patient carried p.L26 R and p.S169 fs.Immunofluorescence showed abnormal mitochondrial networks.p.L26 R and p.S169 fs mutations can lead to abnormal distribution of mitochondrial networks.Western Blotting results indicated that mutations of p.L26 R and p.S169 fs lead to reduction of protein expression level of Gdap1 and Drp1.Bioluminescence assay showed decreased mitochondrial ATP production in mutational fibroblast.Splicing reporter minigene assay represented the identification of pathogenic splicing mutations(c.490+1G>A).Conclusions: Our study confirmed the pathogenicity of novel variants in GDAP1,and preliminarily described the effect of GDAP1 mutations on mitochondrial function.