| Background and purposeCharcot-Marie-Tooth(CMT)disease refers to a group of sensorimotor inherited neuropathies,with a prevalence estimated at up to 40 individuals in every 100,000.The classic CMT phenotype reflects length-dependent axonal degeneration characterized by progressive muscle weakness and atrophy of the distal extremities,distal sensory loss,reduced or absent deep tendon reflexes,and foot deformities.The disease is highly clinically heterogeneous.On the basis of nerve conduction studies and nerve pathology,CMT is divided into three main subtypes:demyelinating CMT(CMT1)with the median motor nerve conduction velocity(MNCV)below 25m/s,axonal CMT(CMT2)with MNCV above 45m/s,and intermediate CMT with MNCV between 25 and 45m/s.To date,CMT has mutated more than 100 genes.However,in the two Chinese CMT cohort studies reported in2019,the molecular diagnostic rates reached 63.8% and 84.4%,respectively,and20% –40% of CMT patients had no genetic cause.there is still no effective pharmacologic treatment for any form of CMT so far.Recent research studies are focused on developing new treatment strategies,and multiple clinical trials are ongoing or being planned.Some have achieved good preliminary results,but no clinical trial has been performed in CMT patients of Chinese populations yet.To fill this gap and increase the understanding of CMT subtypes,we assessed 202 unrelated CMT patients in baseline to provide a comprehensive overview of the clinical,genetic,and electrophysiology profile of Charcot-Marie-Tooth disease in China.Based on the cohort,rigorous clinical trials such as drug development can be conducted in the future MethodA CMT cohort was established,and number NCT04010188 was registered on Clinicaltrials.gov in September 2019.All patients were corresponded with the standards for the diagnosis of distal symmetrical peripheral neuropathy published by the American Academy of Neurology,American Association of Neuromuscular and Electrodiagnostic Medicine,and American Academy of Physical Medicine and Rehabilitation in 2019.Patients those diagnosed as toxic and inflammatory peripheral neuropathy and secondary chronic peripheral neuropathy caused by other diseases were excluded.Neurologic examinations including clinical presentations,physical examinations,laboratory tests,Electromyography(EMG),and CMT neuropathy score(CMTNS),the Overall Neuropathy Limitations Scale(ONLS),Six-Minute Walk Test,Nine Hole Peg Test were conducted by two fixed clinicians.All clinical data is entered and managed through the "Yiduyun" data platform.For the CMT patients to be diagnosed,the most common genetic mutation screening is carried out according to the classification: 17p11.2 fragment copy number detection for CMT1 patients;MFN2 exon detection for CMT2 patients;patients;GJB1 exon detection exon detection for intermediate CMT.Patients with negative results were further tested for pathogenic mutations by whole exon sequencing,and the detected candidate genes were screened and verified.The two new sites of ATP1A1,c.620C> T(p.S207F)and c.2629G> A(p.G877S),were further tested in vitro to investigate its pathogenicity.Skin biopsy established the fibroblast cell line of ATP1A1 patients.Fluorescence quantitative PCR and Western blotting were used to analyze the expression levels of ATP1A1 gene m RNA and protein.The ATP1A1 new mutation site plasmid were constructed,further transfected into 293 T cells,and intervene with cycloheximide(CHX)and inhibitor MG132 to detect the expression level and stability of the mutant protein.ResultA total of 202 CMT cases have been collected,distributed from 137 families,most of them from southern of China.The male to female ratio is 1.4: 1.Most patients have their first symptoms at 16-20 years of age,but the peak age at diagnosis is 26-38 years old.In the typical peripheral nervous system symptoms,patients with the three type have typical manifestations of muscle weakness and/or muscular atrophy and paresthesia,but the proportion of people using orthotics and walking aids is low.Clinical symptoms outside the peripheral nervous system are only found in CMT2 and intermediate CMT,which have implications for disease typing and selection of genetic testing methods.The three subtypes of nerve conduction study showed different degrees of incubation period extension and conduction velocity and amplitude reduction,of which CMT1 type was the most serious.A needle electromyography suggests that the incidence of positive sharp waves is related to the severity of the disease.A total of 78.71%(159/202)of the cases in this study were genetically diagnosed.The 17p11.2 repeat accounted for 35.15% of all CMT cases and76.34% of CMT1 cases.MFN2 is the most common pathogenic gene of CMT2 type,accounting for 57.69% of all cases with positive gene diagnosis.In the intermediate CMT,the mutation rate of GJB1 is as high as 57.45%.At the same time,13 new mutation sites were detected.According to ACMG standards and guidelines,2 were classified as "pathogenic",7 were classified as "probable pathogenic",and the remaining 4 were classified as "ambiguous" ".It was also found that the two positions of GJB1 and ATP1A1 genes were chimera mutations.In vitro functional experiments suggest that the two new mutation sites c.620C> T(p.S207F)and c.2629G> A(p.G877S)of the ATP1A1 gene both lead to decrease protein expression and affect protein stability,and the mutant protein accelerates degradation by the proteasome pathway.ConclusionWe established a cohort of CMT patients and analyzed the clinical,electrophysiological and genetic mutation characteristics of CMT patients in the database,providing a natural history of CMT patients’ disease,which has enriched the clinical phenotype spectrum of CMT in China to a certain extent.To promote the development of CMT clinical trials and molecular therapies in China.All-exon sequencing can efficiently and accurately locate the rare pathogenic mutation genes and improve the diagnosis rate of CMT patients.This study found14 new disease-causing mutation sites and 2 chimera mutation sites,which enriched the Chinese CMT gene mutation database,and were of great significance for genetic counseling and prenatal diagnosis of CMT.In this study,in vitro functional experiments were used to confirm the pathogenicity of the new ATP1A1 mutation site,expand the ATP1A1 gene pathogenic mutation spectrum,and preliminary explore the effect of ATP1A1 mutation on protein stability. |
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