Phenotypic And Genotypic Features Of X-linked Charcot-Marie-Tooth Disease Type 1 And The Study Of Mutant Cx32 Protein Expression

Background and objiective : Charcot-Marie-Tooth?CMT?is a clinically and genetically heterogeneous group of inherited neuropathies characterized by progressive distal limb muscle weakness,atrophy and distal sensory loss.X-linked Charcot-Marie-Tooth type 1?CMTX1?,which is caused by mutations in the gap junction protein beta-1?GJB1?,also known as connexin 32?Cx32?gene,and is the second most common form of CMT.Despite of researching widely,the clinical and genetic characteristics of the Chinese CMTX1 population have not been reported,and its pathogenesis is currently unclear.In this research,we aimed to analyze and summarize the clinical manifestations as well as genetic features of eight CMTX1 families.Furthermore,we also explored the pathogenesis of GJB1 gene mutations.Methods:We collected eight CMTX1 families.The age of the probands were 11 to 30 years,showing weakness of the distal limbs,muscle atrophy,decreased tendon reflex and decreased sensation.The proband 1 showed paroxysmal aphasia,and the head magnetic resonance showed reversible white matter changes;the proband 5 showed nystagmus and ataxia;the proband 7 had no central nervous system involvement,and the MRI is a change in white matter.Eight patients with probands underwent neurophysiological examination and were further evaluated using the Charcot-Marie-Tooth Disease Rating Scale Version 2?CMTNS2?.At the same time,200 healthy controls were selected and Sanger sequencing was used to analyze the GJB1 gene mutation sequence.The pc3.1-GFP-N1-m Cx32 mutant plasmid was constructed,and the recombinant plasmid was transfected into He La cells.The expression of the mutant Cx32 recombinant protein in He La cells was detected by fluorescent staining.Results:The main symptoms of CMTX1 patients are distal muscle weakness and mild sensory disturbance,which meets the diagnostic criteria of CMTX1.Peripheral neurophysiological examination of all probands suggested that the motor and sensory conduction velocity slowed down,and the amplitude of the compound muscle action potential decreased,further confirming the "intermediate type" of CMT classification,and found that MNCV combined with patients with central nervous system lesions The CMAP amplitude is lower than that of patients with only the peripheral nervous system.The pathological examination of the sural nerve of seven probands?probands 1 to 7?suggested mixed neuropathy.We also found five GJB1 missense variants?c.59T>C?c.379A>T?c.425G>A?c.533A>G?c.580A>G?c.590C>T?,one GJB1 insertion variant?c.403₄04ins T?and one GJB1 synonymous variant?c.30C>T?were detected eight families GJB1,and two mutations were located at the N-terminus,one mutation was located in the intracellular loop,two mutations were located in the third transmembrane domain,and two mutations was located in the fourth transmembrane domain.Analysis of the clinical phenotype of CMTX1 and the location of GJB1 mutation revealed that the distribution of Cx32 mutant protein was not associated with CNS symptoms.The expression of recombinant Cx32 protein in He La cells,wildtype Cx32 protein on the surface of He La cell membrane,punctate and small clusters,expressed in the cytoplasm and perinuclear cells;C30T,T59 C,G425A mutations in spots and small clumps distribution in the membrane and cytoplasm,Cx32 protein expression decreased;403₄04ins T,A533 G,A580G mutations were flaky,no clump-like substances,cell expression decreased;mutant A379 T mainly accumulated in the cytoplasm,The nucleus was squeezed at one end of the cell;the A533 G mutation was only expressed in the perinuclear nucleus;the C590 T expression was similar to the wild type,with a dot-like distribution on the membrane and in the cytoplasm,but less than the wild type.Conclusion:The clinical manifestations of CMTX1 are mainly myasthenia in the distal extremity,which may be associated with central nervous system involvement,but there is no correlation between the clinical phenotype and the location of the mutation site of the transmembrane structure.Neurophysiological examination revealed that the myelin and axon were involved at the same time,showing an intermediate type of change.Pathological features thin myelin sheath,onion ball,regenerative cluster,some may be accompanied by inflammatory cell infiltration.The pathogenesis of CMTX1 can be caused by Cx32 mutations.Cx32 protein is completely or partially retained in cells,and it is difficult to reach the membrane and form gap junction protein plaques or form gap junctions without gap junction function.Whether the GJB1 gene mutation affects the gap junction plate and its functional changes remains to be further studied.