A Novel Mutation In GJB1(c.185 G>C) In A Family With X-linked Charcot-marie-tooth Disease:Pedigree Analysis And Functional Research

X-linked Charcot-Marie-Tooth disease(CMTX)is one of the most common types of hereditary motor sensory neuropathy with a high degree of clinical and genetic heterogeneity.Usually this syndrome presents in the first 2 decades of the life in most of the patients,characterized by progressive symmetrical limb far-end weakness and muscle atrophy,sensory abnormalities and absent deep tendon reflexes.It progress gradually,resulting in severe disability and affecting quality of life.CMTX can be divided into six types according to genetic patterns and mutant genes,CMTX1 is caused by mutations in gene of gap junction beta 1(GJB1)located in chromosome Xq13.1 encoding the gap junction beta 1 protein connexin 32(Cx32).CMTX1 accounts for 90%of CMTX,and has become the second most common genetic variant of CMT.CMTX1 is X-linked dominant inheritance.The clinical features of CMTX 1 are similar with other types of Charcot-Marie-Tooth disease(CMT),however,some patients get the central nervous system involved;males are more severely than females.At present,there is no effective therapy available for CMTX1.Gene diagnosis can provide a reliable basis for genetic counseling,which can inform patients of the chance that they will pass CMTX1 on to their children.To date,more than 400 distinct mutations in the GJB1 gene have been identified,but the relationship between genotype with phenotype and the specific pathogenic mechanism of different mutant forms is still unclear.So it is worth performing careful clinical analysis,genetic testing and function research of novel mutation in GJB1 gene to the CMTX1 family,which can provide important clues to the pathogenesis of CMTX1.Objectives:We analyzed the clinical and genetic characteristics of a CMTX1 family.Genetic analysis was performed to identify the causing gene mutation.GJB1(c.185 G>C)plasmid was build to confirm the influence on GJB1 gene functions and the pathogenesis of this new gene mutation.The potential mechanism was discussed.Methods:1.Detailed clinical data and lab results of the patient,the affected members and other family members were collected.Genetic characteristics of the affected family were analyzed.2.The peripheral blood samples of 5 members of the affected family were collected and DNA was extracted.High throughput sequencing was used for screening the potential pathogenic genes of the proband.Sanger sequencing was provided to the 5 members of the family.3.GJB1(c.185 G>C)mutant plasmid and wild-type plasmid were transfected into Schwann cells individually.Functional experiments were performed to examine the role of the mutant gene on Schwann cells.Results:1.In this study,the symptoms of peripheral neuropathy in male patients were more obvious,on the other hand female patients were mild.The electrophysiological examination was in accordance with intermediate type.Pedigree analysis was consistent with X dominant genetic characteristics.One of the family members suffered from transient central nervous system dysfunction accompanied by white matter changes observed by MRI after travelling to high altitude area.2.Gene analysis revealed that mutation of GJB1 gene c.185(G>C)presented in 3 members of this family,which was not been reported before.3.GJB1 mRNA expression and Cx32 protein expression decreased in Schwann cells transfected with GJB1(c.185 G>C)mutant plasmid.No gap junction plaques were detected in the cell membrane surface.Intercellular calcium ions flow was reduced.The proliferation of Schwann cells decreased,however the apoptosis increased.PMP22 and MPZ genes,which are associated with the formation of myelin,were down regulated and the corresponding protein expression decreased.Conclusions:1.Male patients showed a more severe phenotype than female in CMTX1 patients,electrophysiological examination was in accordance with intermediate type,some members of the CMTX1 family may manifest with the central nervous system abnormalities.2.Mutation of GJB1 gene 185(G>C)can lead to CMTX1.3.GJB1 gene 185(G>C)mutation affects the formation of gap junction plaques and peripheral nerve myelin sheath result in peripheral neuropathy.