Genetic Characteristics Of Early Onset Charcot-Marie-Tooth Disease And The Functional Study Of A Novel MPZ Mutation

Part1 Genetic characteristics of early onset Charcot-Marie-Tooth diseaseObjectives:Charcot-Marie-Tooth disease is an inherited group of peripheral neuropathies with genetically heterogeneous.Charcot-Marie-Tooth disease is a complex molecular disorder with over a 1000 different putative mutations in 80 disease-associated genes.Due to the development and application of next-generation sequencing,whole exon sequencing,multiplex ligation-dependent probe amplification and other technologies,we make our knowledge of the disease much deeper.But on the other hand,with the increased complexity of sequencing technology,this paradigm shift in genetic testing has been accompanied by new challenges in sequence interpretation.Based on the phenotype and genotype of each patient,mutation analysis of disease genes and identification of diagnostic procedures for gene diagnosis are particularly important in today's clinical work.That is helpful for the complete understanding of the disease and can provide guarantee for the follow-up prevention and treatment.Methods:Collected the clinical data of children with Charcot-Marie-Tooth disease diagnosed in Children's Hospital,Zhejiang University School of Medicine from April 2014 to March 2018,including sex,age of visiting doctor,age of onset,symptoms of onset,family history,nervous system signs,electromyogram and Charcot-Marie-Tooth disease related genes.Result:1.In this study,14 cases of Charcot-Marie-Tooth disease were diagnosed in childhood during these 4 years.The ratio of male to female was 2.5:1?10:4?.The age distribution ranged from 1.8 to 9 years.Among them,13 cases started symptoms less than 2 years old,6 cases delayed developmental milestone.2.According to the electromyography,10 children were classified in Type 1,1 child in dominant intermediate type and 3 children in Type 2.The compound muscle action potential of median nerve in 6 of 10 children with Type 1 was lower than normal.Also the sensory nerve conduction velocity and sensory nerve action potentials of median nerve were not elicited.3.The gene detection results of these 14 patients showed that 11 cases were known mutations and 3 cases were new mutations.The mutations included PMP22 duplication mutation?6 cases?,MPZ insertion mutation and point mutation?4 cases?,MFN2 point mutation?3 cases?and NEFL point mutation?1 case?.6 of them had positive family history.Three new mutations were MPZ c.103₁04insTGGTTTACACCG?p.D35delins VVYTD?,MPZ c.394C>G?p.P132A?and MFN2 c.326A>G?p.K109R?.4.Child with heterozygous mutation of the MPZ gene c.103₁ 04 insTGT TACACCG?p.D35 delins VVYTD?had a positive family history.This MPZ insertion mutation was rated as "potentially pathogenic".Child with of MPZ c.394C>G?p.P132A?heterozygous mutation was de novo.This MPZ point mutation was rated as"possibly pathogenic".Child with MFN2 c.326A>G?p.K109R?heterozygous mutation was also de novo.This MFN2 mutation rated as "pathogenic".5.Fourteen patients with Charcot-Marie-Tooth disease were confirmed in this article.6 cases of Type 1A were caused by PMP22 gene duplication mutation.4 cases of Type 1B were caused by MPZ gene mutation.3 cases of Type 2A were caused by MFN2 gene mutation.And 1 case of dominant intermediate type caused by NEFL gene mutation.Conclusion:Combined with the clinical manifestations,neuromuscular electrophysiological characteristics,genetic pattern,next-generation sequencing results,further classification of the novel variants according to American College of Medical Genetics and Genomics standards and guidelines,we finally confirmed the phenotype and genotype of CMT of those 14 patients.Part 2 The functional study of a novel MPZ mutationObjectives:MPZ c.103₁04insTGGTTTACACCG?p.D35delinsVVYTD?mutation was a new MPZ mutation.According to the guidelines of the The American College of Medical Genetics and Genomics,population data and co_segregation data are still insufficient to determine the pathogenicity of the new mutation.In this paper,the functional study of this new MPZ mutation was performed to clarify its pathogenicity.Methods:1.In silico analyses:The visualization of the structure and the consequences of the mutations on the 3D structure of the MPZ extracellular domain were carried out using Swiss-Model.2.Construction of plasmids:The wild type plasmid?wtMPZ?and mutant type plasmid?insMPZ?of MPZ were constructed and the plasmids were verified by double enzyme digestion and sequencing analysis.3.Transient transfection:HEK293FT cells and sNF96.2 cells were both transient transfected with both wild-type MPZ and mutant MPZ plasmid.4.2-Deoxy-D-glucose?2-DG?treatment;We treated 24-hour transfected cells with 2 mM 2-DG for 16 h before fixation,staining or Western-blot.For R.T-PCR,24-hour transfected cells were treated 24 h with 2 mM 2-DG.For apoptotic assay,cells were treated with 2 mM 2-DG or same volume of serum-free DMEM for 12 h.5.Experimental groups:?1?wild type group?wtMPZ group???2?wild type +2-DG group?wtMPZ+2-DG group?,?3?mutation group?insMPZ group???4?mutation +2-DG group?insMPZ +2-DG group?.6.Experimental methods:Intercellular adhesion experiments were carried out in each group,and cells aggregation was observed under microscope.The expression levels of GRP78 and Cleaved Caspase 3 in each group were detected by immunofluorescence.The expression mRNA and protein levels of GRP78,CHOP,ATF6 and sXBP-1 in the cells of each group were detected by real-time PCR and Western blot.And the numbers of apoptotic cells was detected by flow cytometry.Result:1.In silico analyses showed this insertion p.D35delinsVVYTD was within the immunoglobulin-like extracellular domain of P0.The mutation caused an increase in the(3-sheet of the secondary structure of P0.This resulted in an alteration of the secondary structure of PO.2.Cell adhesion assay:insMPZ group of HEK293 cells reduced adherence.HEK293 cells transfected with insMPZ formed aggregates significantly larger than cells expressing wild-type PO?P = 0.0003?.3.insMPZ mutant enhanced the expression of GRP78 and sXBP-1.Immunofluorescence,western blotting and RT-PCR all detected an increase of GRP78 in sNF96.2 cells and HEK293 cells in insMPZ group compared with wtMPZ group.After the treatment of 2-DG,the elevation of GRP78 only appeared in wtMPZ.The expression of the CHOP and ATF6 protein and mRNA level had no change between insMPZ and wtMPZ group in HEK293 cells.After the treatment of 2-DG,both protein and mRNA level of CHOP had no change,the protein level of ATF6 elevated in insMPZ group but the mRNA level of ATF6 had no change.Western blotting and RT-PCR demonstrated an upregulation of sXBP-1 in insMPZ group compared with wtMPZ?p=0.018?.Still only wtMPZ group showed response to 2-DG treatment?p=0.013?.4.insMPZ mutant increased apoptosis in Schwann cells and was sensitive to 2-DG treatment.Immunofluorescence demonstrated insMPZ increased cleaved caspase 3 level in sNF96.2 cells.After the treatment of 2-DG,cleaved caspase 3 was significantly increased in insMPZ group.Flow cytometric analysis confirmed that insMPZ induce apoptosis,while compared with wtMPZ?p=0.006?.Also with 12 hrs treatment of 2-DG,both groups induced significantly apoptosis in sNF96.2 cells.The percentage of apoptosis cells after 2-DG treatment was especially high in mutant MPZ group?p=0.001?.Conclusion:1.In silico analyses showed this insertion p.D35delinsVVYTD was within the immunoglobulin-like extracellular domain of P0.insMPZ mutation caused an increase in the p-sheet of the secondary structure of P0.This resulted in an alteration of the secondary structure of P0.2.insMPZ mutant largely reduced intercellular adherence.insMPZ could start endoplasmic reticulum stress and trigger IRE1 arm of the UPR.insMPZ increased apoptosis in Schwann cells and was sensitive to 2-DG treatment.