Pathogenic Gene Mutation And Molecular Mechanism Of An X-linked Hypohidrotic Ectoderm Dysplasia Family

Objective:Ectodermal dysplasia(ED)is a series of clinical and genetic heterogeneous diseases,which have more than 200 different clinical phenotypes.It is characterized by abnormal development of ectodermal structures such as such as teeth,hair,and sweat glands.Hypohidrotic ectodermal dysplasia(HED)is the most common,less with autosomal dominant and recessive HED.Compared with autosomal recessive inheritance and dominant inheritance,X-linked hypohidrotic ectodermal dysplasia(X-linked HED,XLHED)is the most common with Hypodontia/Oligodontia teeth,Absent/sparse hair,Anhidrosis/hypohidrosis,and characteristic facial features.The main reason is ectodysplasin A(EDA)gene mutation.The objective of this research was to describe the molecular and clinical characteristics of a Chinese XLHED male and his family.whole exome resequencing(WES)and Sanger sequencing were combined to verify the mutation site of the pathogenic gene causing XLHED and determine the mutation type of the patient,thus broadening the mutation spectrum of XLHED caused by EDA mutation.In addition,the relationship between phenotype and genotype was studied,and the previous research results were summarized and compared.Methods:Genomic DNA was extracted from the peripheral blood of the proband and its family members,and WES was used to detect the pathogenic genes,then EDA mutation site was verified by Sanger sequencing.Seven bioinformatics software,Mutation Taster,Poly Phen-2,FATHMM,SIFT,REVEL,LRT,and GERP,were used to predict the pathogenicity of the mutation sites,and combined with the homologous comparison of 9 species to predict the changes of mutant proteins,the pathogenic mutation sites were initially determined.In this study,the cell function of the missense mutation c.1119G>C/p.M373 I was identified.As the first step,wild-type EDA eukaryotic expression vector was constructed,and the mutant EDA eukaryotic expression vector was obtained after the mutation site was induced to the wild-type EDA eukaryotic expression vector using the point mutation kit.Non-specific control,wild type and mutant EDA eukaryotic expression vectors were transfected into HEK293 T cells of human embryonic kidney cells.Culture medium with appropriate concentration of plasma and antibiotics.Total RNA was extracted 24 hours later and total protein was extracted 48 hours later.The expressions of EDA and EDA proteins were detected by fluorescence quantitative PCR(q PCR)and Western blotting.The transcriptional activity of nuclear factor-κB(NF-κB)was detected using a luciferase assay.Graph Pad was used to statistical analysis,and P<0.05 was considered statistically significant.Result:By whole exome sequencing and Sanger sequencing,the proband with XLHED was identified a novel EDA mutation,c.1119G>C(p.M373I),that affected the molecular analysis of transmembrane protein exon8 mutations,inherited from the mother.Multi-species homology comparison showed that it was located in the region of highly conserved protein.Bioinformatics analysis software predicted the mutation to be harmful.He showed a severe multiple-tooth loss,with over 20 permanent teeth missing and sparse hair and eyebrows,skin dry,thin,and itching.Furthermore,his sweating function was abnormal to a certain extent.And the patient had more typical facial features,such as saddle nose.The functional study showed this novel mutation lead to a significant decrease in the EDA expression level and transcriptional activity of NF-κB.The possible pathogenic mechanism of XLHED caused by this mutation was described,which provided theoretical basis for early diagnosis,prenatal diagnosis and targeted therapy of XLHED patientsConclusion:In this study,a novel EDA mutation site causing XLHED was identified,extend the range of EDA mutations in XLHED patients,and the association between the expression and genotype was confirmed by cell functional studies.Our finding provides the basis and idea for further exploring the pathogenesis of XLHED.And it play an important role to promote genetic intervention in prenatal diagnosis.Significance: To provide molecular biological basis for prenatal diagnosis,early diagnosis and related treatment of XLHED patients.This study provides evidence and ideas for further exploring the pathogenesis of XLHED,and is helpful to promote prenatal diagnosis and genetic intervention.