Genetic Study Of Congenital Tooth Agenesis Raised By EDA/EDAR Gene Mutation

Objective:Tooth development is a complex process of interaction between epithelium and mesenchyme,and many transcription factors and signaling molecules are involved.Hypoplasia of teeth is the most common craniofacial congenital malformation in humans,including abnormalities in the number,shape and structure of teeth.Abnormal number of teeth,namely congenital tooth agenesis,is the most common developmental abnormality in human dentition.In the population,the proportion of congenital missing single teeth can be as high as 20%.Excluding the proportion of missing third molars,the incidence of permanent tooth missing is 1.6%-9.6%,and the incidence of congenital missing deciduous teeth is low.Except for the third molars,the second premolars and lateral incisors are most easily affected.According to the number of missing teeth,it is generally divided into hypodontia(a small number of missing teeth),oligodontia(a majority of missing teeth),and anodontia(complete mouthless teeth).Among them,a small number of missing teeth means that the number of missing teeth is less than 6;most missing teeth means that the number of teeth is congenital missing 6 or more(not including the third molar).Congenital tooth loss can be caused by environmental or genetic factors.Clinically,sporadic cases and family inheritance cases can be seen,the latter can be autosomal dominant inheritance,autosomal recessive inheritance or X chromosome linked inheritance.Congenital tooth loss not only affects the patient's chewing function,but also affects their pronunciation,appearance and mental health.According to whether it is accompanied by other tissue and organ changes,congenital tooth loss can be divided into syndrome type and non-syndromic type.Ectodermal dysplasia syndrome(ectodermal dysplasia syndrome,EDs)is the most common syndrome-type congenital tooth loss in clinical practice.In addition to congenital lack of teeth,children are also accompanied by developmental disorders of other ectodermal tissues,which are divided into two types:hypohidrosis and nonsweat.A large number of studies have shown that there are more than 20 genes related to congenital dentition.It is generally believed that PAX9,MSX1,AXIN2 and EDA genes are closely related to non-syndromic congenital dentition,and the most common syndrome congenital dentition-outside Hypodermal dysplasia syndrome,its pathogenic genes are mainly EDA,EDAR and EDARADD.In this study,we collected congenital edentulous families through clinical examination,detected relevant candidate genes,and preliminarily predicted their functional changes,and explored the gene mutation sites and pathogenesis of patients with congenital edentulous.Methods:1.Collect the families of patients with ectodermal dysplasia and congenital tooth loss,ask for medical history and make a family profile after clinical examination,collect blood samples,perform second-generation whole exome sequencing(WES)screening,and analyze the screening results And verification.2.Collect blood samples from patients with EDAR gene C.175-2A>G,extract RNA and perform Sanger sequencing after reverse transcription to analyze the changes in the splicing site,and RT-PCR to detect the effects of mutations on the transcription products.3.The detected EDAR gene C.175-2A>G new mutation site is designed to introduce mismatch primers,and the DNA of 100 normal human blood samples is detected by polyacrylamide gel electrophoresis method,SNP is excluded,and it is determined that it is pathogenic New mutation site.4.Analysis of the influence of pathogenic gene mutations on protein function: use the functional analysis website SIFT and polyphen-2 to evaluate protein function changes;use the Translate website(https://web.expasy.org/translate/)to analyze the protein translation results.Refer to NCBI database,use UGENE software to reference related homologous proteins for amino acid conservation analysis;use PSIPRED website to analyze protein secondary structure changes;use Swiss-model to simulate protein three-dimensional structure,analyze wild-type and variant EDA protein three-dimensional structure changes and their Impact on function.Results:1.In this study,six Chinese Han families with congenital dentition were screened for four known pathogenic variants and two new variants,and a family map was drawn.The EDA,EDAR,and WNT10 A genes are among those involved.2.In the first family,a missense mutation in the EDA gene c457C>T,a recognized pathogenic variant site,was screened.The mutation will result in p.Arg153 Cys.The ACMG grading guideline classifies it as congenital tooth loss and X-linked hypohidrosis.The pathogenic gene of germ layer dysplasia,clinical manifestations include loss of a majority of teeth,decreased sweating,and sparse hair,conservative analysis findings show that this site is strongly conserved among species and protein analysis.This mutation can impair the hydrolysis and processing of EDA protein products,as well as the release of the TNF homology domain.3.In the second family,a known pathogenic variant was discovered.The proband carries the EDA gene missense variant c.895G>A.The variant can cause p.Gly299 Ser.The ACMG grading guideline classifies it as congenital tooth loss,and the X-linked hypohidrosis ectodermal dysplasia is clinically manifested as loss of majority teeth,low sweating,and sparse hair.According to the findings of the conservative analysis,this locus is highly conserved among species.4.In the third Family,A new deletion mutation in the EDA gene,c.954 del C deletion mutation,was discovered,which can cause p.Ala318 fs,which is determined to be the cause of congenital tooth loss and X-linked hypohidrosis ectodermal dysplasia according to the ACMG grading guidelines.The clinical manifestations are toothlessness,sparse hair,less sweating,and reduced vision.The results of a conservative analysis show that this site is highly conserved among species.According to protein analysis,this mutation will lead to a frameshift of amino acid coding and early termination of protein-coding.5.In the fourth Family,a known missense mutation c.511C>T in the WNT10 A gene was discovered,which can cause p.Arg171 Cys.It has been determined to be a pathogenic gene for congenital tooth loss,and it is inherited in an autosomal dominant manner,according to ACMG grading guidelines.The clinical manifestation is that the majority of the teeth are missing.Protein analysis revealed that Cys substitution might eliminate the electrostatic interaction between R171 and adjacent residues.6.In the fifth family,a new missense variant c.175-2A>G in the EDAR gene was discovered.According to the ACMG grading guidelines,it was determined to be a pathogenic gene for congenital tooth loss and hypohidrosis ectodermal dysplasia,autosomal dominant Inheritance.This mutation causes abnormal splicing during EDAR gene mRNA transcription,skipping the 22 bp of the initial part of exon4,and RT-PCR detection indicates that the mRNA transcript at the new splicing site is degraded,affecting protein expression and function.This can lead to congenitally missing teeth and HED.7.In the sixth Family,a known missense variant c.442T>C in the EDAR gene,which caused p.Cys148 Arg,which according to ACMG grading guidelines was caused congenital tooth loss and X-linked hypohidrosis-type ectodermal dysplasia.Disease gene.Conclusion:1.In this study,two known mutation sites of the EDA gene,one new mutation site of the EDA gene,one new mutation site of the EDAR gene,and one known mutation site of the EDA gene were discovered in six Chinese Han families with congenital dentition.The mutation site and a known mutation site in the WNT10 A gene2.The EDA gene c.954 del C(p.Ala318fs)heterozygous deletion mutation causes congenital dentition and HED.3.The EDAR gene c.175-2A>G heterozygous mutation causes congenital tooth loss.4.This study found new pathogenic mutation sites for congenital tooth loss and ectodermal dysplasia through high-throughput sequencing technology,enriched the gene mutation database,and analyzed the abnormal protein function caused by the diseasecausing gene mutation.Family members of patients with this disease provided genetic counseling and eugenics guidance.