Functional Study And Susceptible Tooth Position Of Non-syndromic Tooth Agenesis Caused By EDAR Mutations

Background:Genetic factors implicated in tooth development play a major role in both nonsyndromic and syndromic tooth agenesis.Compared to a majority of syndromic tooth agenesis which exhibit explicit etiologies,the pathogenic genes in non-syndromic tooth agenesis have not yet been fully identified.To the best of our knowledge,the genes,such as PAX9,MSX1,EDA,AXIN2,WNT10A,EDAR,KDF1,GREM2,LRP6,and WNT10B,etc.,have been associated with non-syndromic tooth agenesis thus far.Among them,EDAR,EDARADD,KRT17,IKBKG,LRP6,and WNT10B,etc.,have been rarely associated with non-syndromic tooth agenesis.The EDA/EDAR/NF-κB pathway has been previously found to be required for normal embryogenesis,particularly in the development of tooth,hair,skin,and other ectodermal organs.In this pathway,EDAR functions as a core member,interacting with its ligand,EDA,and its adaptor,EDARADD,which results in the downstream activation of NFκB signaling.Mutations in members of the EDA/EDAR/NF-κB pathway,such as ectodysplasin A(EDA),ectodysplasin A receptor(EDAR),and EDAR associated death domain(EDARADD),are associated with HED-related tooth agenesis.Although approximately 70 EDAR mutations have been identified in autosomal dominant or recessive HED patients,only 11 mutations have been identified in non-syndromic tooth agenesis.Previously,we showed an association between EDAR polymorphisms and non-syndromic tooth agenesis in a Chinese population.Therefore,we hypothesize that EDAR is a reliable causative gene for non-syndromic tooth agenesis,and the EDAR mutation spectrum and the phenotypic variability of EDAR-associated non-syndromic tooth agenesis need to be further explored and study the functional mechanism of EDAR mutations.At the same time,genotypephenotype relation analysis of large sample size was used to identify the susceptible positions of non-syndromic tooth agenesis related to EDAR mutation,to further explore the pathogenic causes of tooth agenesis.Objective:In this study,we performed mutation screening in patients with non-syndromic tooth agenesis to detect novel EDAR mutations.To determine the mutation detection rate of EDAR gene in non-syndromic tooth agenesis.The effect of EDAR mutations on NF-κB signaling pathway activity was further studied by functional studies.The susceptible positions of non-syndromic tooth agenesis with EDAR mutation were statistically analyzed and report susceptible positions comparison between EDAR-and EDA-related tooth agenesis.Methods:A cohort of 112 unrelated patients with non-syndromic tooth agenesis(61 males and 51 females between 3 and 42 years of age)from 2008 to 2019,were recruited to participate in this study.EDAR mutations were detected by whole exome sequencing(WES)and mutation screening.And then the pathogenicity of the detected EDAR mutations were analyzed by mutation analysis,familial co-segregation analysis,conservation analysis,and threedimensional structural analysis.To study the effect of EDAR mutations on NF-κB signaling pathway activity,the wild-type and all mutant EDAR expression plasmids were constructed to transfected transiently to the HEK-293T cell.Western Blot experiment were conducted to detect the content p-p65 and p-IκBα and the NF-κB signaling activity.Immunoprecipitation was taken to explore the affinity between mutant EDAR and EDA or EDARADD.To study the tooth agenesis susceptible positions caused by EDAR mutations,we obtained data on 17 patients(9 males and 8 females)with defined EDAR mutations from our medical record database and 13 patients with detailed tooth agenesis sites reported in 4 previous studies.We also included data on 84 patients with EDA-mutations(69 males and 15 females)with detailed documentation of tooth agenesis sites(or with panoramic film)from fourteen studies.All the patients included were non-syndromic.Statistical analysis was performed using the Chisquared χ2 test using Prism 8.0 P value of<0.05 was considered statistically significant.Results:In this study,we performed mutation screening in a cohort of 112 unrelated patients with non-syndromic tooth agenesis,and identified 5 novel mutations(c.404G>A,c.871G>A,c.43G>A,c.1072C>T,and c.1109T>C)and 2 known mutations(c.1138A>C and c.319A>G)in EDAR.The results of evolutionary conservation analysis revealed that the EDAR amino acid residues Vall5,Cys135,Ala291,Cys352,Arg358,Val370,and Ser380 were highly conserved across several species.Tertiary structural prediction suggested that the conformational changes observed in the mutants might impair EDAR function.Western Blot experiments showed that the content p-p65 and p-IκBα in the mutant group was lower than that in the wild group,indicating that mutations impair the signal transduction of the EDA/EDAR/NF-κB pathway.Immunoprecipitation showed that the mutations reduced the affinity between EDAR and EDA or EDARADD.Statistical analysis was used to find a unique tooth agenesis pattern in the permanent dentition associated with EDAR mutations:the mandibular second premolars and maxillary lateral incisors were the most affected(58.33%and 50.00%),while the maxillary central incisor was the least affected(1.67%).And to EDA mutations in non-syndromic tooth agenesis,maxillary lateral incisors,mandibular LI,and mandibular CI had the highest missing rate(73.81%,75.00%and 76.79%),and maxillary first molars had the lowest missing rate(7.14%).Conclusions:In this study,5 novel EDAR mutations responsible for non-syndromic oligodontia were identified,enriching the literature on EDAR mutation spectrum.To analyze the potential pathogenic mechanism of EDAR mutations causing non-syndromic tooth agenesis by functional studies.Furthermore,this study is the first to characterize the EDARrelated tooth agenesis susceptible positions,systemically comparing the similarities and differences of the EDAR-and EDA-related susceptible positions in non-syndromic tooth agenesis,and Our findings provide new evidence for the genotypic study of non-syndromic tooth agenesis,and facilitate the diagnosis,treatment,genetic counseling,and prenatal diagnosis of this rare congenital anomaly by health providers.