A Study On The Susceptibility Of BMP2 And BMP4 Gene Polymorphisms To Congenital Tooth Loss In Chinese Han Population

Part 1 Association of BMP2 and BMP4 polymorphisms with isolated tooth agenesis in a Chinese Han population:a case–control studyTooth agenesis is a common craniofacial anomaly.Excluding the third molar,the incidence of missing permanent teeth varies from 2.6%to 11.3%depending on demographic and geographic profiles.The development of human dentition depends on a series of epithelial-mesenchymal reciprocal interactions mediated by signaling molecules and their receptors.These comprise of the transforming growth factor??TGF??,bone morphogenetic proteins?BMP?,fibroblast growth factors?FGF?,epidermal growth factor?EGF?,the hedgehog?Hh?and wingless?Wnt?families.Based on the complexity of tooth development,the etiology and pathogenesis of tooth ageneis have not been clearly elucidated.Early in 1975,reseachers found that tooth agenesis is a multigenic disease.So far,genetic variations among the genes MSXI,PAX9,EDA,EDARADD had been identified to be associated with tooth agenesis.Single nucleotide polymorphisms is widely in the general people,since its large amount and allele easy to identify,SNPs is often used to study the association between genes and diseases.BMPs is an important family of redundant activators for tooth development.During early development,BMP2 and BMP4 are expressed in high amounts from the enamel knot and play a role in cell proliferation and apoptosis,position and number of cusps,and tooth patterning.BMP2 and BMP4 SNPs were observed in Mexican oligodontia families.The polymorphisms in the BMP4 gene was associated with isolated human tooth agenesis in a Brazil population.To evaluate the association of the polymorphisms in the BMP4 gene?rs17563?and the BMP2 gene?rs3178250,rs15705,rs235768,rs235769?with isolated human tooth agenesis in Chinese population,444 control individuals without tooth agenesis and335 cases with tooth agenesis were evaluated using a case-control design.All the participants of the study were recruited through the Stomatological Hospital of JiangSu Province and nanjing First People's Hospital,JiangSu.Genotyping of the selected polymorphisms for BMP4 and BMP2 were carried out by real-time PCR using the Taqman assay method from genomic DNA isolated from venous blood samples of all individuals.In the control group,the genotype frequencies were in HWE,suggesting good homogeneity within the study subjects.There was no significant difference on gender distribution and age between cases and controls.In the overall analysis,significant statistical differences were found in genotype frequencies between tooth agenesis and control samples in rs3178250 and rs15705?P=0.039 and P=0.046,respectively?.In the subtype analysis,recessive model distribution of rs3178250 and rs15705 in mandibular incisor agenesis were different from the control,and recessive model of the mandibular incisor were associated with increased risk of tooth agenesis(rs3178250,P_rec=0.020,OR_rec=1.60,95%CI_rec=1.08-2.37;rs15705,P_rec=0.024,OR_rec=1.57,95%CI_rec=1.06-2.37).In rs235768,there was difference in recessive model distribution between the mandibular incisor agenesis cases and the control,and AA genotype was protective against mandibular incisor agenesis(P_hom=0.03,P_rec=0.033,OR_rec=0.36,95%CI_rec=0.14-0.95).For BMP4?rs17563?,the allele C was associated with decreased risk of APTA compared with the control(P_Allele=0.018,OR=0.37,95%CI=0.15-0.87).These results indicate that polymorphisms in the BMP2 and BMP4 gene contribute to tooth agenesis.Nonetheless,the extent to which this polymorphism may actually contribute to the tooth agenesis status should be clarified.Part 2 Functional study of genetic variants of BMP2 with susceptibility of tooth agenesis in a Chinese population The gene BMP2 located on 20p12,16 kbp length with 3 exons.The gene BMP2 is a member of BMP signal pathway,of which the encoded protein belong to the TGF? superfamily.The TGF? superfamily is involved in the tooth development,research suggested that the members expressed in rat incisor pulp.The enamel knot enamel acts as a signaling or organizing center in developing tooth,during which BMP2 is expressed.Bmp2 is specifically expressed in the epithelium between E11.0 and E12.0 when tooth morphogenesis is first apparent and has a distinct molecular action on the jaw mesenchyme.BMP2 plays an important role in the differentiation of odontoblast cells,induction and formation of alveolar bone and regeneration of periodontal tissue.Besides the developing tooth bud,BMP2 was found localized in jaw bone,striated and smooth muscle,spinal cord and nasal tracheal and oesophagal epithelium.In our previous research,we found that SNP?rs15705and rs3178250?located in 3'UTR of BMP2 were associated with a significantly decreased tooth agenesis risk of mandibular incisor in a chinese population.The bioinformatic method was performed to predict the miRNAs of binding sites.The miRNA mimics were transfected independently to the 293 A and COS7 cells.To evaluate whether rs15705 and rs3178250 variants might affect the binding sites of miRNAs,the luciferase reporter palsmids of BMP2 3'UTR were cotransfected with miRNA mimics in COS7 and 293 A cells.The results of RT-PCR study showed that the miRNA mimics could bind to the BMP2 gene and reduce the levels of BMP2 mRNA of the two cells.The results of vitro experiment showed that the wild-type and mutant-type of two SNPs changed the binding capability of miRNAs.When we cotransfected the miRNA and BMP2 mRNA,relatively decreased expression of BMP2 were observed in mutant type of two SNPs.In conclusion,rs15705 and rs3178250 of BMP2 3'UTR may associate with the risk of tooth agenesis through enhancing miRNA binding sites.