The Axis Inhibition Protein 2 Polymorphisms And Nonsyndromic Orofacial Clefts Susceptibility In A Chinese Han Population

Orofacial clefts are common congenital birth malformations in human beings with an incidence of 1/700-1,000 live births worldwide.However,according to the data provided by Chinese Birth Defects Monitoring Network(CBDMN),Chinese infants appear to have a relatively higher risk of orofacial clefts with a birth prevalence of 1.42/1000.Non-syndromic orofacial clefts(NSOC)account for nearly 70%of the orofacial clefts,which are identified as those presenting without any other syndromes.During the early stages of orofacial development,the complete fusion failure between any of the independently differentiated orofacial primordia can result in NSOC.According to the cleft location,NSOC are usually divided into cleft lip only(CLO),cleft lip with palate(CLP)and cleft palate only(CPO).Meanwhile,CLP and CLO are usually collectively defined as cleft lip with or without cleft palate(CL/P).Depending on the results from recent epidemiological and genetic studies,CL/P and CPO should be separated in studies because of their distinct embryonic development process.In the past few decades,many studies indicated that NSOC was a multifactorial congenital birth defect.There are two major factors,environmental element and genetic element,which are considered as the etiology of NSOC.Furthermore,with the increasing attention to genetic risk factors,researches on candidate genes and genome-wide association studies for novel polymorphisms or regions revealed that several genetics factors contributed to the risk of NSOC,such as MSX1,IRF6,MTHFR,BMP4 and members of the FGF and Wnt gene families.As a major negative regulator of the Wnt signaling pathway,the axis inhibition protein 2(AXIN2),the gene of which is mapped to 17q23-q24,plays a significant role in the regulation of the stability of P-catenin in the Wnt pathway,which plays a key role in craniofacial morphogenesis.The goal of this study was to investigate the potential relationship between AXIN2 polymorphisms and the risks of non-syndromic orofacial clefts(NSOC)in a Chinese Han population.The protein encoded by this gene is a member of the fibroblast growth factor(FGF)family.in humans,22 members of the FGF family have been identified.FGF family members possess broad mitogenic and cell survival activities,and are involved in a variety of biological processes,including embryonic development,cell growth,morphogenesis,tissue repair,tumor growth and invasion.This protein functions as a modifier of endothelial cell migration and proliferation,as well as an angiogenic factor.It acts as a mitogen for a variety of mesoderm-and neuroectoderm-derived cells in vitro,thus is thought to be involved in organogenesis.Fibroblast growth factor 5(FGF5)is widely expressed in embryonic but scarcely in adult tissues.And it also associated with hair and skin development.This study was an ongoing hospital-based case-control study approved by the Institutional Review Board of Nanjing Medical University.The details of the subject's recruitment described previously.All the samples were collected from genetically unrelated Han Chinese people in Jiangsu Province and its surrounding areas,which consisted of a large sample size of 599 NSOC cases and 602 healthy controls recruited from three affiliated hospitals of Nanjing Medical University:theStomatological Hospital of Jiangsu Province,Xuzhou First People's Hospital and Nanjing Children's Hospital.Then,we recorded the general information of every participant,including gender,age,ethnicity,health status and family history of clefts and other congenital anomalies.Four polymorphisms of AXIN2(rs2240307,rs11867417,rs2240308 and rs7591)were selected.And three polymorphisms of FGF5 were also selected,which located in 3'UTR region of FGF5.The single nucleotide polymorphisms(SNPs)were genotyped by a custom-by-design 2×48-Plex SNP scanTM Kit.And this kit was developed based on the patented SNPs genotyping technology by Genesky Biotechnologies Inc.,on the basis of double ligation and multiplex fluorescence PCR.Weak associations were found between these four SNPs and the risk of NSOC.Further stratified analysis showed that the overall genotype frequencies of rs2240307 were different between the cleft palate only(CPO)group and the control group(P =0.048),and GG genotype markedly contributed to the susceptibility to CPO(OR =3.22,95%CI = 1.13-9.18).The similar effect was also observed on GA/AA genotype compared with GG homozygote(OR=0.30,95%CI=0.11-0.84).The results of LD analysis between each pair of SNPs revealed that two SNPs(rsl 1867417 and rs7591)were in a LD block(r2>0.8).But no statistically significant was found between cases and controls from haplotype analysis in these two loci.For rs4690150 and rs6838203,there was no statistical difference between NSOC case group and control group(P>0.05).Another polymorphism,rs3733336,showed different result.There was statistical difference between NSOC case group and control group(P=0.004).GA and GG genotype might contribute to a lower risk of NSOC(OR=0.74,95%CI:0.58,0.94,OR= 0.48,95%CI:0.28,0.83,respectively),as well as GA/GG genotype(OR= 0.70,95%CI=0.56,0,89).In the further stratified analysis by clefts subgroups,we found that GA genotype contributed to decrease risk of CPO and CL/P,respectively.And GG genotype could reduce risk of CLO and CL/P.Meanwhile,GA/GG genotype appeared to be protective factors of all the subgroups of NSOC.But weak associations were found between rs4690150 and rs6838203and the risk of any NSOC subgroups.We found that SNP(rs3733336)located in 3'UTR of FGF5 was associatedwith a significantly decreased NSOC risk.Furthermore,the bioinformatics prediction suggested that this SNP was located at about 30bp downstream of the binding sites of miR-145 in 3'UTR of FGF5.To evaluate whether rs3733336 variant might affect the binding site of miRNAs,we used luciferase reporter palsmids of FGF5 3'UTR(containing rs3733336 A or G allele)that were cotransfected with miR-145 mimics in cos7,C2C12 and 293A cells.The results of vitro experiment showed that both the variant A and G allele of rs733336 changed the binding capability of miRNAs.The result of RT-PCR study showed that miR-145 mimics could reduce the levels of FGF5 mRNA of these three cells.The Additional experiments with 55 NSOC tissues revealed that the levels of FGF5 mRNA expression in NSOC tissues were associated with different genotypes.The borderline results gave us a hint that rs2240307 contributed to the susceptibility to CPO in a Chinese Han population.Meanwhile,rs3733336,the polymorphism which located in the 3'UTR region of FGF5,was associated with the risk of NSOC.It may alter expression of FGF5 though breaking miRNA binding sites and contribute to the suppression of NSOC,which was conductive to improving our awareness of the causes of NSOC.