Associations Of SNPs In MicroRNA Processing Genes And MSX1 With Nonsyndromic Oral Clefts Susceptibility

Orofacial clefts are major congenital abnormalities,characterized fractures of normal facial tissues.The incidence of orofacial clefts is almost 1-7/1000 live births.And the susuceptibility of it is various according to ethnic groups,or countries.Asian population is the most prone to this disease.There are three main types of orofacial clefts:cleft lip only(CLO),cleft lip with palate(CLP),and cleft palate only(CPO).Non-syndromic orofacial clefts(NSOC)are cases without any other developmental abnormalities,which constitute the majority of orofacial clefts.NSOC has a complex etiology that composed of both genetic and environmental factors.Among all the risk factors,the genetic factor takes a critical role in the embryonic development of NSOC.Single nucleotide polymorphism(SNP)are the main components of human genome DNA variations,and has been considered as the main type of human genetic variations.SNPs of miRNA processing genes may be responsible for aberrant splicing,altered protein binding sites,or different gene expression.Previous studies had shown these SNPs affected the genesis and progression of various common diseases,including colorectal cancer,and Parkinson disease.microRNAs(miRNAs)are endogenous,short(20-23 nucleotide),noncoding RNAs participated in the regulation of differentiation,proliferation,apoptosis or metabolism.And miRNAs usual bind with the target genes(3’ untranslated regions),and act as negatively regulators in different developmental processes.Genetic variants of SNPs in miRNA processing genes may affect the interaction between mRNA and target miRNA by altering the binding sites,and cause multiple functional processes.Therefore,SNPs in miRNAs may affect the risk of human diseases though regulating the expression of the miRNAs.And the functions of the SNPs in miRNA processing genes have been confirmed by considerable researches on common human diseases like cancer and cardiovascular disease.According to the rencent studies,miRNAs played important roles in craniofacial development.For instance,a recent study showed that miRNA-23b and miRNA-133b participated in midfacial developm ental process.Given the importance of miRNA in the development of craniofacial structures,genetic variants of miRNA processing genes may also affect the risk of NSOC.SNPs in miRNA genes may modulate different biological processes via affecting the miRNA target selection and/or procession.Accumulated researches were studied about the function of SNPs from miRNA genes in biological process,and recognized that the majority of these SNPs might affect the occurrence and development of common tumors,like breast cancer and breast cancer.In the last few years,more importance was gradually attached to the indispensable role of miRNAs in orofacial development.For instance,Dicer--one of miRNA processing genes is associated with mammalian craniofacial malformations,and loss of its expression could lead cleft palate and midfacial hypoplasia.In addition,Warner et al found that miRNAs also involve in the development of murine upper lip,thus may causing the occurrence of cleft lip.However,few studies nevertheless had referred to the relationship between the SNPs within miRNA genes and the susceptibility of NSOC.Muscle segment homeoboxl(MSX1)gene is located at 4p16.1,encoding a DNA-binding sequence,and is expressed in spatially restricted regions of the head during critical stages of early development.It has a highly conserved structural organization,and its abundant expression could induce the interactions between cells at the embryonic stage.In the recent decades,association studies of candidate gene have convinced as an expedient tool to investigate the genetic part of common complex diseases,including NSOC.Mutations in MSX1 are associated with autosomal dominant hypodontia,tooth agenesis and other common diseases.And MSX1 also has been demonstrated to be a favorable candidate gene for susceptibility to NSOC.Recently,MSX1 was extensively studied,and has been conviced as an important factor in craniofacial development.In mice models,lack of MSX1 expression might contribute to cleft palate,and abnormal tooth development.Furthermore,recent work based on an Iranian population has shown the associassion between rs12532(located in 3’-UTR of MSX1)and NSOC susceptibility.However,the results were different among various populations.In the first section of this study,we genotyped 12 potentially functional SNPs from seven miRNA processing genes(GEMIN3,DROSHA,DGCR8,GEMIN4,PIWIL1,XPO5,and DICER)in a case-control study of 602 NSOC cases and 605 controls.And in the second section,the association between rs12532 in 3’untranslated region(UTR)of MSX1 and NSOC was investigated.Part ⅠAssociations between Single nucleotide polymorphisms in microRNA processing genes and risk of non-syndromic orofacial cleftsMiRNAs play important roles in the craniofacial development.And miRNA processing genes are indispensable in the miRNAs mature processes.Thus,we hypothesized that single nucleotide polymorphisms(SNPs)of miRNA processing genes may be associated the risk of non-syndromic orofacial clefts(NSOC).In this study,12 potential functional SNPs of seven miRNA processing genes(GEMIN3,DROSHA,DGCR8,GEMIN4,PIWIL1,and XPO5)were selected,and we evaluated the associations between these SNPs with risk of NSOC in a case-control study.We found that three SNPs were associated with the susceptibility of NSOC.Logistic regression analysis indicated that 3SNPs might affect the risks of NSOC,and we found that rs417309 and rs493760 were related to a reduced risk of NSOC(rs417309:P=0.049,OR=0.65,95%CI=[0.43,0.99]for heterozygous comparison;rs493760:P=0.020,OR=0.53,95%CI=[0.31,0.90]for homozygote comparison).On the contrast,rs10719 was associated with an increasing risk of NSOC(P=0.022,OR=1.32,95%CI=[1.04,1.68]for heterozygous comparison;P=0.035,OR=1.27,95%CI=[1.02,1.60]for dominant comparison).And the stratified analysis of NSOC subgroups was performed.The results indicated that rs493760 had protective effects on CLP and CPO(CLP:allelic comparison,minor vs major,OR=0.77,95%CI=[0.61,0.97];genotypic comparison,homozygote comparison,OR=0.43,95%CI=[0.21,0.88].CPO:genotypic comparison,homozygote comparison,OR=0.89,95%CI=[0.85,0.92].).Conversely,positive associations were found between rs10719 and subgroups of NSOC(CLP:genotypic comparison,heterozygous comparison,OR=1.50,95%CI=[1.12,2.00],dominant genetic comparison,OR=1.39,95%CI=[1.05,1.83]).In addition,rs10719 and rs493760 were both in DROSHA,and we conducted LD and haplotype analysis via SHEsis software.The results shown in Table 5 revealed that D’was 0.586,and r2 was 0.044 between the two SNPs.Taken together,our results provided the further evidence that rs10719 and rs493760 might contribute to the risk of NSOC,and suggested potential genetic basis and ma-chanisms of NSOC.Part ⅡAssociations between a miRNA-binding-site SNP of MSX1 and susceptibility of non-syndromic orofacial cleftsIn vertebrate and mammalian,MSX1 is expressed in craniofacial tissues and limb buds.Genetic variations in MSX1 could influence the occurrence and development of tooth agenesis and NSOC in human.However,the researches about the roles of MSX1 gene in the susceptibility of NSOC showed different results among various populations.In our study,the association between rs12532,rs3821947,rs3821949,and rs4464513 with NSOC were investigated in a case-control study,including 602 NSOC cases and 605 healthy controls.The results indicated that rs 12532 in 3’-untranslated region(UTR)of MSX1 could affect the susceptibility of NSOC.The individuals with rs12532 AA genotype contributed to a decreased risk of NSOC(AA vs.GG:OR=0.69,95%CI=[0.49,0.98]),especially in the CLP subgroups(A vs.G:OR=0.80,95%CI=[0.66,0.99];AA vs.GG,OR=0.58 95%CI=[0.37,0.91]).Hsa-miRNA-3649(miR-3649)was predicted as a binding miRNA with rs12532,and rs12532 might affect the binding ability of the miRNA.The results of luciferase activity assay showed that the expression level of rs12532 A allele was lower than that of the G allele(293A:P<0.001,COS7:P<0.001).In addition,the in vivo study showed that the expression of MSX1 of the individuals carrying the rs12532 GG genotype was higher than that of those carrying the A A genotype.And the result of miR-3649 showed an inverse correlation.In conclusion,our findings indicate that rs12532 in 3’ UTR of MSX1 could regulate the expression of MSX1 via the interaction between miR-3649 and contributed to the susceptibility of NSOC.