Genetic Variation Of Chromosome And Genetic Susceptibility Of Non-syndromic Cleft Lip And Palate In Chinese Population

Cleft lip and palate(CLP)is a common congenital malformation in China,which is manifested as abnormal facial structure fusion,with the incidence of 1-7/1000,and the risk of different regions and races is heterogeneous.Asians are the most susceptible population to this disease.CLP is caused by abnormal fusion of facial processes.If the growth and development of facial processes are affected during the process of facial fusion,means facial processes cannot be fused or completely fused,formed various facial deformities.For example,the cleft lip will happen if the globular process and maxillary process can not be fused.If one side palatal process and the opposite side one can not combined or partially fused,the cleft palate will form.No-syndromic orofacial cleft(NSOC)is the most important type of cleft lip and palate.Its pathogeny is more complex,including genetic and environmental element.Genetic factors account for about 70% of the risk factors affecting the occurrence of NSOC,which plays a key role.According to the different clinical manifestations,NSOC is divided into three types: Cleft lip only(CLO),Cleft lip with Cleft palate(CLP),and Cleft palate only(CPO).The occurrence of NSOC has greatly affected many children with NSOC and their families,both physically and psychologically.They usually need a long-term multidisciplinary sequential therapy,which brings great economic burden to patients,families and society.Nowadays,although there have been many studies using genome-wide association studies(GWAS),linkage analysis and animal experiments revealed many single nucleotide loci associated with the non-syndrome type of cleft lip and palate,genes and pathways.While its etiology and pathogenic is still in the exploratory stage.Although cleft lip and palate families exist,the inheritance pattern does not conform to Mendelian inheritance law.Non-syndromic cleft lip and palate are about 2% to 3% likely to be passed on to their offsprings.However,the interactions between genetic and environmental factors willincrease the risk of non-syndromic cleft lip and palate.For example,pregnant women who smoke during pregnancy are twice as likely to have a cleft lip and palate,but pregnant women who have an abnormal MSX1 allele and who smoke during pregnancy are three times more likely to have a cleft lip and palate.In addition,lack of multivitamins and folic acid during pregnancy and viral infection all increase the risk of cleft lip and palate.Therefore,the research on the pathogenesis of cleft lip and palate is helpful for us to explore the process of the occurrence and development of the disease and provide help in clinical diagnosis and genetic counseling.In recent years,more and more studies have focused on the pathogenesis of cleft lip and palate,while NSOC has genetic heterogeneity.Many genes have been implicated in cleft lip and palate,such as IRF6,TP63,FGF8,MSX1,PTCH1,PAX7,FGFR2,MAFB and FOXE1.Beaty et al.conducted a GWAS on NSOC and found rs13041247 which located near the MAFB gene of chromosome 20q12 was a susceptible locus for non-syndromic cleft lip and palate,which was specially related to the NSOC in Asian population.However,due to the small sample size of the Chinese population in this study,it is difficult to determine whether it is related to the risk of NSOC in the Chinese population.According to a meta-analysis about NSOC revealed that loci always associated with specific population,this is due to the different allele frequency of different ethnic groups.And through the study of genetic susceptibility loci of 8q24 region with Caucasian population in Europe and South America and MAFB gene in Asia group,confirmed the existence of such differences.Therefore,we proposed the hypothesis that the genetic variation of SNP locus rs13041247 located in the 20q12 region may be related to the NSOC susceptibility in Chinese population,and there may be other SNPs located in this region that are correlated with the incidence of NSOC.FOXE1,located at 9q22,is a member of the transcription factor family and plays an important role in embryonic development.FOXE1 was originally thought to be a transcription factor of the thyroid gland,which plays an important role in the growth and development of the thyroid gland.But FOXE1 was found to be present in a wide range of other tissues including the testis,epithelium,brain,heart,lungs,hair follicles,thymus,kidneys,placenta,liver,skeletal muscle,pancreas,colon and small intestine.As early as 1998,clifton-bligh et al.have found that FOXE1 is related to bamforth-lazarus syndrome,a clinical manifestation of which is cleft lip and palate.Moreno et al.found in 2009 that mutations in the 5 ’UTR region of FOXE1 were associated with NSOC.However,it is not clear whether FOXE1 gene is related to the susceptibility of Chinese han population to cleft lip and palate.It is necessary to verify the relationship between FOXE1 polymorphism and NSOC in Chinese han population.Therefore,we proposed hypotheses:(1)rs13041247 and other genetic variants located near the 20q12 region may be associated with NSOC susceptibility in Chinese population.(2)FOXE1 polymorphism is associated with NSOC susceptibility in the Chinese population.Part Ⅰ Associations of SNPs at Chromosome 20q12 and Non-syndromic cleft lip and palate risk in Chinese Han population and the exploration of underlying mechanismThis study selected 24 tag SNPs in 20q12 area,designed two phases among a total of1278 cases and 1295 controls,to explore the correlation between SNPs and the risk of cleft lip and palate syndrome type as well as three kinds of clinical subtypes in Chinese people.Results confirmed that rs13041247 was significantly associated with NSOC in Chinese population(P<0.001).Further stratified analyses with clinical subtypes showed that rs13041247 was associated with CLO and CLP,but not with CPO.Compared with TT genotype,rs13041247 TC genotype could reduce the risk of CLO in Chinese population(P <0.001,OR=0.70,95%CI =[0.55,0.88]).The CC genotype compared with TT genotype significantly reduced the risk of CLO(P<0.001,OR = 0.56,95% CI =[0.41,0.77]).In the CLP group,genotype distribution of case and control are statistically significant(P<0.001).Heterozygote and homozygote comparison results showed that rs13041247 acts as a protector(TC vs.TT: OR = 0.78,95% CI =[0.63,0.96];CC vs.TT: OR=0.53,95% CI=[0.40,0.71]).During the two-stage study,a total of 6 SNPs was found to be related to the incidence of non-syndromic cleft lip and palate in Chinese population,and pairwise conditional analysis showed that rs6129653(P=1.4E-06)was the most powerful and independent locus.The result of heterozygous model(P=0.011,OR [CI]: 1.24 [1.05-1.46]),homozygous model(P=1.5E-06,OR [CI]:1.89 [1.46 2.46])and additive model were(P=1.4E-06,OR [CI]:1.33 [1.18 1.49])showed that the T allele of rs6129653 played a pathogenic role in the pathogenesis of NSOC.In other words,patients with rs6129653 CT genotype were 1.24 times more likely to suffer NSOC than those with CC genotype,and those with TT genotype were 1.89 times more likely to develop NSOC than those with CC genotype.Further stratified analyses found that rs6129653 was related to the incidence of CLO and CLP,but not related to CPO.Bioinformaticsanalysis showed that rs6129653 was located in the transcription enhancement region and correlated with the expression of MAFB in brain tissue.We collected 49 tissues of lip skin and found the expression level of MAFB in TT and CT genotype was significantly lower than that of rs6129653 CC genotype.In vitro gene reporting experiments also found that the transcriptional activity of gene containing T allele was significantly lower than that of C alleles.Based on the results of previous GWAS studies,this study explored the correlation between genetic variants in the 20q12 region and NSOC susceptibility,verified the correlation between rs13041247 and the susceptibility to non-syndromic cleft lip and palate in the Han Chinese population.And found a new independent locus rs6129653 in the 20q12 region which was related to the incidence of NSOC in Chinese population.Stratified analyses showed that rs13041247 and rs6129653 may be associated with CLO and CLP,but not with CPO.Functional annotation results indicated that SNPs located in the 20q12 region may affect the risk of non-syndromic cleft lip and palate in Chinese population by affecting MAFB expression.The results of this study provided a more powerful basis for the genetics of non-syndromic cleft lip and palate,helped to reveal the pathogenesis and future prevention and treatment of non-syndromic cleft lip and palate.Part Ⅱ FOXE1 polymorphisms and non-syndromic orofacial cleft susceptibility in Chinese Han population FOXE1 plays an important role in craniofacial development.The aim of this study was to investigate associations between genetic variants of FOXE1 and the risk of non-syndromic orofacial clefts in Chinese population.Three potentially functional SNPs of FOXE1(rs3758250 and rs907577 in the5’upstream,and rs7043516 in the 3’-UTR)were selected and their associations with non-syndromic orofacial clefts susceptibility were investigated in a case-control study from a Chinese population(602 cases and 605 controls).Associations between the SNPs and risk of non-syndromic orofacial clefts and its subgroups were estimated from unconditional logistic regression analysis.Luciferase reporter assay was conducted to assess SNPs function.Overall,we did not find any of the individual SNP or haplotype was associated with NSOC susceptibility: rs3758250(CC/CT+TT: OR=1.14,95% CI=0.82-1.59;CC+CT/TT: OR=0.50,95% CI=0.12-2.00;C/T: OR=0.93,95% CI=0.68-1.26);rs907577(TT/CT+CC: OR=1.06,95% CI=0.81-1.39,TT+CT/CC: OR=0.50,95%CI=0.15-1.68,T/C: OR=0.98,95% CI=0.77-1.25),rs7043516(AA/AC+CC: OR=1.24,95% CI=0.84-1.81,A/C: OR=1.22,95% CI=0.84-1.77).Nevertheless,in stratified analysis,we found rs7043516,locating in the 3’-UTR of FOXE1,was associated with risk of CLO:(AA/AC+CC: OR=0.51,95% CI=0.28-0.94;A/C: OR=0.53,95%CI=0.29-0.95).Further in vitro luciferase assay indicated that this SNP could contribute to differential binding ability with mi RNA.Taken together,the present study showed that rs7043516 may be considered as a potentially susceptible marker of cleft lip only among Chinese Han populations.