Genome-wide Association Study Of Nonsyndromic Cleft Lip With Cleft Palate In Chinese Han Population

Background:Orofacial clefts are the most common congenital birth defects in humans.Affected children need multistage therapy and multidisciplinary care from birth until adulthood,thus many children and their families are affected psychologically to some extent[1-3].Epidemiology studies suggest that the prevalence of OFC varied across different populations.African-derived populations have the lowest prevalence rates at approximately 1 in 2,500,and European-derived populations have intermediate prevalence rates at approximately 1 in 1,000.Native American and Asian populations(especially Chinese and Japenese),have the highest reported birth prevalence rates at nearly 1 in 500.According to whether the patients have other malformations or anomalies,OFC can be divided into syndromic and non-syndromic forms.Additionally,OFC is further classified into CLP,CLO and CPO based on the anatomical morphology.Because they share common epidemiological patterns and occur during the same embryological period,CLP and CLO are often grouped together as CL/P[4-6].Approximately 70%of CL/P cases and 50%of CPO cases occur as isolated entities with no other abnormal phenotypes are considered to be non-syndromic.The sex ratio of OFC is also varying with severity of the cleft.Consistent observations show that there is a 2:1 male to female ratio for clefts involving the lip,and approximately a 1:2 male to female ratio for cleft palate only[4,6].The lip and palate development needs a complex series of events that require close coordination of programmes.Studies have indicated that the aetiology of OFC is multifactorial,including genetic factors,environmental factors,and also genetic-environmental interaction.So far,Genetic factors are thought to be the most important basis of OFC.Twin studies and segregation analysis supported a genetic component to NSOFC,and presented a significant trend of familial aggregation[7-9].Various genetic approaches,such as candidate genes[10,11],chromosomal anomalies[12],sequencing[13,14],linkage[15,16]and association studies[17-19]also confirmed the genetic effects from different aspects.However,the identification of candidate genes is still complicated by non-Mendelian inheritance patterns,heterogeneity,and limited sample sizes.Epidemiological and experimental data suggested that environmental risk factors might be important in the development of OFC,such as maternal exposure to tobacco smoke,alcohol,poor nutrition,viral infection,medicinal drugs,and teratogens in early pregnancy[4,20].Meanwhile,data suggested that maternal use of multivitamin supplements in early pregnancy has been linked to decreased risk of OFC[21].These evidences from both sides provide certain evidence to the relationship between environmental factors and OFC.Smoking and TGFA[22,23],smoking and TGFB3[24],smoking and IRF6[25],as well as smoking and GSTT1 and NOS3[26]were reported to play an interactional role in NSOFC.Meanwhile,findings on interactions of alcohol and ADH1C in NSOFC were partly confirmed[27].Previous researches have presented us many important clues on the aetiology of OFC,and provide us a solid foundation for further study.However,more studies are needed to elucidate the inconsistent conclusions.As a complex desease caused by multiple factors,the mainly effective research strategies of NSOFC are still focused on genetic factors.Present genetic approaches to NSOFC,such as linkage analysis study,candidate gene association studies,may have the limitation to explore the genetic of complex disease.Therefore,to conduct the GWAS of NSOFC(especially for the three sub-phenotypes)will be of great importance in enhancing our understanding of the pathogenesis and genetic basis of NSOFC.Objectives:To explore associated variants in whole genomewide and identify susceptibility loci/genes for the most serious sub-phenotye(NSCLP)by using GWAS approach in Chinese Han population,and replicate them in other populations.To identify phenotype(NSCLP,NSCLO and NSCPO)specific associated loci/genes by using genotype-phenotype stratification analysis.To prioritize candidate genes by using functionally analysis,and to discuss the relations between these candidate genes/loci and NSOFC by using synthetically analysis.Materials and Methods:(1)2,096 NSCLP cases and 4,051 controls of Chinese Han ancestry were genotyped by using Illumina HumanOmniZhongHua-8 BeadChips,with totally 900,015 SNPs in the GWAS stage.After strict quality control,unqualified samples and SNPs were excluded.(2)Replication stages:?Replication 1:Taking LD blocks into account,we selected 152 top SNPs for a follow up replication in additional 1,346 NSCLP cases and 4,542 controls of Chinese by using Sequenom Mass Array.After quality control,we combined GW AS and replication of the first stage together for statistical analysis.?Replication 2:We selected the 41 significantly associated index SNPs from the above NSCLP results,and replicated them in 1,104 NSCLO cases and 3,312 controls by using Sequenom MassArray.?Replication 3:We then replicated the same 41 index SNPs from replication 2 in 1,104 NSCPO cases and 3,312 controls(shared with replication 2)by using Sequenom MassArray.?Stratification analysis of NSCLP,NSCLO and NSCPO among the 41 index SNPs.?Replication 4,5 and 6:We also checked associations of the newly identified NSCLP loci with totally 24 SNPs(one to two SNPs from each locus)in additional three populations,including 339 NSCL/P cases and 1,318 controls of German,861 NSCL/P trios of Asian ancestry,and 557 NSCL/P trios of European ancestry.(3)Stratification of genotype-phenotype analysis with clinical data.(4)Functional analysis based on the 14 new NSCLP loci:?Regional scatter plot;?Gene annotation and biological description:All genes presented in the associated region in LD with index-SNP(R2>0.7)were annotated for literature review,gene expression,animal model experiments and mutation detected in symdromic OFC by using Pubmed,EMAGE,MGI and OMIM database.Functional annotations of index SNP/gene/protin based on the 26 NSCLP loci:?Annotate the index-SNP or any of the SNPs in LD with this SNP in our data(R2?0.7,totally 135 SNPs)by ENCODE annotations.?Performe a network analysis of notable genes in the 26 NSCLP associated loci.?Carry out a comprehensive evaluation for pleiotropic effects of 26 SNPs associated with risk to NSCLP(± 500kb either side of each index SNP)in the publicly available GWAS catalogue in NHGRI database,and further assess possible independence among these various birth defects/diseases/traits for these particular SNPs.?IHC expression studies in the mouse:Five genes of the most interesting were selected for IHC expression in mice at embryonic stages E13.5 to E16.5.Results:(1)After strict quality control,2,033 NSCLP cases and 4,051 controls with 803,202 SNPs were left for further analysis.(2)Replication stages:?Replication 1:146 SNPs were left for further analysis after strict quality control.We identified 14 new NSCLP-associated loci and confirmed 12 previously reported NSCL/P loci reaching genome-wide significance after Meta analysis.?Replication 2:After quality control,40 SNPs were left for further analysis.Among them 15 SNPs reached significant level,and they were distributed in 2 new loci and 8 previously reported loci.?Replication 3:After quality control,40 SNPs were left for further analysis.Three SNPs reached significant level,and they were distributed in one new locus and 2 previously reported loci.?Stratification analysis:We observed that the numbers of heterogeneous loci between NSCLP and NSCLO,between NSCLP and NSCPO,as well as between NSCLO and NSCPO were 1,8 and 5.?Replication 4,5 and 6:After quality control,the numbers of significant loci were 3(5 SNPs)in German,7(10 SNPs)in Asian ancestry group,5(6 SNPs)in European ancestry group.(3)After stratification analyses,the positive results of genotype-phenotype analyses were present in:1q32.2 locus and gender with NSCLP,as well as 8q21.3 locus and maternal age with NSCLP.(4)Functional analysis based on the 14 NSCLP novel loci:?Regional scatter plot:There were one gene or more than one genes located within 12 loci harboring the association,except two loci.?Manual gene annotation and biological description:We found 28 genes were classified into different biological categories;a total of 20 genes were found related to embryonic development,among which 12 were expressed in the related tissue for OFC;Twelve genes were found to produce cleft lip/palate malformations in mutant mouse models;Seven genes were reported to be associated with 9 recognized malformation syndromes including OFC as a clinical phenotype.Functional analysis of SNP/gene/protein based on the 26 NSCLP novel loci:?Of the 135 SNPs associated with the risk of NSCLP at these 26 loci(R2>0.7 with the index SNPs),33,99 and 113(respectively)were found in known or predicted regulatory elements such as promoters,enhancers or motifs biochemically characterized to regulate transcription for 50 reference genes.?24 genes are highly involved in the pathway network.?Of the 26 genetic risk factors,19 had reported associations with a total of 34 other diseases/traits.?IHC expression studies in the mouse:Positive immunohistochemistry results on mouse embryos were showed for three genes.Conclusions:(1)This is the first NSCLP GWAS of the largest smaple size in Chinese Han.We identified 14 novel NSCLP-associated loci and confirmed 12 previously reported NSCL/P loci in Chinese Han.(2)When comparing NSCLP,NSCLO and NSCPO with each other,there is clear evidence that genetic relationships between NSCLP,NSCLO and NSCPO were different thus they might share some common genetic factors.(3)Replication results of foreign populations suggest that heterogeneity do really exist among different ancestry groups.(4)Genotype-phenotype stratification analyses provide evidence on genetic-enviromental interaction of 1q32.2 locus and gender with NSCLP,as well as 8q21.3 locus and maternal age with NSCLP.(5)Our study provides further genetic evidence that impaired FGF signaling contribute to the pathogenesis of NSCLP.Our current study has advanced the understanding of the genetic architecture controlling the risk of NSOFC and has highlighted potential candidate genes through subsequent genetic and biological analyses.