Association Between Rs2262251 And Genetic Susceptibility To Non-syndromic Cleft Lip And Palate And Its Mechanism Of Action

Orofacial Cleft is one of the most common congenital diseases,accounting for about 1:700 to 4:1000 of the newborns.According to epidemiological investigation,the incidence of cleft lip and palate is 1.663/1000 in China.Children with orofacial cleft usually suffer various probloms,such as sucking,language,infection,hearing,psychological development and social adaptability and so on.Orofacial Cleft brings heavy burden to the children themselves and their families.Therefore,it need to explore the orofacial cleft etiology to improve the measures of prevention and treatment.We expect it could make early prevention from the the risk of suffering from orofacial cleft family.Orofacial Cleft can be divided into syndrome type cleft lip(syndromic orofacial clefts,SOCs)and non syndromic cleft lip and palate(Non-syndromic Orofacial Cleft,NSOC),which has a higher incidence,accounting for 70%.According to epidemiology,NSOC is often divided into CLP,CLO and CPO.The etiology of NSOC is complex,and the pathogenesis is not unclarified.The single nucleotide polymorphisms(SNP),as a single nucleotide substitution,insertion or deletion of the gene polymorphism,is the most common type of variation accounting for about 90%of the total variation.Genome wide association study(GWAS)can be used to detect SNP markers in large populations.So far,at least 9 GWAS studies refered to CL/P,2 GWAS studies for CP,2 GWAS-meta studies,1 imputation study.At GWAS level,there were nearly 30 genes/gene regions detected,providing high quality evidence to explore the susceptibility gene of NSOC.Genetic variations of 4 regions(1q32-41,8q24,17q22 and 10q25.3)were considered to account for the genetic factors of cleft lip and palate from 20-25%in these significant genes/gene regions.Long noncoding RNA(lncRNA)is the largest subclass of noncoding transcripts in humans,ranging in length from two hundred to thousands of nucleotides,with no coding ability.LncRNA had been considered to be the "noise" of the transcription,and is not taken seriously.However,more and more lncRNA have been discovered in recent ten years.It has been proved that these lncRNA play important roles in the normal tissues and many diseases development.As lncRNA is not "noise",so SNPs on the lncRNAs may also change theirs function by affecting the expression and biological function,which has also been confirmed in subsequent studies.However,there are no reports about lncRNA and SNP related to the development of cleft lip and palate by now.Our research group has published the first GWAS study of cleft lip and palate about Chinese population,and found a new SNP site rs8049367,which is located between the CREBBP-ADCY9 gene region.With further analysis,it found that rs8049367 strongly linked with rs2262251,suggesting that rs8049367 may play an important role in the development of NSOC through rs2262251.Firstly,we using TaqMan methodology to genotype rs2262251 among 1321 NSOC cases and 1274 control samples in an ongoing hospital-based case-control study.There was no difference between cases and controls(P=0.064).We divided these NSOC cases into three subgroups:CLP 656 cases(49.66%),CLO 445(33.69%)and CPO 220(16.65%).Then,according to additional biochemical analysis(dual-luciferase assays,qRT-PCR,Western blot,cell scratch and cell flow cytometry et al.),we explored the mechanism of rs2262251 on the development of NSOC.The genotyping results showed that rs226225 G>C was significantly associated with NSOC(P=0.002).Compared to GG genotype,CC genotype could reduce 0.36 times suffering the risk of NSOC,and GC/CC,GG/GC,GG/GC/CC genotype decreased 0.18 times,0.32 times,0.18 times.Stratified analysis showed that CC,GC/CC,GG/GC and GG/GC/CC genotypes may reduce the risk of CL/P and CLP;CC,GC/CC and GG/GC/CC genotypes could decrease the risk of CLO;but there was no genotye effecting the risk of CPO.Over expression of rs2262251 G(lnCRNAG122[G])can significantly reduce G0/G1 phase cells,promote cell proliferation and inhibit of apoptosis,and these effects was significantly inhibited after lncRNAG122[G]was knockdown.The functional studies verified that miR-744-5p could interactive with rs2262251 C(lncRNAG122[C])in a variant-specific manner.Moreover,we confirmed that miR-744-5p could bind to 3'UTR of IQSEC2.Take all the things together,the existence of rs2262251 effected lncRNAG122 second level structure and free energy change.As miR-744-5p could specific bind with rs2262251 on lncRNAG122 and lncRNAG122 could regulate IQSEC2 through miR-744-5p as miR-744-5p's 'miRNA sponge'.They effected as 'lncRNAG122-miR-744-5p-IQSEC2' pathway to regulate NSOC development.