Association Between IRF6, MSX1 SNPs And Nonsyndromic Cleft Lip With Or Without Cleft Palate In The West Chinese Population

Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is one of the most common birth defects, but its etiology is largely unknown. It is very likely that both genetic and environmental factors contribute to this malformation. Over the past decade, genetic linkage and association analyses have provided evidence to support the involvement of several genes and chromosomal regions, among them loci at 1q32 (IRF6), 2p13 (TGFA), 4p16 (MSX1), 6p23-25, 14q24 (TGFB3), 17q21 (RARA), and 19q13 (BCL3, TGFB1) have the most supporting data. But many difficulties have been encountered in replicating previous findings. Inconsistent results have accumulated from genetic studies of the NSCL/P complex traits. This is not unexpected; given the complicated heterogeneous nature of isolated CL/P and the number of confounding factors. The results of multiple genome scans and a subsequent meta-analysis have significantly advanced our knowledge by revealing novel loci. Furthermore, candidate gene approaches have identified important roles for IRF6 and MSX1.One gene contributing to NSCL/P, IRF6, has been identified. It was initially targeted for investigation after mutations were detected in the gene in patients with Van der Woude syndrome (VWS OMIM 119300). An interesting approach to study genetics of NSCL/P is to evaluate genes known to contribute to syndromic forms of CL/P and especially to those phenotypically closest to NSCL/P. The best phenocopy for isolated clefts is VWS. It is a dominantly inherited disorder in which pits of the lower lip (85% of cases) and hypodontia (25% of cases) are the only features distinguishing it from NSCL/P. Recently, IRF6 was shown to harbor mutations in patients with VWS. Variation at the IRF6 locus is responsible for 12% of the genetic contribution to NSCL/P at the population level and triples the recurrence risk for a child with a cleft in some families. Similar results have been since reproduced in additional populations:Another gene contributing to isolated cleft lip or palate, MSX1, has also been identified. It was initially targeted for investigation after a nonsense mutation was detected in the gene, which segregated with cleft lip or palate in a large family. The occurrence of cleft palate in MSX1 knockout mice aided the identification of a MSX1 mutation cosegregating with tooth agenesis, CL/P, and/or CPO. Approximately 2% of patients with NSCL/P have missense mutations in the coding sequence or in highly conserved regulatory elements of MSX1. MSX1 has also been implicated as a causative gene in cleft palate in case- control studies and by animal models.In our project, we have recruited 332 NSCL/P patients, their parents (289 mothers, 243 fathers and 206 complete trios), and 174 normal match controls. All Subjects were recruited between 2004 and 2006 from the Cleft lip and palate Surgery Department of West China Stomotology College of Sichuan University. Two genes---IRF6 and MSXI were choosed as the candidate predisposing genes. Within each gene, 3 singificant representative SNPs wese selected as the studied loci. Venous blood samples were drawn from the whole participants and DNA was extracted by phenol chloroform extraction protocol and was amplified using the PCR. Genotyping for each SNP carried out by restriction digestion of PCR products. Family-Based Association Analysis (Transmission Disequilibrium Test, TDT) and case-control study were used to study the association between IRF6 and MSXI gene SNPs (single-marker and haplotype) and NSCL/P in the west Chinese population. On the basis of both genetic and environmental factors contribute to the NSCL/P, using the case-control study method, we further investigate the effects of the environmental exposure and IRF6, MSX1 gene polymorphisms to NSCL/P. Multifactor Logistic regression was used to analyses the results of investigation and detection. In order to approach the role IRF6 and MSXI gene mutation and enviromental factors play in the NSCL/P and build up data for Chinese group gene databank.In our study, strong evidence of overtransmission of IRF6 gene rs2235375 and rs2235371 (V274I) two SNPs loci G allele was found in the west Chinese population (P=0.0000). There were significant differences in the frequency distributions of both genotypes and alleles among probands as compared with the control group. Ours results support the previous findings of Zucchero and Srichomthong.At IRF6 gene rs2235371 locus, results of the TDT showed a highly significant (P＜0.001) association between cleft lip or palate and the G allele, but not between cleft palate alone and the G allele. In 4 kinds of genetic models (the additive,dominative,recessive,genotype), the FBAT analysis also appeared a stronger association between the rs2235375 and rs2235371markers and the NSCL/P.But interestingly, for the IRF6 gene rs2013162 marker, no significant association was obtained in our samples, which very differed from previous found. This is not surprising given that two different ancestral populations were studied.There are 5 haplotypes constructed by the 3 SNPs loci in IRF6 gene showed significant results. However, among the whole significant results of the haplotypes, the frequent G allele for marker rs2013162 was always found to be carried. The most significant results were obtained for the G-G-G haplotype (P=0.000525).Pairwise linkage disequilibrium (LD) measures across the 3 markers of IRF6 gene showed a significant association.At MSXI gene rs3821949 and rs12532 two SNPs loci, no significant differences in genotype and allele frequencies between patients and controls were observed.At MSXI gene Q147I locus, no TT genotype variations were detected in our study group inclouding the controls. TDT and Haplotype analyses based on these markers were unable to support the association of variation in MSX1 gene with risk of oral clefting. Suggesting no transmission distortions occur in MSXI gene Q147I, rs3821949 and rs12532 loci.The environmental exposure factors, such as, father smoking, mother abortion times, history of disease, overdose and vitamin supplement in pregnancy, and IRF6 rs2235371 GG genotypes were related significantly with NSCL/P( P＜0. 05), their OR were 0.407, 7.509, 58.531, 62.608, 0.017, 2.246 respectively.Based on these findigs, we found strong evidence that IRF6 mutations make a contribution to NSCL/P in west Chinese population. DNA-sequence variants associated with IRF6 are major contributors to NSCL/P. The contribution of variants in single genes to cleft lip or palate is an important consideration in genetic counseling. Replication of positive findings in complex diseases is has been difficult. That six different studies have yielded significant results for an association between IRF6 and NSCL/P suggests that this is an important gene and clearly warrants further study.Moreover, in west Chinese population no significant associations were found between NSCL/P and variants at MSX1 gene Q147I, rs3821949 and rs12532 three SNPs loci. We were unable to confirm the involvement of the MSX1 gene in NSCL/P. But up to now, no one MSX1 gene mutation locus has been identified making a contribution to NSCL/R Our study on the association between MSX1 and NSCL/P is a significant exploration and trial.Father smoking, mother abortion times, history of disease, overdose and vitamin supplement in pregnancy, and polymorphism of IRF6 rs2235371 was the important susceptible factors, suggesting that the NSCL/P occurrence be related with affection both of environmental exposure and gene polymorphisms.