We conducted a genome-wide association study for nonsyndromic cleft lip with or without cleft palate (NSCL/P) in 401 affected individuals and 1,323 controls, with replication in an independent sample of 793 NSCL/P triads. We report two new loci associated with NSCL/P at 17q22 (rs227731, combined P = 1.07 × 10 −8 , relative risk in homozygotes = 1.84, 95% CI 1.34-2.53) and 10q25.3 (rs7078160, combined P = 1.92 × 10 −8 , relative risk in homozygotes = 2.17, 95% CI 1.32-3.56).NSCL/P is one of the most common human birth defects. In European populations, NSCL/P has a prevalence ranging from 1 in 700 to 1 in 1,000. We recently reported a susceptibility locus for NSCL/P at chromo some 8q24.21 from a genome wide association study in 224 individuals with NSCL/P (cases) and 383 population based controls 1 . This locus is the second susceptibility locus to have been unequivocally identified for NSCL/P to date, the first being the IRF6 locus 2 .To identify additional cleft susceptibility loci, we enlarged our sample by genotyping an additional set of 177 NSCL/P cases and adding the genotypes of 940 population based controls of central European origin. Genotyping was performed using Illumina BeadChips (Human610 Quad and HumanHap 550k).Following quality control (Supplementary Methods and Supplementary Fig. 1), association analysis of 521,288 SNPs having a minor allele frequency (MAF) of ≥1% in controls was performed in 399 cases and 1,318 controls.After excluding markers from the previously described 8q24.21 locus, 20 SNPs with P < 10 −5 remained. Five chromosomal loci (8q12.3, 10q25.3, 13q31.1, 15q13.3 and 17q22) were located within these 20 top SNPs, and the associations at these loci were further supported by at least three more SNPs with P < 10 −4 ( Supplementary Fig. 2 and Supplementary Table 1). Two additional regions were considered to be promising NSCL/P susceptibility loci (6p22.1, 11q14.2), as they contained at least four markers with P < 10 −4 .To replicate the genome wide association study (GWAS) findings, we selected the 20 top SNPs (P < 10 −5 ) as well as additional backup markers for each of the seven previously mentioned loci, resulting in two replication assays. We included additional SNPs with P < 10 −4 in the two replication assays, giving highest priority to SNPs with the lowest P values. Thus, a total of 56 markers were genotyped in a replication sample of 793 NSCL/P triads of European origin. Genotyping using matrix assisted laser desorption/ionization time of flight (MALDI TOF) mass spectrometry (Sequenom Inc.) was successful for 45 markers (representing 32 different loci), which were then analyzed by the transmission disequilibrium test in 665 triads (128 triads were excluded after quality control, Supplementary Methods).Of the 45 SNPs successfully genotyped, 11 (representing six differ ent loci) showed P < 0.05 in the replication sample (Supplementary Table 2). Two of these SNPs remained significant after correction for multiple testing by a conservative Bonferroni procedure (17q22: rs227731, P corr ...

We conducted a genome-wide association study involving 224 cases and 383 controls of Central European origin to identify susceptibility loci for nonsyndromic cleft lip with or without cleft palate (NSCL/P). A 640-kb region at chromosome 8q24.21 was found to contain multiple markers with highly significant evidence for association with the cleft phenotype, including three markers that reached genome-wide significance. The 640-kb cleft-associated region was saturated with 146 SNP markers and then analyzed in our entire NSCL/P sample of 462 unrelated cases and 954 controls. In the entire sample, the most significant SNP (rs987525) had a P value of 3.34 x 10(-24). The odds ratio was 2.57 (95% CI = 2.02-3.26) for the heterozygous genotype and 6.05 (95% CI = 3.88-9.43) for the homozygous genotype. The calculated population attributable risk for this marker is 0.41, suggesting that this study has identified a major susceptibility locus for NSCL/P.

Variants in the interferon regulatory factor 6 (IRF6) gene have repeatedly been associated with non-syndromic cleft lip with or without cleft palate (NSCL/P). A recent study has suggested that the functionally relevant variant rs642961 is the underlying cause of the observed associations. We genotyped rs642961 in our Central European case-control sample of 460 NSCL/P patients and 952 controls. In order to investigate whether other IRF6 variants contribute independently to the etiology of NSCL/P, we also genotyped the non-synonymous coding variant V274I (rs2235371) and five IRF6-haplotype tagging single nucleotide polymorphisms (SNPs). A highly significant result was observed for rs642961 (P = 1.44 x 10(-6)) in our sample. The odds ratio was 1.75 [95% confidence interval (CI): 1.38-2.22] for the heterozygous genotype and 1.94 (95% CI: 1.21-3.10) for the homozygous genotype, values that are similar to those reported in a previously published family-based study. Our results thus confirm the involvement of the IRF6 variant, rs642961, in the etiology of NSCL/P in the Central European population. We also found evidence suggestive of an independent protective effect of the coding variant V274I. In order to understand fully the genetic architecture of the IRF6 locus, it will be necessary to conduct additional SNP-based and resequencing studies using large samples of patients.

Non-syndromic cleft lip with or without cleft palate (NSCL/P) is one of the most common birth defects and has a multifactorial etiology that includes both genetic and environmental components. MYH9, the gene coding for the heavy chain of non-muscle myosin II, has been considered as a good candidate gene in NSCL/P on the basis of its expression profile during craniofacial morphogenesis. Reports in an Italian sample, as well as in an ethnically mixed North American sample, of a positive association between single-nucleotide polymorphisms in the MYH9 gene and NSCL/P have provided further support for the role of MYH9 in the development of NSCL/P. In the present study, we aimed to replicate these findings by conducting a family-based association study with seven single nucleotide polymorphisms in MYH9 using a sample of 248 NSCL/P patients and their parents. Single marker analysis resulted in a highly significant association for rs7078. In haplotype analysis, the most significant result was obtained for the SNP combination (rs7078; rs2071731; rs739097; rs5995288). Our results thus confirm the potential involvement of MYH9 in the etiology of NSCL/P in our patients of Central European origin, although further studies are warranted to determine its exact pathogenetic role.