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.

We have conducted the first meta-analyses for nonsyndromic cleft lip with or without cleft palate (NSCL/P) using data from the two largest genome-wide association studies published to date. We confirmed associations with all previously identified loci and identified six additional susceptibility regions (1p36, 2p21, 3p11.1, 8q21.3, 13q31.1 and 15q22). Analysis of phenotypic variability identified the first specific genetic risk factor for NSCLP (nonsyndromic cleft lip plus palate) (rs8001641; PNSCLP = 6.51 × 10−11; homozygote relative risk = 2.41, 95% confidence interval (CI) 1.84–3.16).

Nonsyndromic cleft lip with/without cleft palate (nsCL/P) and nonsyndromic cleft palate only (nsCPO) are the most frequent subphenotypes of orofacial clefts. A common syndromic form of orofacial clefting is Van der Woude syndrome (VWS) where individuals have CL/P or CPO, often but not always associated with lower lip pits. Recently, ∼5% of VWS-affected individuals were identified with mutations in the grainy head-like 3 gene (GRHL3). To investigate GRHL3 in nonsyndromic clefting, we sequenced its coding region in 576 Europeans with nsCL/P and 96 with nsCPO. Most strikingly, nsCPO-affected individuals had a higher minor allele frequency for rs41268753 (0.099) than control subjects (0.049; p = 1.24 × 10(-2)). This association was replicated in nsCPO/control cohorts from Latvia, Yemen, and the UK (pcombined = 2.63 × 10(-5); ORallelic = 2.46 [95% CI 1.6-3.7]) and reached genome-wide significance in combination with imputed data from a GWAS in nsCPO triads (p = 2.73 × 10(-9)). Notably, rs41268753 is not associated with nsCL/P (p = 0.45). rs41268753 encodes the highly conserved p.Thr454Met (c.1361C>T) (GERP = 5.3), which prediction programs denote as deleterious, has a CADD score of 29.6, and increases protein binding capacity in silico. Sequencing also revealed four novel truncating GRHL3 mutations including two that were de novo in four families, where all nine individuals harboring mutations had nsCPO. This is important for genetic counseling: given that VWS is rare compared to nsCPO, our data suggest that dominant GRHL3 mutations are more likely to cause nonsyndromic than syndromic CPO. Thus, with rare dominant mutations and a common risk variant in the coding region, we have identified an important contribution for GRHL3 in nsCPO.

Although no conclusions regarding the prevalence of chromosomal or other anomalies in patients with a cleft lip with or without cleft palate in the general population could be drawn, the study revealed a strong relationship between the type of facial cleft, associated malformations, chromosomal abnormalities and fetal outcome.