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Genome-wide association studies (GWAS) have generated unprecedented insights into the genetic etiology of orofacial clefting (OFC). The moderate effect sizes of associated noncoding risk variants and limited access to disease-relevant tissue represent considerable challenges for biological interpretation of genetic findings. As rare variants with stronger effect sizes are likely to also contribute to OFC, an alternative approach to delineate pathogenic mechanisms is to identify private mutations and/or an increased burden of rare variants in associated regions. This report describes a framework for targeted resequencing at selected noncoding risk loci contributing to nonsyndromic cleft lip with/without cleft palate (nsCL/P), the most frequent OFC subtype. Based on GWAS data, we selected three risk loci and identified candidate regulatory regions (CRRs) through the integration of credible SNP information, epigenetic data from relevant cells/tissues, and conservation scores. The CRRs (total 57 kb) were resequenced in a multiethnic study population (1061 patients; 1591 controls), using single-molecule molecular inversion probe technology. Combining evidence from in silico variant annotation, pedigree- and burden analyses, we identified 16 likely deleterious rare variants that represent new candidates for functional studies in nsCL/P. Our framework is scalable and represents a promising approach to the investigation of additional congenital malformations with multifactorial etiology.

BackgroundNonsyndromic cleft palate only (nsCPO) is a common and multifactorial form of orofacial clefting. In contrast to successes achieved for the other common form of orofacial clefting, that is, nonsyndromic cleft lip with/without cleft palate (nsCL/P), genome wide association studies (GWAS) of nsCPO have identified only one genome wide significant locus. Aim of the present study was to investigate whether common variants contribute to nsCPO and, if so, to identify novel risk loci. MethodsWe genotyped 33 SNPs at 27 candidate loci from 2 previously published nsCPO GWAS in an independent multiethnic sample. It included: (i) a family-based sample of European ancestry (n=212); and (ii) two case/control samples of Central European (n=94/339) and Arabian ancestry (n=38/231), respectively. A separate association analysis was performed for each genotyped dataset, and meta-analyses were performed. ResultsAfter association analysis and meta-analyses, none of the 33 SNPs showed genome-wide significance. Two variants showed nominally significant association in the imputed GWAS dataset and exhibited a further decrease in p-value in a European and an overall meta-analysis including imputed GWAS data, respectively (rs395572: P-MetaEU=3.16 x 10(-4); rs6809420: P-MetaAll=2.80 x 10(-4)). ConclusionOur findings suggest that there is a limited contribution of common variants to nsCPO. However, the individual effect sizes might be too small for detection of further associations in the present sample sizes. Rare variants may play a more substantial role in nsCPO than in nsCL/P, for which GWAS of smaller sample sizes have identified genome-wide significant loci. Whole-exome/genome sequencing studies of nsCPO are now warranted.

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, similar to 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 x 10(-2)). This association was replicated in nsCPO/control cohorts from Latvia, Yemen, and the UK (p(combined) = 2.63 x 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 x 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.

BACKGROUND: Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is one of the most common of all congenital anomalies, and has a multifactorial etiology involving both environmental and genetic factors. Recent genome-wide association studies (GWAS) identified strong association between a locus on chromosome 10q25.3 and NSCL/P in European samples. One gene at 10q25.3, the ventral anterior homeobox 1 (VAX1) gene, is considered a strong candidate gene for craniofacial malformations. The purpose of the present study was to provide further evidence that VAX1 is the causal gene at the 10q25.3 locus through identification of an excess of rare mutations in patients with NSCL/P. METHODS: The 5'UTR, complete coding regions, and adjacent splice sites of the two known VAX1 isoforms were sequenced in 384 patients with NSCL/P and 384 controls of Central European descent. Observed variants were investigated with respect to familial cosegregation or de novo occurrence, and in silico analyses were performed to identify putative effects on the transcript or protein level. RESULTS: Eighteen single-base variants were found, 15 of them rare and previously unreported. In the long VAX1 isoform, predicted functionally relevant variants were observed more often in NSCL/P cases, although this difference was not significant (p = 0.17). Analysis of family members demonstrated incomplete cosegregation in most pedigrees. CONCLUSION: Our data do not support the hypothesis that highly penetrant rare variants in VAX1 are a cause of NSCL/P. To determine whether VAX1 is the causative gene at 10q25.3 further research, in particular into the biologic function of its long isoform, is warranted. Birth Defects Research (Part A), 2012. (c) 2012 Wiley Periodicals, Inc.