Identification of causal genes for nonsyndromic orofacial clefts using Whole Exome Sequencing

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Orofacial clefting (OFC) is one of the most common congenital malformations worldwide. In the absence of any surgical intervention, neonatal mortality is reported to be high. Formal genetic / epidemiological studies have shown that the two most common subtypes of OFC, namely nonsyndromic cleft lip with or without cleft palate (nsCL/P) and cleft palate only (nsCPO), have a multifactorial aetiology in which both genetic and environmental factors contribute to the phenotype. The heritability of nsCL/P has been estimated to be more than 90%.Genome-wide association studies (GWAS) and subsequent meta-analyses have recently led to major breakthroughs in deciphering the genetic aetiology of nsCL/P, with 15 susceptibility loci for nsCL/P being identified. However, a large proportion of the nsCL/P heritability still remains to be explained. For nsCPO, the success of GWAS has been limited. The fact that both types of nonsyndromic OFC are often sporadic and the very low rate of reproduction due to high neonatal mortality suggest that a substantial proportion of clefting is caused by individual and rare de novo mutations. GWAS, which are primarily designed to target common variants, are unlikely to detect such alterations.The aim of this study is the identification of causative de novo mutations, risk genes, and functional pathways for nonsyndromic OFC, using a whole exome sequencing (WES) approach. From our large cohort of nonsyndromic OFC patients we will select 50 triads with nsCL/P and 50 triads with nsCPO (parents unaffected respectively) as a discovery cohort. Exome capture will be performed using Agilent SureSelectv5+UTR, targeting 74 Mb of the human genome. WES will be run on the in-house Illumina HiSeq2500 sequencing platform using paired-end technology. Stringent parameters for read alignment, mapping and variant calling will be applied, and variants will only be retained if they are not inherited from either parent. We anticipate that this approach will yield about 150 de novo candidate variants/genes. In order to identify the most promising candidates for follow-up (priority candidate genes), detected variants will be analysed and evaluated by different statistical approaches, such as burden and pathway analyses, locus-specific mutation rates and detrimental scores. Priority candidate genes (estimated number: 50-60) will then be re-sequenced on the Illumina MiSeq system, in a large independent confirmation cohort of 300 individuals with nsCL/P and 300 with nsCPO. The data generated will provide the basis for further in-depth analyses, such as combining with WES data from another European group. Additionally, candidate genes will be checked for association / sub-phenotype specific effects of common variants in our nsCL/P GWAS meta-analyses data.We anticipate that this study's approach will uncover the spectrum of susceptibility variants/genes for nonsyndromic clefting, and substantially improve our understanding of craniofacial development.