Deleterious coding variants in multi-case families with non-syndromic cleft lip and/or palate phenotypes

www.nature.com/scientificreports OPEN received: 16 November 2015 accepted: 06 July 2016 Published: 26 July 2016 Deleterious coding variants in multi-case families with non-syndromic cleft lip and/or palate phenotypes Reuben J. Pengelly1, Liliana Arias2, Julio Martínez2, Rosanna Upstill-Goddard1, Eleanor G. Seaby1, Jane Gibson3, Sarah Ennis1, Andrew Collins1 & Ignacio Briceño2 Nonsyndromic Cleft Lip and/or Palate (NSCLP) is regarded as a multifactorial condition in which clefting is an isolated phenotype, distinguished from the largely monogenic, syndromic forms which include clefts among a spectrum of phenotypes. Nonsyndromic clefting has been shown to arise through complex interactions between genetic and environmental factors. However, there is increasing evidence that the broad NSCLP classification may include a proportion of cases showing familial patterns of inheritance and contain highly penetrant deleterious variation in specific genes. Through exome sequencing of multi-case families ascertained in Bogota, Colombia, we identify 28 nonsynonymous single nucleotide variants that are considered damaging by at least one predictive score. We discuss the functional impact of candidate variants identified. In one family we find a coding variant in the MSX1 gene which is predicted damaging by multiple scores. This variant is in exon 2, a highly conserved region of the gene. Previous sequencing has suggested that mutations in MSX1 may account for ~2% of NSCLP. Our analysis further supports evidence that a proportion of NSCLP cases arise through monogenic coding mutations, though further work is required to unravel the complex interplay of genetics and environment involved in facial clefting. Cleft lip and/or palate (CLP) phenotypes are among the most frequent birth defects occurring at rates of 1/500– 1/2500 births1. A proportion of cases present with syndromic disease (CLP in addition to a spectrum of additional phenotypes) mostly caused by rare mutations in single genes that often show Mendelian patterns of inheritance. However up to 70% of cases show phenotypes lacking any additional cognitive or craniofacial abnormalities, referred to as nonsyndromic cleft lip and/or palate (NSCLP). Such phenotypes are regarded as genetically complex arising through the interplay of numerous genetic and environmental factors. Increased understanding of the underlying aetiology of NSCLP phenotypes (both genetic and environmental) is needed to ultimately develop strategies for prevention, and improve treatment and prognosis. NSCLP has a significant genetic basis, for example, the first degree relatives of affected individuals have a 30–40 fold elevated risk and phenotype concordance for monozygotic (MZ) twins is 40–60%, compared to 5% for di-zygotic twins1. Genetic studies including linkage analysis, genome-wide association (GWAS), and GWAS-based meta-analysis, have yielded reproducible evidence for the involvement of several genes and gene regions. Collins et al.2, listed 16 genes and gene regions which have been firmly implicated in NSCLP through linkage and association analysis. Several of these are broad regions where the underlying causal variant(s) have yet to be pinpointed, however, polymorphisms in genes such as IRF6 are strongly associated with NSCLP3 and more minor roles have been established for MSX14,5, PVRL1, FGFR2, PAX7, NOG and SPRY2 among others6. Exome sequencing presents opportunities to identify rare coding variation that may contribute to risk of NSCLP phenotypes. If NSCLP is entirely multifactorial, the contribution of rarer variants may be largely polygenic and mediated by numerous variants of very small individual effect. In this case, causal genes may only be detectible through the analysis of large numbers of patients using, for example, burden tests7. However, there is 1 Genetic Epidemiology and Genomic Informatics, Faculty of Medicine, University of Southampton, Southampton, UK. 2Department of Biomedical Sciences, Medical School, Universidad de La Sabana, Bogota, Colombia. 3Centre for Biological Sciences, Faculty of Natural & Environmental Sciences, University of Southampton, Southampton, UK. Correspondence and requests for materials should be addressed to R.J.P. (email: ) Scientific Reports | 6:30457 | DOI: 10.1038/srep30457 1 www.nature.com/scientificreports/ Figure 1. Pedigrees of families analysed. +symbol indicates that the individual has been exome sequenced (sequenced cases: two families with one family member; two families with parent and offspring; two families with sib pair; one family with avuncular pair). growing evidence for involvement of rare variants of larger effect in NSCLP including, for example, truncating mutations in the ARHGAP29 gene8 and mutations in the IRF6 gene, which is also known to contain mutations involved in malformation syndromes that include CLP such as Van der Woude9. We consider here a number of NSCLP families with multiple affected individuals and undertake exome sequencing to investigate the contribution of rare variants in genes previously associated with any form of clefting phenotype. Materials and Methods Exome sequences of twelve individuals from seven multi-case families (CL1-CL7) with NSCLP phenotypes were obtained. All experimental protocols were approved by the Research Ethics Committee at the Universidad de La Sabana, Bogota; informed consent was obtained for all participants and research was conducted in accordance with the Declaration of Helsinki. Families included between two and six individuals with isolated NSCLP (Fig. 1). Most individuals have unilateral CLP but several individuals have the more severe bilateral phenotype. DNA samples were extracted from blood collected at Operation Smile, Bogota, Colombia and exomes were captured using the Agilent SureSelect v5 (51 Mb) kit and sequenced on a HiSeq 2000. Read depth coverage statistics for all 12 exome sequences are given in Supplementary Table 1, and indicate ~85–97% coverage of exon targets at >20 fold depth across all samples. Orthogonal genotyping was performed for a panel of 24 SNPs to validate sample identity after processing10. To understand the spectrum of potentially damaging variation, we considered the list of 865 genes previously implicated in any form of CLP phenotype presented by Pengelly et al.11 (Supplementary Table 2). Examining rare variation in genes in this comprehensive list enables evaluation of whether known CLP genes contain variation which may underlie more familial forms of NSCLP. Furthermore, because each exome contains a very large number of putatively damaging variants including those completely unrelated to the clefting phenotypes (including potential incidental findings), this strategy focussing only on genes previously implicated in any form of clefting is a practical route to identifying causal variation in these families. The list is derived in part (363 genes out of the 865) Scientific Reports | 6:30457 | DOI: (...truncated)