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

口面裂是世界上最常见的先天性畸形之一。据报道，在没有任何手术干预的情况下，新生儿死亡率很高。正式的遗传/流行病学研究表明，OFC的两种最常见亚型，即伴有或不伴有腭裂的非综合征性唇裂（nsCL/P）和单纯腭裂（nsCPO），具有多因素的病因学，遗传和环境因素都对表型有影响。据估计，nsCL/P的遗传率在90%以上。最近，全基因组关联研究（GWAS）和随后的荟萃分析在破译nsclc /P的遗传病因方面取得了重大突破，鉴定出了15个nsclc /P的易感位点。然而，nsCL/P遗传率的很大一部分仍有待解释。对于非scpo， GWAS的成功是有限的。事实上，这两种类型的非综合征性OFC通常是散发的，而且由于新生儿死亡率高，繁殖率很低，这表明很大一部分唇裂是由个体和罕见的新生突变引起的。GWAS主要是针对常见变异设计的，不太可能检测到这种改变。本研究的目的是使用全外显子组测序（WES）方法鉴定非综合征性OFC的致病新生突变、风险基因和功能途径。从我们的大型非综合征性OFC患者队列中，我们将选择50例患有非scl /P的三联体和50例患有非scpo的三联体（父母分别未受影响）作为发现队列。外显子组捕获将使用Agilent SureSelectv5+UTR进行，目标是74 Mb的人类基因组。WES将在Illumina内部HiSeq2500测序平台上运行，使用对端技术。将应用读取对齐、映射和变量调用的严格参数，并且只有当变量不从父节点继承时才会保留它们。我们预计这种方法将产生大约150个新的候选变异/基因。为了确定最有希望进行随访的候选基因（优先候选基因），检测到的变异将通过不同的统计方法进行分析和评估，例如负担和途径分析、位点特异性突变率和有害评分。优先候选基因（估计数量：50-60）将在Illumina MiSeq系统上重新测序，在300名nsCL/P患者和300名nsCPO患者的大型独立确认队列中进行。生成的数据将为进一步深入分析提供基础，例如与来自另一个欧洲小组的WES数据相结合。此外，候选基因将在我们的nsCL/P GWAS荟萃分析数据中检查常见变异的关联/亚表型特异性效应。我们期望这项研究的方法将揭示非综合征性唇裂的易感性变异/基因谱，并大大提高我们对颅面发育的理解。

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.