Nonsyndromic cleft lip with or without cleft palate (NSCLP) is a common birth defect for which only ~ 20% of the underlying genetic variation has been identified. Variants in noncoding regions have been increasingly suggested to contribute to the missing heritability. In this study, we investigated whether variation in craniofacial enhancers contributes to NSCLP. Candidate enhancers were identified using VISTA Enhancer Browser and previous publications. Prioritization was based on patterning defects in knockout mice, deletion/duplication of craniofacial genes in animal models and results of whole exome/whole genome sequencing studies. This resulted in 20 craniofacial enhancers to be investigated. Custom amplicon-based sequencing probes were designed and used for sequencing 380 NSCLP probands (from multiplex and simplex families of non-Hispanic white (NHW) and Hispanic ethnicities) using Illumina MiSeq. The frequencies of identified variants were compared to ethnically matched European (CEU) and Los Angeles Mexican (MXL) control genomes and used for association analyses. Variants in mm427/MSX1 and hs1582/SPRY1 showed genome-wide significant association with NSCLP (p ≤ 6.4 × 10-11). In silico analysis showed that these enhancer variants may disrupt important transcription factor binding sites. Haplotypes involving these enhancers and also mm435/ABCA4 were significantly associated with NSCLP, especially in NHW (p ≤ 6.3 × 10-7). Importantly, groupwise burden analysis showed several enhancer combinations significantly over-represented in NSCLP individuals, revealing novel NSCLP pathways and supporting a polygenic inheritance model. Our findings support the role of craniofacial enhancer sequence variation in the etiology of NSCLP.

中文翻译：

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非综合征性唇裂和腭裂的颅面增强子变异的证据。

非综合征性唇裂伴或不伴腭裂 (NSCLP) 是一种常见的先天缺陷，仅约 20% 的潜在遗传变异已被确定。越来越多的人认为非编码区域的变异会导致遗传力缺失。在这项研究中，我们调查了颅面增强剂的变化是否有助于 NSCLP。使用 VISTA Enhancer Browser 和以前的出版物识别候选增强器。优先排序基于敲除小鼠的模式缺陷、动物模型中颅面基因的缺失/复制以及全外​​显子组/全基因组测序研究的结果。这导致要研究 20 种颅面增强剂。设计了基于扩增子的定制测序探针，并使用 Illumina MiSeq 对 380 个 NSCLP 先证者（来自非西班牙裔白人 (NHW) 和西班牙裔的多重和单纯性家族）进行测序。将鉴定出的变异频率与种族匹配的欧洲 (CEU) 和洛杉矶墨西哥 (MXL) 对照基因组进行比较，并用于关联分析。mm427/MSX1 和 hs1582/SPRY1 中的变体显示出与 NSCLP 的全基因组显着关联（p ≤ 6.4 × 10-11）。计算机分析表明，这些增强子变体可能会破坏重要的转录因子结合位点。涉及这些增强子以及 mm435/ABCA4 的单倍型与 NSCLP 显着相关，尤其是在 NHW 中（p ≤ 6.3 × 10-7）。重要的，分组负担分析显示，几种增强子组合在 NSCLP 个体中显着过度，揭示了新的 NSCLP 途径并支持多基因遗传模型。我们的研究结果支持颅面增强子序列变异在 NSCLP 病因学中的作用。