Meta-analysis Reveals Genome-Wide Significance at 15q13 for Nonsyndromic Clefting of Both the Lip and the Palate, and Functional Analyses Implicate GREM1 As a Plausible Causative Gene

Nonsyndromic orofacial clefts are common birth defects with multifactorial etiology. The most common type is cleft lip, which occurs with or without cleft palate (nsCLP and nsCLO, respectively). Although genetic components play an important role in nsCLP, the genetic factors that predispose to palate involvement are largely unknown. In this study, we carried out a meta-analysis on genetic and clinical data from three large cohorts and identified strong association between a region on chromosome 15q13 and nsCLP (P = 8.13×10−14 for rs1258763; relative risk (RR): 1.46, 95% confidence interval (CI): 1.32–1.61)) but not nsCLO (P = 0.27; RR: 1.09 (0.94–1.27)). The 5 kb region of strongest association maps downstream of Gremlin-1 (GREM1), which encodes a secreted antagonist of the BMP4 pathway. We show during mouse embryogenesis, Grem1 is expressed in the developing lip and soft palate but not in the hard palate. This is consistent with genotype-phenotype correlations between rs1258763 and a specific nsCLP subphenotype, since a more than two-fold increase in risk was observed in patients displaying clefts of both the lip and soft palate but who had an intact hard palate (RR: 3.76, CI: 1.47–9.61, PdiffGrem1-deficient mice, wild type embryonic palatal shelves developed divergent shapes when cultured in the presence of ectopic Grem1 protein (P = 0.0014). The present study identified a non-coding region at 15q13 as the second, genome-wide significant locus specific for nsCLP, after 13q31. Moreover, our data suggest that the closely located GREM1 gene contributes to a rare clinical nsCLP entity. This entity specifically involves abnormalities of the lip and soft palate, which develop at different time-points and in separate anatomical regions.非综合征性口面裂（Nonsyndromic orofacial clefts）是一类常见的出生缺陷，病因具有多因素性。其最常见的类型为唇裂，可分为伴腭裂的唇裂（nsCLP）与不伴腭裂的唇裂（nsCLO）两类。尽管遗传因素在nsCLP的发生中发挥重要作用，但介导腭裂易感性的遗传因子在很大程度上仍未明确。 本研究针对三个大型队列的遗传与临床数据开展荟萃分析，发现15号染色体q13区域的一段序列与nsCLP存在显著关联：rs1258763位点的P值为8.13×10⁻¹⁴，相对危险度（RR）为1.46，95%置信区间（CI）为1.32~1.61；而该区域与nsCLO无显著关联（P=0.27，RR=1.09，95%CI：0.94~1.27）。 该最强关联的5kb区域定位于Gremlin-1（GREM1）基因下游，后者编码骨形态发生蛋白4（BMP4）通路的分泌型拮抗剂。我们的实验证实，在小鼠胚胎发生过程中，Grem1基因在发育中的唇与软腭中表达，但在硬腭中无表达。这与rs1258763与特定nsCLP亚表型之间的基因型-表型关联相符：在同时表现为唇裂与软腭裂但硬腭完整的患者中，发病风险升高超过2倍（RR=3.76，95%CI：1.47~9.61）。此外，在Grem1缺陷小鼠模型中，体外培养的野生型胚胎腭突在异位Grem1蛋白的作用下形态出现显著分化（P=0.0014）。 本研究鉴定出15q13区域的一段非编码区，是继13q31区域之后第二个针对nsCLP的全基因组显著性特异性位点。此外，我们的数据提示，紧邻该区域的GREM1基因参与了一种罕见的临床nsCLP表型：该表型仅累及唇与软腭的发育异常，二者在不同的时间点与独立的解剖区域中形成。