A common cis-regulatory variant impacts normal-range and disease-associated human facial shape through regulation of PDKCC during chondrogenesis [HiChIP]

Genome-wide association studies (GWAS) identified thousands of genetic variants linked to phenotypic traits and disease risk. However, mechanistic understanding of how GWAS variants influence complex morphological traits and can, in certain cases, simultaneously confer normal-range phenotypic variation and disease predisposition, is still largely lacking. Here, we focus on rs6740960, a single nucleotide polymorphism (SNP) at the 2p21 locus, which in GWAS studies has been associated both with normal-range variation in jaw shape and with an increased risk of non-syndromic orofacial clefting. Using in vitro derived embryonic cell types relevant for human facial morphogenesis, we show that this SNP resides in an enhancer that regulates chondrocytic expression of PKDCC - a gene encoding a tyrosine kinase involved in chondrogenesis and skeletal development. In agreement, rs6740960 SNP is sufficient to confer a large difference in acetylation of its cognate enhancer preferentially in chondrocytes. By deploying dense landmark morphometric analysis of skull elements in mice, we show that changes in Pkdcc dosage are associated with quantitative changes in maxilla, mandible, and palatine bone shape that are concordant with the facial phenotypes and disease predisposition seen in humans. We further demonstrate that the frequency of the rs6740960 variant strongly deviated among different human populations, and that the activity of its cognate enhancer diverged in hominids. Our study provides a mechanistic explanation of how a common SNP can mediate normal-range and disease-associated morphological variation, with implications for the evolution of human facial features. Overall design: We performed H3K27ac HiChIP experiments in order to identify enhancer-promoter 3D interactions in in vitro derived cranial neural crest cells (CNCCS) and cranial chondrocytes.

全基因组关联研究（Genome-wide Association Studies, GWAS）已鉴定出数千个与表型性状及疾病风险相关的遗传变异。然而，目前学界仍普遍缺乏对GWAS变异如何调控复杂形态性状，且在部分情境下可同时介导正常范围表型变异与疾病易感性的机制性认知。本研究聚焦于2p21位点的单核苷酸多态性（Single Nucleotide Polymorphism, SNP）rs6740960：该位点在GWAS分析中既与颌骨形态的正常范围变异相关，也与非综合征性口面裂的发病风险升高存在关联。本研究采用体外诱导获得的、与人类面部形态发生相关的胚胎细胞类型，证实该SNP位于一个调控PKDCC基因软骨细胞表达的增强子（enhancer）区域内；PKDCC编码一种参与软骨发生（chondrogenesis）与骨骼发育的酪氨酸激酶（tyrosine kinase）。验证结果显示，rs6740960 SNP足以显著改变其同源增强子的乙酰化（acetylation）水平，且该效应优先出现于软骨细胞中。本研究通过对小鼠颅骨骨骼开展致密地标形态计量分析，证实Pkdcc基因剂量的改变与上颌骨（maxilla）、下颌骨（mandible）及腭骨（palatine bone）形态的定量变化存在关联，且该变化与人类中观察到的面部表型及疾病易感性相契合。本研究还证实，rs6740960变异的等位基因频率在不同人类人群中存在显著差异，且其同源增强子的活性在人科（hominids）动物演化过程中发生了分化。本研究为常见SNP如何介导正常范围及疾病相关的形态变异提供了机制性解释，同时也为人类面部特征的演化研究提供了新的理论参考。整体实验设计：本研究开展了H3K27ac HiChIP实验，以鉴定体外诱导获得的颅神经嵴细胞（Cranial Neural Crest Cells, CNCCs）及颅骨软骨细胞中的增强子-启动子三维相互作用。