Integrative craniofacial genomics - From GWAS to function

非综合征性唇裂伴或不伴腭裂(nsCL/P)是一种常见的人类先天缺陷，由颅面发育过程中的分子紊乱引起。最近，主要通过全基因组关联研究，在识别nsCL/P遗传危险因素方面取得了突破性进展。到目前为止，已经建立了16个风险基因座，其中大多数映射到已知基因之外的非编码区。虽然这表明已鉴定的变异体具有调节作用，但对其潜在的生物学机制的了解仍然有限，主要是因为很少获得相关的生物材料。这一Emmy-Noether应用的主要目的是以nsCL/P作为模式性状来促进对胚胎颅面发育的理解。该项目将(I)通过整合全基因组遗传和功能数据集来确定新的易感区域，以及(Ii)利用遗传学、生物信息学和功能方法来表征已知和新发现的风险基因座。功能数据将包括对人类和小鼠胚胎发育过程中不同的组蛋白修饰标记、转录因子结合图谱和表达水平的分析。为此目的，拟议的合作伙伴目前正在生成有价值的数据集，并将由国际财团(如FaceBase)提供的数据加以补充。这种方法将导致可能的因果调节元件和候选基因的发现。已确定的区域将在1500名nsCL/P患者和1500名对照中使用一种新的靶向下一代测序方案，即分子倒置探针进行重新测序。在艾美诺德实验室建立这项技术将允许对大量队列进行具有时间和成本效益的测序，并可以扩展到对体细胞变异和其他表型的分析。随后，确定的变异将通过桑格测序得到确认，并将评估共分离或从头开始状态。在队列水平上，负担分析将提供对功能相关区域内罕见变异在患者中普遍过度表达的洞察力。对于确认的变种，将以斑马鱼为模式生物进行体内功能分析。候选调控元件将通过Tol2介导的转座被插入到一期胚胎中，并将在受精后五天内监测荧光模式。此外，候选基因将使用吗啉或Crispr/Cas9-技术进行敲除。这个艾美奖-诺乙特小组的结果将为研究颅面发育的分子途径提供新的见解。更广泛地说，它们将有助于理解非编码区的调控架构，以及它们在胚胎发育和疾病中的作用。

Nonsyndromic cleft lip with or without cleft palate (nsCL/P) is a common human birth defect resulting from molecular disturbances during craniofacial development. Recent breakthroughs in the identification of genetic risk factors for nsCL/P have been achieved, mainly via genome-wide association studies. Sixteen risk loci are established to date, and the majority of these map to non-coding regions outside known genes. Although this suggests that the identified variants have a regulatory effect, understanding of the underlying biological mechanisms remains limited, mainly due to little access to relevant biological material.The main aim of this Emmy-Noether application is to advance understanding of embryonic craniofacial development using nsCL/P as a model trait. The project will (i) identify novel susceptibility regions via the integration of genome-wide genetic- and functional datasets, and (ii) characterize known and newly identified risk loci using genetics, bioinformatics and functional approaches. Functional data will include the analysis of different histone modification marks, transcription factor binding profiles, and expression levels during human and murine embryonic development. For this purpose, valuable datasets are currently being generated by the proposed collaboration partners and will be complemented by data from international consortia (e.g., FaceBase). This approach will lead to the discovery of putative causal regulatory elements and candidate genes. The identified regions will be re-sequenced in large cohorts of 1,500 nsCL/P patients and 1,500 controls using a novel targeted next-generation sequencing protocol, i.e., molecular inversion probes. Establishing this technology in the Emmy-Noether lab will allow time- and cost-effective sequencing of large cohorts, and can be extended to the analysis of both somatic variation and other phenotypes. Subsequently, identified variants will be confirmed by Sanger sequencing, and co-segregation or de novo status will be assessed. At a cohort-level, burden analyses will provide insights into the general overrepresentation of rare variants in patients, within functionally relevant regions. For confirmed variants, functional analyses will be conducted in vivo using the zebrafish as a model organism. Candidate regulatory elements will be inserted into one-stage embryos using Tol2-mediated transposition, and fluorescence patterns will be monitored up to five days post-fertilization. In addition, candidate genes will be knocked-down using either morpholinos or Crispr/Cas9- technology.The results of this Emmy-Noether group will provide novel insights into the molecular pathways underlying craniofacial development. More generally, they will facilitate understanding of the regulatory architecture of non-coding regions, and their role in embryonic development and disease.