Integrative craniofacial genomics - From GWAS to function

非综合征性唇裂伴或不伴腭裂（nsCL/P）是一种常见的人类出生缺陷，由于颅面发育过程中的分子干扰而导致。最近在确定nsCL/P的遗传风险因素方面取得了突破，主要是通过全基因组关联研究。到目前为止，已经确定了16个风险位点，其中大多数定位在已知基因以外的非编码区。虽然这表明，已确定的变体具有调节作用，了解潜在的生物学机制仍然有限，主要是由于很少获得相关的生物材料。这艾美奖-诺特应用的主要目的是推进了解胚胎颅面发育使用nsCL/P作为模型性状。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，（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。功能数据将包括分析不同的组蛋白修饰标记，转录因子结合谱，以及在人类和小鼠胚胎发育过程中的表达水平。为此目的，拟议的合作伙伴目前正在生成宝贵的数据集，并将得到国际财团（例如，FaceBase）。这种方法将导致推定的因果调节元件和候选基因的发现。将使用新的靶向下一代测序方案，在1，500名nsCL/P患者和1，500名对照的大型队列中对鉴定的区域进行重新测序，即，分子倒置探针在Emmy-Noether实验室建立这项技术将允许对大型队列进行时间和成本效益的测序，并可以扩展到体细胞变异和其他表型的分析。随后，将通过桑格测序确认鉴定的变体，并评估共分离或从头状态。在队列水平上，负担分析将提供对功能相关区域内患者中罕见变异的普遍过度表达的见解。对于确认的变体，将使用斑马鱼作为模型生物体在体内进行功能分析。候选调控元件将使用Tol 2介导的转座插入到一阶段胚胎中，并且将在受精后长达五天监测荧光模式。此外，候选基因将使用morpholinos或Crispr/Cas9技术进行敲除。Emmy-Noether小组的结果将为颅面发育的分子途径提供新的见解。更一般地说，它们将有助于理解非编码区的调控结构及其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.