Rapid Identification and Validation of Human Craniofacial Development Genes

描述（由申请人提供）：新的基因组测序技术的出现使得人类发育障碍中基因发现的任务变得非常高效。同时，模型生物（特别是斑马鱼）基因靶向方面的进展，使得对人类候选基因（包括导致颅面疾病的基因）进行半高通量验证和分析变得可行。此应用程序适用于新的辐条项目 FaceBase 2 将利用新技术的融合来识别和功能验证涉及人类颅面发育新方面的大约两打基因。具体来说，我们将利用波士顿儿童医院 (BCH) 和沙特阿拉伯费萨尔国王专科医院和研究中心 (KFSHRC) 已确定的颅面畸形集合，其中近亲结婚的高发生率使得自合性作图和隐性因果位点的识别高度可行。我们将把 FaceBase 的工作扩展到目前对腭发育疾病的关注之外，将涉及颅面复合体其他组成部分的相对广泛的颅面疾病纳入其中。此外，使用FaceBase已经编译的资源，包括小鼠和斑马鱼的详细基因表达数据、增强子分析和全基因组关联研究，结合现有数据和公开数据集，将进一步促进这些新验证基因的功能注释。为了向其他 FaceBase 研究人员和整个社区提供有价值的可交付资源，我们将追求三个具体目标。在目标 1 中，我们将确定并招募患有多种可能由单基因病因引起的颅面畸形的患者。这些患者不仅会在 BCH 和 KFSHRC 转诊中心进行识别，还会从其他临床研究人员甚至可能是 FaceBase Biorepository 处招募。在目标 2 中，将根据识别因果变异的遗传可能性，优先考虑对患者进行进一步研究。然后，我们将对先证者和可能的其他家庭成员进行全外显子组，在某些情况下进行全基因组序列 (WES/WGS) 分析，使用 aCGH 确保基因组完整性和自合性映射（如果适用）。现有的最先进的计算管道将用于导出一组有限的潜在因果 DNA 序列变异和候选基因。最后，在目标3中，在无法从已知功能和表达数据轻易确定因果关系的情况下，我们将寻求额外的独立验证案例，同时采用由高通量基因表达分析、吗啉代敲低、诱变和转基因组成的快速分析策略来制备GOF和LOF等位基因。结果将转发给 FaceBase 2 协调中心，向社区提供的关键成果是经过验证的人类颅面发育调节基因的基因列表和一组相应的斑马鱼突变体，可以广泛共享以进行进一步的详细研究。

DESCRIPTION (provided by applicant): The advent of new genomic sequencing technologies has made the task of gene discovery in human developmental disorders highly efficient. Simultaneously, advances in gene targeting in model organisms, specifically in zebrafish, have made semi-high throughput validation and analysis of human candidate genes feasible, including those responsible for craniofacial disorders. This application for a new spoke project in FaceBase 2 will take advantage of this convergence of new technologies to identify and functionally validate approximately two dozen genes involved in novel aspects of human craniofacial development. Specifically, we will take advantage of already ascertained collections of craniofacial dysmorphoses from Boston Children's Hospital (BCH) and from King Faisal Specialist Hospital and Research Center (KFSHRC) in Saudi Arabia, where the high incidence of consanguinity makes autozygosity mapping and the identification of recessive causal loci highly feasible. We will extend the work of FaceBase beyond its current focus on disorders of palatal development by including a relatively wide range of craniofacial disorders that involve other components of the craniofacial complex. In addition, use of resources already compiled by FaceBase, including detailed gene expression data in mouse and zebrafish, enhancer analyses, and genome wide association studies, in combination with the present data and publicly available datasets, will further facilitate the functional annotation of these newly validated gene. To provide valuable deliverable resources to other FaceBase investigators and to the community at large, we will pursue three Specific Aims. In Aim 1, we will ascertain and recruit patients with a wide range of craniofacial dysmorphoses of likely monogenic etiology. These patients will not only be identified at the BCH and KFSHRC referral centers, but also solicited from other clinical investigators and potentially even the FaceBase Biorepository. In Aim 2, patients will be prioritized for further study based on the genetic likelihood of identifying a caual variant. We will then perform whole exome and in some cases whole genome sequence (WES/WGS) analysis, on the proband and potentially other family members, using aCGH to ensure genomic integrity and autozygosity mapping where applicable. An existing state-of-the-art computational pipeline will be used to derive a limited set of potentially causal DNA sequence variants and candidate genes. Lastly, in Aim 3, in cases where causation cannot be readily established from known function and expression data, we will seek additional independent confirmatory cases and, in parallel, employ a rapid analysis strategy consisting of high-throughput gene expression analysis, morpholino knockdown, and mutagenesis and transgenesis to prepare GOF and LOF alleles. The results will be forwarded to the FaceBase 2 Coordinating Center, with the key deliverables to the community being a validated gene list of human craniofacial developmental regulatory genes and a set of corresponding zebrafish mutants that can be widely shared for further detailed study.