Rapid Identification and Validation of Human Craniofacial Development Genes

描述(申请人提供)：新的基因组测序技术的出现使人类发育障碍的基因发现任务变得高效。同时，在模式生物中，特别是在斑马鱼中进行基因打靶的进展，使得对人类候选基因的半高通量验证和分析成为可能，包括那些导致头面部疾病的基因。这是一个新的分支项目的申请 FaceBase 2将利用这种新技术的融合来识别和功能验证大约24个涉及人类头面部发育新方面的基因。具体地说，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)已经确定的颅面畸形的收集，在这些地方，血缘关系的高发生率使得同合子图谱和隐性原因基因座的识别高度可行。我们将扩大FaceBase的工作范围，使其超越目前对腭部发育障碍的关注，包括涉及颅面复合体其他组成部分的相对广泛的头面部疾病。此外，利用FaceBase已经汇编的资源，包括详细的小鼠和斑马鱼基因表达数据、增强子分析和全基因组关联研究，结合目前的数据和公开可用的数据集，将进一步促进对这些新验证的基因的功能注释。为了向其他FaceBase调查人员和整个社区提供有价值的交付资源，我们将追求三个具体目标。在目标1中，我们将确定和招募具有可能单基因病因的广泛的颅面畸形患者。这些患者不仅将在BCH和KFSHRC转诊中心被识别，而且还将从其他临床研究人员，甚至可能是FaceBase生物信息库获得。在目标2中，将根据识别Caual变体的遗传可能性对患者进行进一步研究的优先顺序。然后，我们将对先证者和潜在的其他家庭成员进行完整的外显子组和在某些情况下的全基因组序列(WES/WGS)分析，在适用的情况下使用aCGH来确保基因组完整性和自合性作图。现有的最先进的计算管道将被用来获得一组有限的潜在因果DNA序列变体和候选基因。最后，在目标3中，在已知功能和表达数据不能很容易确定原因的情况下，我们将寻找额外的独立确证病例，并同时采用快速分析策略，包括高通量基因表达分析、吗啉基因敲除以及突变和转基因，以准备GOF和LOF等位基因。结果将被转发到FaceBase 2协调中心，向社区提供的关键成果是一份经过验证的人类颅面发育调控基因的基因清单，以及一组可以广泛分享的相应斑马鱼突变体，用于进一步的详细研究。

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.