Identification of human orofacial enhancers and their role in orofacial clefts

描述(申请人提供)：口腔裂隙是最常见的人类出生缺陷之一，但其潜在原因仍很大程度上未知。全基因组关联研究表明，这种缺陷是遗传的，然而，绝大多数关联属于基因之外，这表明基因调控缺陷是主要原因。这项拟议的工作旨在通过识别和表征在人类口腔面部早期发育中活跃并在受影响的个体中被干扰的基因调控序列，来确定非综合征性唇腭裂(NSCLP)的遗传原因。直接在人类胚胎组织中使用功能基因组学方法进行的初步研究已经确定了数千个潜在的口腔面部调控序列，其中包括几个与 单核苷酸多态与NSCLP密切相关。这项“独立之路”奖项的申请包括一份由候选人贾斯汀·科特尼博士转变为独立研究人员的有指导的职业发展计划，以及一份附带的研究计划，该计划描述了在基因调控序列中识别和表征与疾病相关的变异的拟议实验。考特尼博士是耶鲁大学医学院的博士后研究员，在遗传学系詹姆斯·努南博士的实验室里工作。他在埃默里大学获得遗传学和分子生物学研究生学位的工作是在埃默里大学生物化学系和耶鲁大学遗传学系的杰拉尔德·沙德尔博士的实验室进行的。在那里，他专注于了解一类新的转录因子的两个成员对人类线粒体基因表达和逆行信号的贡献。在努南实验室，科尼博士运用生化和计算方法，直接在胚胎肢体组织中确定了基因调控序列，这些基因调控序列对人类特有的手脚进化做出了贡献。解决胚胎发育中这些进化问题的技术和计算方法可以用来理解任何发育组织中的基因调控，这使科尼博士处于研究非小细胞肺癌病因的独特位置。文中详述的指导和职业发展计划将通过统计遗传学和人类口腔发育方面的额外培训和指导，补充他在基础分子生物学和功能基因组学方面的背景。科特尼博士的目标是成为学术机构跨学科生物科学、发育生物学或类似系的教员，在那里他可以研究发育基因调控功能障碍在人类常见疾病中的作用。该研究计划将利用科特尼博士在对胚胎组织进行功能分析方面的专业知识，识别在咽弓形成到上颚和面部结构融合过程中活跃的基因调控序列。该项目进一步建议确定这些激活的调控元件中的哪些与NSCLP患者的变异和结构变化有关，并确定发育基因调控中断的后果。识别NSCLP患者中常见的基因调控网络将提供可以预测这些缺陷发生的遗传标记，并为未来的预防或治疗措施提供靶点。减轻口裂的影响并防止其发生将增加全球公共卫生，并减轻受影响的个人及其家庭目前面临的巨大经济负担。

DESCRIPTION (provided by applicant): Orofacial clefts are one of the most commonly occurring human birth defects, yet the underlying causes remain largely unknown. Genome wide association studies indicate heritability for such defects, however the vast majority of associations fall outside of genes suggesting defective gene regulation is a major contributor. The proposed work seeks to identify the genetic causes of nonsyndromic cleft lip and palate (NSCLP) by identifying and characterizing gene regulatory sequences that are active in early human orofacial development and disrupted in affected individuals. Preliminary research using functional genomics methods directly in human embryonic tissue has identified thousands of potential orofacial regulatory sequences, including several that are in linkage disequilibrium with single nucleotide polymorphisms strongly associated with NSCLP. This "Pathway to Independence" award application includes a mentored career development plan for transition of the candidate, Dr. Justin Cotney, into an independent investigator, as well an accompanying research plan describing the proposed experiments on identification and characterization of disease-associated variants in gene regulatory sequences. The candidate, Dr. Cotney, is a postdoctoral fellow at Yale University School of Medicine, in the lab of Dr. James Noonan in the Department of Genetics. The work leading to his graduate degree in Genetics and Molecular Biology at Emory University was conducted in the lab of Dr. Gerald Shadel in the Department of Biochemistry at Emory and Department of Genetics at Yale. There he focused on understanding the contributions of two members of a novel class of transcription factors to human mitochondrial gene expression and retrograde signaling. In the Noonan lab, Dr. Cotney applied biochemical and computational methods to identify gene regulatory sequences directly in embryonic limb tissue that have contributed to human-specific evolution of the hand and foot. The techniques and computational methods to address these evolutionary questions in embryonic development can be adapted to understand gene regulation in any developing tissue, placing Dr. Cotney in a unique position to investigate the causes of NSCLP. The mentoring and career development plan detailed within will supplement his background in basic molecular biology and functional genomics with additional training and instruction in statistical genetics and human orofacial development. Dr. Cotney's goal is to become a faculty member in an interdisciplinary bioscience, developmental biology, or similar department at an academic institution, in which he can research the role of dysfunction of developmental gene regulation in common human diseases. The research plan will leverage Dr. Cotney's expertise in functionally profiling embryonic tissue to identify gene regulatory sequences that are active during the formation of pharyngeal arches to fusion of palates and facial structures. The project further proposes to determine which of these activated regulatory elements are burdened with variants and structural changes in NSCLP patients and identify the consequences of disrupted developmental gene regulation. The identification of gene regulatory networks that are commonly perturbed in NSCLP patients will provide genetic markers that can predict the occurrence of these defects and provide targets for future preventative or therapeutic measures. Alleviating the impact of orofacial clefts and preventing their occurrence will increase global public health and lift a large financial burden currently faced by affected individuals and their families.