Analysis of HSCR patient exome sequence data using Zebrafish

使用斑马鱼分析 HSCR 患者外显子组序列数据

• 批准号: 8638444
• 负责人: IAIN T SHEPHERD
• 金额: $ 25.1万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-30 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8638444
• 关键词: 9q31; Accounting; Alleles; Animal Model; Antisense Oligonucleotides; Biological Models; Candidate Disease Gene; Child; Childhood; Clinical; Clinical Data; Code; Collaborations; Collection; Complex; Congenital Megacolon; DNA; DNA Sequence; Data; Defect; Development; Disease; Enhancers; Enteral; Enteric Nervous System; Etiology; Female; Fishes; Gastrointestinal tract structure; Gene Combinations; Genes; Genetic; Genetic Counseling; Genetic Epistasis; Genetic Screening; Genetic Variation; Genomics; Goals; Hereditary Disease; Heritability; Human; Human Genome; Individual; Intestines; Introns; Live Birth; Mediating; Methods; Mutate; Mutation; Nervous system structure; Neurons; Organism; Parents; Patients; Penetrance; Pilot Projects; Population; Predisposition; Probability; Recurrence; Resources; Risk; Risk Factors; Sampling; Scientist; Sex Characteristics; Sex Ratio; Siblings; Syndrome; Testing; Variant; Waardenburg-Shah syndrome; Zebrafish; base; disease phenotype; exome; exome sequencing; gene discovery; genetic pedigree; genome-wide linkage; human disease; knock-down; loss of function; male; man; mutant; nervous system development; novel; positional cloning; public health relevance; research study

PROJECT SUMMARY The goal of this project is to demonstrate that the zebrafish can be used to rapidly determine the functional significance of newly identified gene variants in Hirschsprung disease (HSCR) patients. HSCR is a pediatric condition where the enteric nervous system (ENS) fails to form properly. The disease occurs once in every 5,000 live births and occurs both familially and sparodically. HSCR is a classic multifactorial genetic disorder, and its genetic basis is only partly understood. So far, geneticists have identified 12 genes with mutations in HSCR patients. Of these genes, ret accounts for >80% of all known mutations. However the genetic basis of the disease in the vast majority of HSCR patients is unknown. Furthermore, there is a high degree of variability in the penetrance of the disease phenotype associated with mutations in the known HSCR genes. Recent studies have identified a ret susceptibility allele in an enhancer of intron 1 of the ret gene that appears to be a significant risk factor in developing the disease but it is not sufficient to cause HSCR on its own. We have developed the zebrafish as a model system to study ENS development. We have shown there is a strong evolutionary conservation in the genes required for ENS development from zebrafish to man. Nearly all of the known HSCR genes have been shown to be required for normal zebrafish ENS development. Significantly, we have shown that antisense knockdown of the zebrafish orthologue of ret leads to intestinal aganglionosis in the fish. In this exploratory proposal, we will utilize the zebrafish model system to functionally analyze HSCR patients gene variants as a method to identify new causative HSCR genes. The proposed study is based on a long-standing collaboration between the Shepherd and Hofstra labs and utilizes the unique resources available to the labs (the collection of 400 Dutch HSCR patient DNA samples) and the expertise found in both the Shepherd (Zebrafish ENS development) and Hofstra (Genetic basis of HSCR) labs. Aim1: Identify gene variants in HSCR patients. There is a need for gene discovery in HSCR patients whose disease is currently not assigned to a specific gene or genes. We will preform exome sequencing on a selected group of HSCR patients who have the highest probability of having a strong loss of function coding mutation in one or more genes not previously implicated in the disease. Aim 2: Assess the functional significance of 10 selected HSCR exome identified genes in zebrafish. We will clone and characterize the expression of zebrafish orthologues of the HSCR variant genes. We will then use anti-sense oligonucleotides to assess the effects of these genes on zebrafish ENS development either alone or in combination with ret mutations in order to test for epistasis. The proposed Aims will identify new genes or gene combinations that cause HSCR. This will benefit patients directly when they undergo genetic screening for the condition and will potentially offer scientists new targets for the development of novel therapies to treat HSCR.

项目摘要 该项目的目标是证明斑马鱼可以用于快速确定功能 新发现的基因变异在先天性巨结肠（HSCR）患者中的意义。HSCR是一种儿科 肠神经系统（ENS）不能正常形成的情况。这种疾病每隔一段时间就会发生一次。 5,000例活产，家庭和零星发生。HSCR是一种典型的多因素遗传性疾病， 它的遗传基础也只是部分被了解。到目前为止，遗传学家已经确定了12个基因突变， HSCR患者。在这些基因中，ret占所有已知突变的80%以上。然而，遗传基础 绝大多数HSCR患者的疾病是未知的。此外， 在与已知HSCR基因突变相关的疾病表型中，最近 研究已经在ret基因的内含子1的增强子中鉴定了ret易感性等位基因， 这是发展疾病的重要风险因素，但本身不足以引起HSCR。 我们已经开发了斑马鱼作为模型系统来研究ENS的发展。我们在那里展示了 是一个强大的进化保守的基因所需的ENS发展从斑马鱼到人。 几乎所有已知的HSCR基因都被证明是正常斑马鱼ENS所必需的 发展值得注意的是，我们已经表明，反义敲低斑马鱼的ret直向同源物， 肠道无神经节细胞症在这个探索性的建议中，我们将利用斑马鱼模型系统， 功能分析HSCR患者基因变异体作为鉴定新的致病HSCR基因的方法。的 拟议的研究是基于Shepherd和Hofstra实验室之间的长期合作，并利用 实验室可利用的独特资源（收集了400份荷兰HSCR患者DNA样本）和 Shepherd（斑马鱼ENS开发）和Hofstra（HSCR的遗传基础）实验室的专业知识。 目的1：确定HSCR患者的基因变异。需要在HSCR患者中发现基因， 疾病目前没有被分配到一个特定的基因或基因。我们将对选定的 HSCR患者组中具有最高可能性的HSCR患者具有较强的功能丧失编码突变， 一个或多个先前与疾病无关的基因。 目的2：对10个斑马鱼HSCR外显子组基因的功能进行分析。 我们将克隆和表征斑马鱼同源的HSCR变异基因的表达。我们将 然后使用反义寡核苷酸来评估这些基因对斑马鱼ENS发育的影响 单独或与RET突变组合以测试上位性。 拟议的目标将确定新的基因或基因组合，导致HSCR。这将有利于 患者直接当他们接受基因筛查的条件，并可能提供科学家新的 为开发治疗HSCR的新疗法提供了靶点。