Exome screening and directed breeding of rhesus for human genetic disease models

人类遗传病模型的恒河猴外显子筛选和定向育种

• 批准号: 9111085
• 负责人: R. PAUL JOHNSON
• 金额: $ 24.79万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9111085
• 关键词: ABCB1 gene; Affect; Animal Model; Animals; Breeding; CYP21A2 gene; Cataloging; Catalogs; Data; Development; Dideoxy Chain Termination DNA Sequencing; Disease; Disease model; Embryo; Exhibits; Female; Funding; Gene Mutation; Genes; Genetic; Genetic Polymorphism; Genetic Screening; Genome; Genotype; Goals; Health; Hereditary Disease; Heterozygote; Human; Human Genetics; Institutes; Institution; Macaca mulatta; Maryland; Mendelian disorder; Methods; Modeling; Monkeys; Mus; Mutation; Orthologous Gene; Phenotype; Physiology; Population; Pre-Clinical Model; Primates; Proteins; Research; System; Universities; Validation; animal breeding; base; cost; disease phenotype; drug development; exome; exome sequencing; expectation; human disease; loss of function; loss of function mutation; male; mutant; nonhuman primate; offspring; pre-clinical research; screening; therapy development; translational study; treatment strategy

 DESCRIPTION (provided by applicant): There are over 6000 disorders in humans that result from a single gene mutation (Rappaport et al. 2013). For many of these conditions, there is no suitable animal model. Because rhesus macaques are genetically and physiologically similar to humans, they have the potential to offer preclinical models for numerous human conditions. We have preliminary data demonstrating that we can identify loss-of-function mutations in rhesus macaques related to human disease from exome sequencing, validate these results with PCR and Sanger sequencing and establish loss-of-function at the protein level. The overall goal of this proposal is to produce rhesus macaque models of single gene human genetic disorders. To accomplish this, we will identify naturally occurring loss-of-function mutations in rhesus macaques at the Yerkes National Primate Research Center (YNPRC). We already have exome sequence data from 61 animals. We will identify loss- of-function mutations in genes using the GATK pipeline and then validate these mutations with PCR/Sanger sequencing. Because these mutations are likely to be present in the heterozygous state, we propose to genotype 500 breeding rhesus macaques for a subset of the newly identified loci. The loci we choose to genotype will be based on whether the polymorphism leads to a predicted loss of function within genes implicated in human disease. After genotyping and identification of a sufficient number of animals that have the same mutation, we will breed multiple male and female heterozygotes with the expectation that 25% of the resulting offspring will be homozygous for the mutation. We expect that these animals will exhibit a phenotype similar to humans with loss-of-function mutations in the orthologous gene and serve as models for preclinical translational studies to begin to identify possible treatment strategies. When completed, this proposal will produce three distinct deliverables. 1) A catalog of high impact polymorphism present within a subset of the Yerkes rhesus population. 2) "Proof of concept" in an affected offspring that will serve as a model in the study of human single gene disorders. 3) Development of a pipeline to produce additional rhesus models of human genetic disease at Yerkes and other institutions.

 描述（由申请人提供）：人类中有超过6000种疾病是由单基因突变引起的（Rappaport et al. 2013）。对于许多这些条件，没有合适的动物模型。由于恒河猴在遗传和生理上与人类相似，因此它们有可能为许多人类疾病提供临床前模型。我们有初步的数据表明，我们可以从外显子组测序中鉴定与人类疾病相关的恒河猴功能缺失突变，用PCR和桑格测序验证这些结果，并在蛋白质水平上建立功能缺失。这项建议的总体目标是产生单基因人类遗传疾病的恒河猴模型。为了实现这一目标，我们将在耶基斯国家灵长类动物研究中心（YNPRC）鉴定恒河猴中自然发生的功能丧失突变。我们已经有了61只动物的外显子组序列数据。我们将使用GATK管道识别基因中的功能丧失突变，然后通过PCR/桑格测序验证这些突变。因为这些突变很可能存在于杂合状态，我们建议基因型500育种恒河猴的一个子集的新确定的基因座。我们选择基因分型的位点将基于多态性是否导致与人类疾病有关的基因内的预测功能丧失。在对足够数量的具有相同突变的动物进行基因分型和鉴定后，我们将繁殖多个雄性和雌性杂合子，预计产生的后代中有25%将是突变纯合的。我们预计这些动物将表现出与人类相似的表型，在orthoglycoprotein基因中具有功能缺失突变，并作为临床前转化研究的模型，以开始确定可能的治疗策略。该提案完成后，将产生三个不同的可交付成果。1)一个目录的高影响多态性存在于一个子集的耶基斯恒河猴人口。2)在受影响的后代中进行“概念验证”，该后代将作为人类单基因疾病研究的模型。3)在Yerkes和其他机构开发生产人类遗传疾病的其他恒河猴模型的管道。

项目成果

Naturally Occurring Genetic Variants of Human Acetylcholinesterase and Butyrylcholinesterase and Their Potential Impact on the Risk of Toxicity from Cholinesterase Inhibitors.

• DOI: 10.1021/acs.chemrestox.6b00228
• 发表时间: 2016-09-19
• 期刊: CHEMICAL RESEARCH IN TOXICOLOGY
• 影响因子: 4.1
• 作者: Lockridge, Oksana;Norgren, Robert B., Jr.;Johnson, Rudolph C.;Blake, Thomas A.
• 通讯作者: Blake, Thomas A.