Modeling the complex genetics of congenital heart disease in mice

模拟小鼠先天性心脏病的复杂遗传学

基本信息

批准号：
9260066
负责人：
CECILIA W. LO
金额：
$ 76.85万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-04-15 至 2020-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9260066
关键词：
Affect Anatomy Animal Model Autopsy Biological Assay CHD4 gene CHD7 gene CRISPR/Cas technology Cardiac Cardiovascular system Chemicals Cilia Clinical Clinical Research Clustered Regularly Interspaced Short Palindromic Repeats Complex Congenital Abnormality DNA Resequencing Defect DiGeorge Syndrome Echocardiography Ensure Ethylnitrosourea Exhibits Female Fetus Future Gene Targeting Genes Genetic Genetic Epistasis Genetic Predisposition to Disease Genetic Screening Genetic Variation Genome Genotype Heart Abnormalities Histopathology Human Human Genetics Hypoplastic Left Heart Syndrome Image Analysis Inbred Mouse Inbreeding Individual Induced Mutation Knock-in Live Birth MLL2 gene Mendelian disorder Modeling Mus Mutagenesis Mutant Strains Mice Mutation Mutation Analysis Nature Partner in relationship Pathogenesis Pathogenicity Patients Pediatric Cardiac Genomics Consortium Penetrance Phenotype Point Mutation Population Proteins Recovery Recurrence Risk Role Structural Congenital Anomalies Syndrome System Testing Transgenic Mice Ultrasonography Validation actionable mutation cardiovascular imaging ciliopathy congenital heart disorder design disease diagnosis disease phenotype disease-causing mutation exome sequencing experience fetal follow-up genetic analysis genetic approach genetic linkage analysis genetic pedigree genome editing imaging modality indexing male mouse genome mouse model mutant mutant mouse model novel offspring pediatric patients postnatal public health relevance reverse genetics

项目摘要

DESCRIPTION (provided by applicant) Congenital heart disease (CHD) is one of the most prevalent birth defects, affecting up to 1% of live births. While a genetic etiology is indicated by the finding of various syndromic forms of CHD, the genetic causes of CHD is still not well understood. As the genetic variability of the human population confounds human genetic analysis, we recently pursued a large scale ethylnitrosourea (ENU) mutagenesis screen in inbred C57BL6 mice to recover recessive mutations causing CHD. Phenotyping was conducted using noninvasive fetal echocardiography, an imaging modality used clinically for CHD diagnosis. From ultrasound scanning 100,000 fetuses, we recovered >250 CHD mutant mouse lines with a wide spectrum of CHD. We recovered 150 CHD causing mutations in 94 genes using whole exome sequencing analysis. Surprisingly, 50% of the CHD genes are cilia related, indicating a central role for cilia in CHD pathogenesis. Also unexpected was the recovery of many cilia related CHD genes encoding proteins known to physically interact, suggesting the hypothesis that CHD pathogenesis may occur via epistasis in the context of a CHD interactome network. This could contribute to the complex genetics associated with human CHD. To test this hypothesis, we plan to conduct a novel sensitized screen for the first systematic interrogation of the mouse genome for semi-dominant CHD mutations. The screen will be conducted using a driver mutation in Cep290, a cilia-CHD gene recovered in our recessive screen that is also clinically well described to exert genetic modifier effects, both in various human ciliopathies and in mutant mouse models. Our screen will entail ultrasound scanning G2 fetuses for CHD derived from females heterozygous for the Cep290 mutation mated to a G1 male heterozygous for ENU induced mutations. The resulting G2 offspring can only be double heterozygous for the Cep290 and ENU induced mutations (except when the ENU mutation is also in Cep290). Thus any CHD observed would reflect the effects of epistasis. Using this strategy, we expect to screen ~54,000 fetuses from 1800 G1 pedigrees, providing a systems approach to interrogate the role of cilia in the complex genetics of CHD not possible with a recessive screen. Mutation recovery will be carried out using whole mouse exome sequencing followed by genotyping analysis, and further linkage analysis conducted within and across multiple pedigrees. For validation of causal mutations identified in the screen, transgenic mouse models will be generated using CRISPR/Cas9 gene editing, followed by intercrosses with the Cep290 mutation to examine for evidence of epistasis. Overall, this novel sensitized screen will provide the first systematic interrogation of the mouse genome for semi-dominant mutations causing CHD. By using mutation in Cep290 as driver for this sensitized screen, we can further elucidate role of cilia in CHD pathogenesis. The mutations and animal models recovered in this sensitized screen will better model the complex genetics of human CHD and better inform future clinical studies interrogating the genetics etiology of CHD in the human population.

描述（由申请人提供）先天性心脏病 (CHD) 是最常见的出生缺陷之一，影响高达 1% 的活产儿，虽然各种 CHD 综合征形式的发现表明了其遗传病因，但 CHD 的遗传原因仍不清楚。由于人类群体的遗传变异性混淆了人类遗传分析，我们最近在近交 C57BL6 小鼠中进行了大规模乙基亚硝基脲 (ENU) 诱变筛选，以恢复导致使用无创胎儿超声心动图进行表型分析，这是一种临床上用于 CHD 诊断的成像方式，通过对 100,000 个胎儿进行超声扫描，我们发现了超过 250 个具有广泛 CHD 的 CHD 突变小鼠品系。令人惊讶的是，50% 的 CHD 基因与纤毛相关，表明纤毛在 CHD 发病机制中发挥着核心作用。同样出乎意料的是，许多纤毛相关的 CHD 基因编码已知的物理相互作用蛋白，这表明 CHD 发病机制可能通过 CHD 相互作用组网络中的上位性发生，这可能有助于与人类 CHD 相关的复杂遗传学。为了检验这一假设，我们计划进行一种新颖的敏化筛选，首次系统地询问小鼠基因组中的半显性 CHD 突变。该筛选将使用 Cep290（一种纤毛-CHD 基因）的驱动突变进行。在我们的隐性筛选中恢复，临床上也被充分描述为在各种人类纤毛病和突变小鼠模型中发挥遗传修饰作用。我们的筛选将需要对来自与 G1 交配的 Cep290 突变杂合的雌性的 CHD 的 G2 胎儿进行超声扫描。 ENU 诱导突变的雄性杂合子所产生的 G2 后代只能是 Cep290 和 ENU 诱导突变的双杂合子（除非 ENU 突变也存在）。因此，观察到的任何 CHD 都将反映上位性的影响，我们期望从 1800 个 G1 谱系中筛选出约 54,000 个胎儿，从而提供一种系统方法来探究纤毛在 CHD 复杂遗传学中的作用，这是不可能的。突变恢复将使用全小鼠外显子组测序进行，然后进行基因分型分析，并在多个内部和多个之间进行进一步的连锁分析。为了验证筛选中发现的因果突变，将使用 CRISPR/Cas9 基因编辑生成转基因小鼠模型，然后与 Cep290 突变交叉以检查上位性证据。总体而言，这种新颖的致敏筛选将提供第一个系统性的。通过使用 Cep290 突变作为致敏筛选的驱动因素，我们可以进一步阐明纤毛在导致 CHD 的半显性突变中的作用。 CHD 发病机制。在这种敏化筛选中恢复的突变和动物模型将更好地模拟人类 CHD 的复杂遗传学，并更好地为未来探究人类 CHD 遗传学病因学的临床研究提供信息。