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）是最普遍的先天缺陷之一，影响了1％的活产。虽然遗传病因是通过发现各种综合征形式的冠心病来表明的，但冠心病的遗传原因仍未得到充分了解。随着人口的遗传变异性混淆了人类的遗传分析，我们最近追求了近交C57BL6小鼠中大规模的乙基硝基库（ENU）诱变筛查，以恢复导致CHD的隐性突变。使用非侵入性胎儿超声心动图进行表型，这是一种临床用于CHD诊断的成像方式。从超声扫描100,000胎胎儿中，我们以较广泛的冠心病恢复了> 250冠冠心突变的小鼠系。我们使用整个外显子组测序分析回收了150个CHD，从而在94个基因中引起突变。出乎意料的是，50％的冠心基因与纤毛相关，表明纤毛在CHD发病机理中起着核心作用。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的蛋白质，这表明在CHD相互作用网络的背景下，可能通过epitipasis发生CHD发病机理的假设。这可能有助于与人冠心有关的复杂遗传学。为了检验这一假设，我们计划进行新的敏感筛选，以首先对小鼠基因组进行半主导冠心病突变的系统询问。屏幕将使用CEP290中的驱动突变进行，这是在我们的隐性筛查中回收的纤毛CHD基因，在临床上也很好地描述了在各种人纤毛病和突变小鼠模型中执行遗传修饰效应的效果。我们的屏幕将需要超声扫描G2胎儿的CHD源自CEP290突变伴侣的CHD，以造成ENU诱导的突变的G1雄性杂合子。对于CEP290和ENU诱导的突变，所得的G2后代只能是双重杂合的（除非ENU突变也在CEP290中）。任何观察到的CHD都将反映上毒的作用。使用这种策略，我们期望从1800 G1的血统中筛选约54,000个胎儿，提供一种系统方法来审问纤毛在隐性筛查中不可能的CHD复杂遗传学中的作用。突变恢复将使用整个小鼠外显子组测序进行，然后进行基因分析分析，并在多种血统内进行进一步的连锁分析。为了验证筛网中确定的因果突变，将使用CRISPR/CAS9基因编辑产生转基因小鼠模型，然后进行CEP290突变的间交叉，以检查关生症的证据。总体而言，这种新型敏感的屏幕将为小鼠基因组提供第一次系统询问，以使其半主导突变引起CHD。通过在CEP290中使用突变作为此敏感筛选的驱动力，我们可以进一步阐明纤毛在CHD发病机理中的作用。在此敏感筛查中恢复的突变和动物模型将更好地模拟人类冠心病的复杂遗传学，并更好地为未来的临床研究提供了询问人群中CHD遗传学病因的遗传学病因。