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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，影响多达1%的活产婴儿。虽然CHD的各种综合征形式的发现表明遗传病因学，但CHD的遗传原因仍然没有得到很好的理解。由于人类群体的遗传变异性混淆了人类遗传分析，我们最近在近交系C57 BL 6小鼠中进行了大规模的乙基亚硝基脲（ENU）诱变筛选，以恢复导致CHD的隐性突变。采用无创性胎儿超声心动图进行表型分型，这是一种临床上用于CHD诊断的成像方式。通过超声扫描100，000个胎儿，我们回收了>250个CHD突变小鼠系，这些突变小鼠具有广泛的CHD谱。我们使用全外显子组测序分析在94个基因中恢复了150个CHD引起的突变。令人惊讶的是，50%的CHD基因与纤毛相关，表明纤毛在CHD发病机制中起着核心作用。同样出乎意料的是许多纤毛相关CHD基因的恢复，这些基因编码已知物理相互作用的蛋白质，这表明CHD发病机制可能在CHD相互作用组网络的背景下通过上位发生的假设。这可能有助于与人类CHD相关的复杂遗传学。为了验证这一假设，我们计划进行一种新型的致敏筛选，首次系统地询问小鼠基因组中的半显性CHD突变。筛选将使用Cep 290中的驱动突变进行，Cep 290是在我们的隐性筛选中回收的纤毛-CHD基因，其在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剂作用。我们的筛查将需要对来自Cep 290突变杂合子女性与ENU诱导突变杂合子G1男性交配的G2胎儿进行CHD超声扫描。所得的G2后代只能是Cep 290和ENU诱导突变的双杂合（除非ENU突变也在Cep 290中）。因此，观察到的任何CHD都反映了上位性效应。使用这种策略，我们预计将从1800个G1家系中筛选约54，000个胎儿，提供一种系统的方法来询问纤毛在CHD复杂遗传学中的作用，这是隐性筛选不可能实现的。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，并在多个谱系内和之间进行进一步的连锁分析。为了验证筛选中鉴定的因果突变，将使用CRISPR/Cas9基因编辑生成转基因小鼠模型，然后与Cep 290突变杂交以检查上位性的证据。总的来说，这种新的敏化屏幕将提供第一个系统的审讯小鼠基因组的半显性突变引起冠心病。通过使用Cep 290中的突变作为这种敏化筛选的驱动器，我们可以进一步阐明纤毛在CHD发病机制中的作用。在这种致敏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，并更好地为未来的临床研究提供信息，以询问人群中CHD的遗传学病因。