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.

Public Health Relevance

The genetic etiology of congenital heart disease is still poorly understood. As mice have the same cardiovascular anatomy and almost identical genome as human, they make ideal animal models for interrogating the genetic basis for congenital heart disease. Using cutting edge forward and reverse genetic approaches with genome editing and a sensitized mutagenesis screen, we will interrogate the complex genetics of CHD pathogenesis in mouse models. These studies will advance our understanding of the complex genetics of human CHD.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Genetics of Health and Disease Study Section (GHD)
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Schramm, Charlene A
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University of Pittsburgh
Anatomy/Cell Biology
Schools of Medicine
United States
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