Congenital heart disease (CHD) affects up to 1% of live births, but its genetic basis is still not well understood. Human studies to unravel the genetic causes of CHD is challenging given genetic diversity of the human population. Genetic analysis in mice is advantageous given the mouse genome is completely sequenced, and inbred mice provide animals that are genetically identical. Knockout mouse studies have identified many genes that can cause CHD. However, ftjnctional redundancies may mask gene function or early embryonic lethality may preclude assessment of CHD. We propose a complementary approach with forward genetic screening with ethylnitrosourea mutagenesis to recover mutations causing CHD. We previously showed noninvasive mouse fetal echocardiography is highly effective for high throughput Cardiovascular phenotyping. Our screen recovered many genes encoding proteins in the cilia or centrosome, suggesting the cilium is a central disease pathway in CHD. To recover mutations causing CHD, we plan to use noninvasive fetal echocardiography to screen 100,000 mouse fetuses from 4000 pedigrees to achieve an estimated five-fold genome coverage (Aim 1). We will use a two-tier approach with high throughput targeted and whole genome DNA sequencing to identify the mutations (Aim 2). For genes suspected to have a role in the cilium, zebrafish will be used for rapid morpholino knock-down to analyze the motiie/nonmotile functions of the cilia and possible disruption of left-right patteming related to cilia defects (Aim 3). Mouse embryonic fibroblasts and tissues derived from mutant embryos will be used to evaluate cell-intrinsic function related to the cilium and centrosome (Aim 4). To elucidate the role of the cilia in cardiac morphogenesis, mutant embryos will be examined for heart looping, deployment of extracardiac cell populations to the heart, outflow tract and chamber septation. Cilia mediated sonic hedgehog and non-canonical Wnt signaling also ^ili be examined (Aim 5). In summary, the proposed studies will help elucidate the genetic basis for CHD. Many new CHD mouse models will be generated and the role of the cilium and other pathways playing important roles in CHD will emerge with the identification of a core set of genes critically involved in CHD.

Public Health Relevance

;The identification of a core set of genes involved in congenital heart disease can provide the basis for future translational studies with human subjects to elucidate the complex genetics of human congenital heart disease. This could include the design of diagnostic chips for genotyping patients with congenital heart disease and examining for correlation between genotype with disease phenotype and long term outcome.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project--Cooperative Agreements (U01)
Project #
5U01HL098180-05
Application #
8518108
Study Section
Special Emphasis Panel (ZHL1-CSR-B (S1))
Program Officer
Schramm, Charlene A
Project Start
2009-09-30
Project End
2015-07-31
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
5
Fiscal Year
2013
Total Cost
$1,605,322
Indirect Cost
$418,116
Name
University of Pittsburgh
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
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San Agustin, Jovenal T; Klena, Nikolai; Granath, Kristi et al. (2016) Genetic link between renal birth defects and congenital heart disease. Nat Commun 7:11103
Li, You; Klena, Nikolai T; Gabriel, George C et al. (2015) Global genetic analysis in mice unveils central role for cilia in congenital heart disease. Nature 521:520-4
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Hjeij, Rim; Onoufriadis, Alexandros; Watson, Christopher M et al. (2014) CCDC151 mutations cause primary ciliary dyskinesia by disruption of the outer dynein arm docking complex formation. Am J Hum Genet 95:257-74

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