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Congenital heart disease (CHD) is the most common birth defect in live births with an estimated incidence of 1%. Although advancements in surgical care have significantly improved patient outcomes, CHD is still a major contributor to morbidity and mortality. The link between genetics and CHD has been well established following population-based studies and single-gene knockout approaches in animal models. Although a subset of CHD can be attributed to a few key genetic contributors, the genetic underpinnings of most CHD cases remain unresolved. Therefore, uncovering novel genetic contributors to CHD is of critical importance.Large scale sequencing approaches of patients with CHD have identified numerous potentially damaging genetic variants. However, gene prioritization and experimental validation for the majority of these candidate genes are lacking. Model organism studies, specifically those performed in mice, have been instrumental in uncovering the developmental impact that genetic contributors have on the developing heart. The growing availability of genomic technologies allows us the opportunity to exploit mouse models of CHD as a tools to facilitate gene discovery. We propose that transcriptomic profiles derived from the normal developing heart and animal models of CHD can be used to prioritize candidate genes that contribute to CHD phenotypes in patients and also identify novel genes and molecular pathways critical for heart development.To better understand the genetic contributors of tetralogy of Fallot (TOF), we designed a gene prioritization pipeline that made use of transcriptomic data derived from a highly penetrant mouse model of outflow tract (OFT) malformations and genetic variant information derived from patients with TOF. The common OFT is the anatomical precursor of the aorta, pulmonary artery, and semilunar valves. Disruption of the normal development of the OFT contributes to OFT malformations such as TOF. We performed bulk RNA-Sequencing of a highly penetrant mouse model of cardiac OFT malformations and identified 1,352 differentially expressed genes (DEG) between mutants and controls. We then categorized these DEGs according to cell identity clusters obtained from scRNA-Sequencing transcriptomes and determined, which genes possessed a strong human homolog. By making use of publicly available whole-exome sequencing data derived from patients with TOF, we tested for overlap of the differentially expressed genes with candidate genetic variants by hypergeometric testing. We were able to conclude that there was an enrichment of genes found to be differentially expressed in a CHD-disease mouse model and genes with predicted damaging variation in patient samples. This work supports the use of mouse models to facilitate gene prioritization and offers a working pipeline for gene discovery in multiple developmental diseases.Gene prioritization is the first step towards the better characterization the genetic contributors to CHD, such as TOF. However, experimental validation of bioinformatic findings is required to determine the functional significance of those prioritized genes within developmental processes. We utilized this approach to characterize a novel gene within the developing heart. scRNA-Seq transcriptomes of the embryonic mouse heart have been previously generated and are available for public use. By probing these transcriptomes for the gene expression pattern of axon guidance family members, which are a known yet understudied family of genes in heart development, we were able to uncover a novel signaling ligand in the developing heart called Netrin-1. This bioinformatic discovery was validated by making use of reporter lines and immunofluorescence staining which showed Netrin-1 was expressed in the developing heart from embryonic (E) day E9.5 – E14.5. Netrin-1 was observed to be exclusively expressed in the developing trabecular myocardium. Netrin-1-/- were observed to suffer from embryonic lethality by E10.5 yet the cause of lethality remains unclear. Conditional deletion of Netrin-1 in developing cardiomyocytes does not contribute to embryonic lethality however, neural tube defects were observed in animals lacking Netrin-1 in the developing hindbrain. Despite strong and consistent expression of Netrin-1 in the developing heart, its functional significance is still unclear. These findings are a reminder of the importance of experimental validation when bioinformatic findings are equivocal.Together, these approaches can be used to prioritize and implicate novel genes involved in the development of CHD. An increased knowledge of the genetic contributors of CHD is needed to better understand disease etiology and ultimately to facilitate discovery of therapeutic gene targets. These are all required to improve outcomes for CHD patients around the world. |
