Congenital heart disease (CHD) is genetically complex, with most cases likely reflecting combined effects of variation at multiple genes. Current methods, both statistical and experimental, for discovering causal alleles of genes that produce disease phenotypes only in combination, are very limited. These limitations are a major reason why the genetic origins of most CHD remain unknown, and there is a pressing need to overcome them. To address this need, we propose a novel approach, which exploits genetic syndromes known as ?transcriptomopathies,? in which transcription is globally, yet subtly, disrupted. In one of these, Cornelia de Lange Syndrome (CdLS), small changes (mostly <1.5-fold) occur in transcript levels for up to 1,000 genes in each cell. This results in a reproducible spectrum of birth defects that includes CHD. CdLS is most commonly caused by haploinsufficiency for NIPBL, a gene that encodes a cohesin-regulatory protein, and mouse and zebrafish models of Nipbl-haploinsufficiency replicate CdLS phenotypes. Such animal models provide a means to identify how sets of perturbations to gene expression which do not individually cause disease, can act collectively to produce heart defects?thus modeling the multi-genic causation of CHD. Our investigation of such models suggests that causal events likely occur early in embryogenesis during the initial morphogenesis of the heart. Recently, using mouse models in which Nipbl-haploinsufficiency can be switched on and off in different embryonic cell lineages, we discovered that risk for atrial septal defects (ASDs), the main form of CHD in Nipbl+/- mice, is controlled by non-additive interactions between gene expression changes in cardiomyocytes and their progenitors; cells derived from endoderm, endocardium or endothelium; and cells of the rest of the embryo. We now propose to exploit the genetic manipulability of this system to identify both the morphogenetic abnormalities and the individual gene expression changes that collectively produce ASDs. By analyzing early morphogenesis in globally- and conditionally-mutant embryos we will test several hypotheses concerning the role of progenitor cell proliferation, patterning and migration, with a particular focus on the progenitors of the second heart field, which preliminary results suggest may be central to ASD causation. By using a variety of Cre recombinase-expressing mice to conditionally create, and rescue, Nipbl- haploinsufficiency in a variety of lineages, we will investigate the roles of distinct subpopulations of cells in both increasing and decreasing ASD risk. Finally, we will use single-cell RNA sequencing of early embryos to identify gene expression changes associated with Nipbl-haploinsufficiency in those cells responsible for causing ASDs. The result of these studies will be to develop novel, directly testable hypotheses about mechanisms that underlie multifactorial, and multi-genic, CHD.
One reason why genetic contributions to congenital heart disease have been especially difficult to identify is that most heart defects are likely to result from combinations of subtle genetic variants, rather than single-gene disorders. We propose to exploit a mouse model of a class of genetic disorders known as ?transcriptomopathies??in which subtle changes in gene expression occur throughout the genome?to identify how combinatorial genetic variation can give rise to atrial septal defects, one of the most common forms of congenital heart disease.
