A system approach to the analysis of Heterotaxy Candidate Genes

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PROJECT SUMMARY Congenital malformations are the major cause of infant mortality in the US. However, our understanding of the genetic causes of congenital malformations is severely limited negatively impacting our ability to care for these patients. To discover the genetic causes, we and others have employed human genomics analyses on patients. In particular, we have focused on Heterotaxy (Htx), a disorder of left-right patterning. Normally, our internal organs are asymmetrically distributed along the left-right axis and failure of this process can lead to severe disease including congenital heart disease, gut malrotation, and immune deficiencies. Human genetic analysis of these patients has identified many candidate genes, but the functional relevance is unclear since a plausible disease pathogenesis mechanism is unknown for nearly all of the candidate genes. In the previous grant period, we proposed a gain of function screen where we overexpressed Htx candidate genes in Xenopus looking for potent phenotypes. While this strategy has limitations, we discovered multiple genes that gave interesting phenotypes that led to exciting new insights into Wnt and Cilia signaling pathways. Funded by the grant proposal, we also developed new techniques for assaying cilia based extracellular fluid flow and notably CRISPR based F0 gene editing. The development of these new technologies has transformed the strategy that we will employ for gene screening in the subsequent grant period. Importantly, we will use F0 CRISPR based loss of function screening which is rapid, inexpensive, and highly effective. Our “next generation” screen will also include an investigation not only of cardiac looping but additional steps in the LR signaling cascade. Finally, we will investigate patient variants and connect with researchers across the world using GeneMatcher to exploit our high-throughput animal model to test additional patient variants and increase the number of experimentally tested alleles in our system. Finally, in a subsequent Aim, we will place Htx candidate genes into four key pathways critical for LR patterning: Cilia, Wnt, Notch, and TGF-β signaling. We have considerable experience in each of these pathways converting novel unexplored genes into molecular mechanisms in each of these pathways. Therefore, our previous experience demonstrates that we are especially well suited to translate patient driven gene discovery into molecular mechanisms.