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-ß 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