Identification of genetic modifiers of GATA6 in human congenital heart diseases

PROJECT SUMMARY Outflow tract (OFT) anomalies comprise more than 30% of congenital heart diseases (CHD) and are life- threatening in the absence of surgical interventions.

GATA6 heterozygous mutations cause OFT and aortic arch patterning anomalies in humans, which are not shown among GATA4 patients who exhibit mainly septal defects.

However, GATA6 mutations cause a broad range of phenotypes, ranging from normal cardiac development to truncus arteriosus (TA), the most severe form of OFT defect, indicating that the severity of CHD in GATA6 heterozygous patients is affected by other genetic modifiers.

To identify potential genetic modifiers affecting the severity of GATA6 mutations, I analyzed the whole- exome sequencing data from the Pediatric Cardiac Genomics Consortium and found that GATA6 patients with TA harbor heterozygous mutations of POR, the cytochrome p450 oxidoreductase, implying that POR and GATA6 may interact during OFT development.

Retinoic acid (RA) levels are upregulated in Por -/- embryos, indicating that the activity of RA 4-hydroxylase (CYP26) reduced due to Por mutations.

Also, the lethality of Por -/- embryos can be rescued by reducing RA levels, implying that the RA pathway is involved in Por functions during OFT development.

Moreover, recent studies showed that RA can directly regulate GATA6 protein expression, suggesting a potential molecular link among GATA6, POR, and RA.

In mice, neural crest cell (NCC)-specific deletion of Gata6 is sufficient to cause OFT and aortic arch anomalies, and ectopic expression of Gata6 in NCCs prevents aortic arch from regression, suggesting that Gata6 plays an important role in aortic arch patterning which is governed by NCC-derived vascular smooth muscle cells (VSMCs).

Furthermore, TA probands with GATA6 mutations also share mutations of several myopathy genes, implying these genes may also influence GATA6 heterozygosity.

To test this possibility, I will perform CRISPR genetic screening with single-cell technology (Perturb-seq) in WT and GATA6 -/+ human pluripotent stem cells during NCC-derived VSMC differentiation.

In summary, I will test the hypothesis that the severity of OFT defects in GATA6 heterozygous patients is affected by other genetic factors.

I will 1) determine whether POR is a candidate modifier gene for GATA6 in vivo, 2) investigate whether RA signaling modulates GATA6 heterozygosity in NCC-derived VSMCs, and 3) identify potential modifiers of GATA6 using CRISPR genetic screening.

This proposal will be the first attempt to identify gene-gene interactions with GATA6 in a systemic manner and will provide important insights into the genetics of complex traits of human diseases.

The combination of cutting-edge techniques in the Srivastava lab and the collaborative environment in Gladstone Institutes will advantage me to accomplish this proposed work, which will provide the foundation of my independent research in the future.