The Role of DAAM1 in Congenital Heart Disease

Alyx Posorske, IU School of Medicine, Indianapolis, IN, United States; James Zwiezynski, IU school of Medicine, United States; Laura Haneline, Indiana University School of Medicine; Riley Children’s Hospital, United States; Weinian Shou, IU School of Medicine, United States; Stephanie M. Ware, IU School of Medicine, Indianapolis, IN, United States; Matthew Durbin, IU School of Medicine, United States

Background: Congenital heart disease (CHD) is the most common birth defect, yet most etiologies remain unknown. DAAM1 is a protein whose loss-of function leads to CHD in mice and humans. DAAM1 binds the central cytoplasmic protein DISHEVELLED2 (DVL2) of the noncanonical Wnt/planar cell polarity signaling pathway (PCP). DVL2 and PCP are necessary for driving the migration of second heart field (SHF) cells and cardiac neural crest cells (NC) and disruption leads to CHD. A recent study demonstrates bimodal signaling, with dual positive and negative regulation of DVL2 by DAAM1 and another PCP protein, VANGL2, during neurulation.

Objectives: We hypothesize that bimodal signaling between DAAM1, DVL2 and VANGL2 is necessary for SHF and NC cell migration during cardiac development, and disruption leads to human CHD.

Design/Methods: We tested the hypothesis by intercrossing heterozygous loss of function mice (Daam1+/gt, Dvl2+/∆, Vangl2+/∆) to generate cohort litters of embryos haploinsufficient for one, two, or all three PCP proteins of interest. We performed histological analysis at E14.5 to identify cardiac defects. We also tested the hypothesis the pathway is necessary for cardiac NC cell and SHF cell migration by intercrossing Dvl2flox/flox mice along with Wnt1-Cre (NC) and Mef2-Cre (SHF) mice, to conditionally delete these cell populations. We analyzed the global transcriptome using RNA-Seq in the outflow tract of the E12.5 embryos followed by pathway analysis.

Results: Preliminary analysis of 42 embryos reveals a trend of decreased survival of the compound heterozygous embryos, including Dvl2+/∆, Daam1+/gt, Vangl2+/∆ (number observed, 1 versus expected, 5.3) and Dvl2∆/∆, Daam1+/gt, Vangl2+/∆ (frequency observed 0 versus expected 2.7). The one compound heterozygous embryo recovered had CHD (a membranous VSD). Preliminary analysis of differential gene expression in the global transcriptome in Mef2-Cre;Dvl2∆/∆ embryos reveals 205 genes were significantly altered (p < 0.05). Ingenuity pathway analysis revealed significant alteration in processes important for cardiac development including epithelial to mesenchymal transition and PCP as top pathways altered.

Conclusion: Future analysis currently underway will test the hypothesis that disruption of DAAM1 is important for human CHD, by analyzing 320 CHD patient exomes for novel variants in DAAM1. The experiments will establish DAAM1’s role interacting with DVL2 and VANGL2, and DAAM1’s in CHD in humans.