964C Poster - 15. Models of human disease
Saturday April 09, 1:30 PM - 3:30 PM

Authors:
Tanja Nielsen ¹; Anais Kervadec ²; Xin-Xin I. Zeng ¹; Analyne Schroeder ¹; Jeanne L. Theis ³; Timothy M. Olson ³; Karen Ocorr ¹; Paul Grossfeld ⁴; Alexandre R. Colas ²; Georg Vogler ¹; Rolf Bodmer ¹

Affiliations:
1) Development, Aging and Regeneration Program, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA; 2) Human Genetics Program, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA; 3) Division of Pediatric Cardiology, Department of Pediatric and Adolescent Medicine, Mayo Clinic, Rochester, MN, USA,; 4) Department of Pediatrics, UCSD School of Medicine, La Jolla, CA, USA

Keywords:
j. cardiovascular disease; z. other (Heart development)

Hypoplastic Left Heart Syndrome (HLHS) represents the most lethal Congenital Heart Disease (CHD) and is characterized by a severely underdeveloped left ventricle. The genetic mechanisms leading to HLHS are poorly understood, but likely of oligogenic origin.
Decreasing sequencing costs have led to the identification of thousands of putative human disease variants. However, establishing genotype-phenotype relationships remains challenging. In vivo functional analysis of patient-derived genes and gene clusters in a high-throughput manner using the fly heart model combined with human iPSC-cardiomyocytes (hiPSC-CMs) and zebrafish, provides a framework for prioritizing and interrogating the contribution of genetic variants in heart disease.
We performed whole genome sequencing in a familial HLHS case and a cohort of 25 HLHS proband-parent trios with poor clinical outcome. Candidate genes identified by Mendelian modeling in the 25 trios were subject to gene network enrichment analysis, revealing an over-representation of ribosomal protein (RP) genes. Segregation analysis in the familial case identified a rare promoter variant affecting RPS15A. Remarkably, patient-derived iPSC-CM proliferation was reduced compared to the parents.
Functional analysis in model systems showed that knockdown (KD) of RPs reduced proliferation in generic hiPSC-CMs and impaired cardiac differentiation in Drosophila resulting in a partial or ‘no’ heart phenotype in adult flies. Furthermore, RpS15Aa KD led to cardioblast misspecification during early cardiogenesis. Functional validation in zebrafish revealed that rps15a KD causes reduced cardiomyocyte numbers and contractility, and defective heart looping, without affecting overall embryonic development. Strikingly, RPS15A KD-induced defects were significantly reversed by p53 KD in hiPSC-CMs or zebrafish, and by myc KD or YAP/yorkie overexpression in flies, dependent on yorkie’s co-factor TEAD/scalloped.
When testing for cardiac-specific RP functions, we found synergistic interactions between RPS15A and cardiac transcription factor tinman/Nkx2.7 and dorsocross/Tbx5a, in both Drosophila and zebrafish suggesting conserved synergism between RPs and cardiogenic genes in both models.
In summary, we conclude that RP genes play a critical role as regulators of cardiac growth and cardiomyocyte proliferation along with cardiogenic transcription factors and we suggest that RP genes represent a novel class of genetic effectors in CHDs, such as HLHS.