Congenital heart diseases (CHD) are the most common human birth defects, affecting approximately 0.8 % of all live births.1 They comprise structural malformations of the heart occurring during development and functional disorders such as cardiomyopathies and arrhythmias. Often a treatment throughout life is required due to impaired cardiac functions through adult stages. The causal genetic underpinning of CHD, which are often complex and oligogenic, are still far from understood, and currently, only 25 % of all CHDs can be explained by gene mutations.2 Therefore, there is a great unmet need to understand the genetic mechanisms leading to developmental defects, and likely the key to fight CHDs lies in understanding the exact molecular mechanisms underlying cardiac development and occurrence of disease. In this work I leverage CHD patient genomic data to identify novel potentially CHD-associated genes, gene networks and pathways, by taking advantage of Drosophila melanogaster as a heart model system. In the first part of this thesis (conducted in the Rickert-Sperling lab) I concentrate on myomesin-2 (MYOM2), a functional component of the M-band of the sarcomeres. MYOM2 was found mutated in patients with Tetralogy of Fallot (TOF) and hypertrophic cardiomyopathy (HCM), which did not carry mutations in any of the known disease genes.3,4 Functional analysis in the Drosophila heart identified CG14964 (which we named MnM for myomesin and myosin binding protein), a so far uncharacterized fly gene, as the likely ortholog for MYOM2 and other myosin binding proteins. Moderate knockdown (KD) of CG14964 in the fly heart led to dilation of the heart, while strong KD caused constrictions. Moreover, genetic interaction studies revealed synergism between CG14964 and myosin heavy chain 6/7 (MYH6/7) fly ortholog Mhc. Overall, these data suggest a critical role for MnM in heart development and thus point towards a potential contribution of MYOM2 variants to CHD manifestations, such as HCM and TOF (Chapter 1). The second part of the thesis (conducted in the Bodmer lab) focusses on the genetic perturbations underlying hypoplastic left heart syndrome (HLHS), which represents the most lethal CHD and is most likely oligogenic in origin. In collaboration with the Mayo Clinic (MN, USA), whole-genome sequencing was performed in a cohort of HLHS proband-parent trios, which revealed enrichment of rare, predicted-damaging variants in LDL receptor-related protein 2 (LRP2).5 Functional analysis in human iPSC-derived cardiomyocytes (hiPSC-CM), in Drosophila and zebrafish hearts with diminished LRP2 function showed a requirement of the receptor for cardiomyocyte proliferation and differentiation.5 In this manuscript and beyond, my contribution was to further study the consequence of systemic LRP2 KD in the fly heart, which leads to cardiac dilation and constrictions. Furthermore, I show a genetic interaction between the multiligand receptor LRP2 and apolipoprotein B (ABOB) and reveal connections with growth associated pathways SHH and Wnt/wg, suggesting that LRP2 potentially regulates cardiac proliferation and differentiation through modulation of these pathways (Chapter 3). Segregation analysis in a familial case within the HLHS cohort from Mayo Clinic furthermore identified a rare promoter variant affecting ribosomal protein RPS15A that segregates with disease. In addition, enrichment analysis in 25 HLHS trios with poor clinical outcome revealed an over-representation of ribosomal protein (RP) gene variants. Functional testing in model systems, showed that KD of RPs reduced proliferation in generic human iPSC-CMs (Colas lab) and impaired cardiac differentiation in Drosophila (my work) resulting in a partial or ‘no’ heart phenotype in adult flies. Furthermore, I found that RpS15Aa KD leads to cardioblasts misspecifications during early cardiogenesis in the fly. Functional validation in zebrafish revealed that rps15a KD causes reduced cardiomyocyte numbers, diminished heart looping, and contractility, without affecting overall embryonic development (Ocorr lab). Strikingly, RPS15A KD-induced defects were significantly reversed by p53 KD in hiPSC-CMs (Colas lab) or zebrafish (Ocorr lab), and by Hippo activation or myc KD in flies (my work). When testing for cardiac-specific RP functions, we found synergistic interactions between RPS15A and cardiac transcription factors, including tinman/NKX2-5 and Dorsocross/TBX5, in both Drosophila and zebrafish (Ocorr lab and my work) suggesting similarly conserved synergistic interactions between RPs and cardiogenic genes in both zebrafish and fly hearts (Chapter 4). Taken together, I conclude that MYOM2, LRP2, and RP genes play a critical role in cardiogenesis and could represent novel candidate genes (MYOM2, LRP2) or an emerging class of genetic effectors (RPs) in heart diseases, such as HCM, TOF, or HLHS. The discovery of disease-causing genes could help define new marker genes for early diagnostic and modeling of patient genotypes has a high potential to push the development of personalized patient care forward.
