Functional analysis across model systems implicates ribosomal proteins in growth and proliferation defects associated with hypoplastic left heart syndrome
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eLife Assessment
This important study applies an innovative multi-model strategy to implicate the ribosomal protein (RP) encoding genes as candidates causing Hypoplastic Left Heart Syndrome. The evidence from the screen in stem cell-derived cardiomyocytes and whole genome sequencing of human patients, followed by functional analyses of RP genes in fly and fish models, is convincing and supports the authors' claims. This work and methodology applied would be of broad interest to medical biologists working on congenital heart diseases.
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Abstract
Abstract
Hypoplastic left heart syndrome (HLHS) is the most lethal congenital heart disease (CHD). The pathogenesis of HLHS is poorly understood, and due to the likely oligogenic complexity of the disease, definitive HLHS-causing genes have not yet been identified. Postulating impaired cardiomyocyte proliferation as a likely important contributing mechanism to HLHS pathogenesis, and we conducted a genome-wide siRNA screen to identify genes affecting proliferation of human iPSC-derived cardiomyocytes (hPSC-CMs). This yielded ribosomal protein (RP) genes as the most prominent class of effectors of CM proliferation. In parallel, whole genome sequencing and rare variant filtering of a cohort of 25 HLHS proband-parent trios with poor clinical outcome revealed enrichment of rare variants of RP genes. In addition, in a familial CHD case we identified a rare, predicted-damaging promoter variant affecting RPS15A that was shared between the HLHS proband and a distant relative with CHD. Functional testing with an integrated multi-model system approach reinforced the idea that RP genes are major regulators of cardiac growth and proliferation, thus potentially contributing to the hypoplastic phenotype observed in HLHS patients. Cardiac knockdown (KD) of RP genes with promoter or coding variants (RPS15A, RPS17, RPL26L1, RPL39, RPS15) reduced proliferation in generic hPSC-CMs and caused malformed hearts, heart-loss or even lethality in Drosophila. In zebrafish, diminished rps15a function caused reduced CM numbers, heart looping defects, or weakened contractility, while reduced rps17 or rpl39 function caused reduced ventricular size or systolic atrial dysfunction of the atrium, respectively. Importantly, genetic interactions between RPS15A and core cardiac transcription factors TBX5 in CMs, Drosocross, pannier and tinman in flies, and tbx5 and nkx2-7 (nkx2-5 paralog) in fish, support a specific role for RP genes in heart development. Furthermore, RPS15A KD-induced heart/CM proliferation defects were significantly attenuated by p53 KD in both hPSC- CMs and zebrafish, and by Hippo activation (YAP/yorkie overexpression) in developing fly hearts. Based on these findings, we conclude that RP genes play novel critical roles in cardiogenesis and constitute an emerging class of gene candidates likely involved in HLHS and other CHDs.
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eLife Assessment
This important study applies an innovative multi-model strategy to implicate the ribosomal protein (RP) encoding genes as candidates causing Hypoplastic Left Heart Syndrome. The evidence from the screen in stem cell-derived cardiomyocytes and whole genome sequencing of human patients, followed by functional analyses of RP genes in fly and fish models, is convincing and supports the authors' claims. This work and methodology applied would be of broad interest to medical biologists working on congenital heart diseases.
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Reviewer #1 (Public review):
Nielsen et al have identified a new disease mechanism underlying hypoplastic left heart syndrome due to variants in ribosomal protein genes that lead to impaired cardiomyocyte proliferation. This detailed study starts with an elegant screen in stem-cell-derived cardiomyocytes and whole genome sequencing of human patients and extends to careful functional analysis of RP gene variants in fly and fish models. Striking phenotypic rescue is seen by modulating known regulators of proliferation, including the p53 and Hippo pathways. Additional experiments suggest that the cell type specificity of the variants in these ubiquitously expressed genes may result from genetic interactions with cardiac transcription factors. This work positions RPs as important regulators of cardiomyocyte proliferation and differentiation …
Reviewer #1 (Public review):
Nielsen et al have identified a new disease mechanism underlying hypoplastic left heart syndrome due to variants in ribosomal protein genes that lead to impaired cardiomyocyte proliferation. This detailed study starts with an elegant screen in stem-cell-derived cardiomyocytes and whole genome sequencing of human patients and extends to careful functional analysis of RP gene variants in fly and fish models. Striking phenotypic rescue is seen by modulating known regulators of proliferation, including the p53 and Hippo pathways. Additional experiments suggest that the cell type specificity of the variants in these ubiquitously expressed genes may result from genetic interactions with cardiac transcription factors. This work positions RPs as important regulators of cardiomyocyte proliferation and differentiation involved in the etiology of HLHS, although the downstream mechanisms are unclear.
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Reviewer #2 (Public review):
Tanja Nielsen et al. present a novel strategy for the identification of candidate genes in Congenital Heart Disease (CHD). Their methodology, which is based on comprehensive experiments across cell models, Drosophila and zebrafish models, represents an innovative, refreshing and very useful set of tools for the identification of disease genes, in a field which are struggling with exactly this problem. The authors have applied their methodology to investigate the pathomechanisms of Hypoplastic Left Heart Syndrome (HLHS) - a severe and rare subphenotype in the large spectrum of CHD malformations. Their data convincingly implicates ribosomal proteins (RPs) in growth and proliferation defects of cardiomyocytes, a mechanism which is suspected to be associated with HLHS.
By whole genome sequencing analysis of a …
Reviewer #2 (Public review):
Tanja Nielsen et al. present a novel strategy for the identification of candidate genes in Congenital Heart Disease (CHD). Their methodology, which is based on comprehensive experiments across cell models, Drosophila and zebrafish models, represents an innovative, refreshing and very useful set of tools for the identification of disease genes, in a field which are struggling with exactly this problem. The authors have applied their methodology to investigate the pathomechanisms of Hypoplastic Left Heart Syndrome (HLHS) - a severe and rare subphenotype in the large spectrum of CHD malformations. Their data convincingly implicates ribosomal proteins (RPs) in growth and proliferation defects of cardiomyocytes, a mechanism which is suspected to be associated with HLHS.
By whole genome sequencing analysis of a small cohort of trios (25 HLHS patients and their parents), the authors investigated a possible association between RP encoding genes and HLHS. Although the possible association between defective RPs and HLHS needs to be verified, the results suggest a novel disease mechanism in HLHS, which is a potentially substantial advance in our understanding of HLHS and CHD. The conclusions of the paper are based on solid experimental evidence from appropriate high- to medium-throughput models, while additional genetic results from an independent patient cohort are needed to verify an association between RP encoding genes and HLHS in patients.
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