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Allele-specific NKX2-5 binding underlies multiple genetic associations with human electrocardiographic traits

ABSTRACT: We conducted a genome-wide analysis to identify regulatory variants affecting the binding of NKX2-5, a core cardiac development transcription factor, and investigated their role in cardiac gene expression and EKG phenotypes. We generated iPSC-derived cardiomyocytes (iPSC-CMs) from a pedigree of seven whole-genome sequenced individuals, and profiled them with a variety of functional genomic assays including RNA-Seq, ATAC-Seq, and ChIP-Seq of both histone modification H3K27ac and NKX2-5. After establishing that iPSC-CMs recapitulated cardiomyocyte-specific expression and epigenetic signatures, and that genetic variants affected the variability of molecular phenotypes across the iPSC-CM lines, we identified heterozygous sites that showed allele-specific effects (ASE). We then investigated NKX2-5 ASE variants in detail by examining whether they altered cardiac TF motifs, and whether they were enriched for eQTLs and EKG GWAS-SNPs. Our data reveal that variation affecting the binding of NKX2-5 and other cardiac TFs likely serves as a molecular mechanism underlying control of numerous EKG loci across the genome, and that fine-mapping approaches, combined with molecular phenotype data from iPSC-CMs, can be used to prioritize causal variants in EKG GWAS loci.

ORGANISM(S): Homo sapiens

PROVIDER: GSE133833 | GEO | 2019/07/12

REPOSITORIES: GEO

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Project description: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 that 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 another familial CHD case of HLHS proband we identified a rare, predicted-damaging promoter variant affecting RPS15A that was shared between the 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 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, 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 critical roles in cardiogenesis and constitute an emerging novel class of gene candidates likely involved in HLHS and other CHDs.

Project description:Background: We had shown that cardiomyocytes (CMs) were more efficiently differentiated from human induced pluripotent stem cells (hiPSCs) when the hiPSCs were reprogrammed from cardiac fibroblasts rather than dermal fibroblasts or blood mononuclear cells. Here, we continued to investigate the relationship between somatic-cell lineage and hiPSC-CM production by comparing the yield and functional properties of CMs differentiated from iPSCs reprogrammed from human atrial or ventricular cardiac fibroblasts (AiPSC or ViPSC, respectively). Methods: Atrial and ventricular heart tissues were obtained from the same patient, reprogrammed into AiPSCs or ViPSCs, and then differentiated into CMs (AiPSC-CMs or ViPSC-CMs, respectively) via established protocols. Results: The time-course of expression for pluripotency genes (OCT4, NANOG, and SOX2), the early mesodermal marker Brachyury, the cardiac mesodermal markers MESP1 and Gata4, and the cardiovascular progenitor-cell transcription factor NKX2.5 were broadly similar in AiPSC-CMs and ViPSC-CMs during the differentiation protocol. Flow-cytometry analyses of cardiac troponin T expression also indicated that purity of the two differentiated hiPSC-CM populations (AiPSC-CMs: 88.23±4.69%, ViPSC-CMs: 90.25±4.99%) was equivalent. While the field-potential durations were significantly longer in ViPSC-CMs than in AiPSC-CMs, measurements of action potential duration, beat period, spike amplitude, conduction velocity, and peak calcium-transient amplitude did not differ significantly between the two hiPSC-CM populations. Yet, our cardiac-origin iPSC-CM showed higher ADP and conduction velocity than previously reported iPSC-CM derived from non-cardiac tissues. Transcriptomic data comparing iPSC and iPSC-CMs showed similar gene expression profiles between AiPSC-CMs and ViPSC-CMs with significant differences when compared to iPSC-CM derived from other tissues. This analysis also pointed to several genes involved in electrophysiology processes to be responsible for the physiological differences observed between cardiac and non-cardiac-derived cardiomyocytes. ¬ Conclusions: AiPSC and ViPSC were differentiated into CMs with equal efficiency. Detected differences in electrophysiological properties, calcium handling activity, and transcription profiles between cardiac and non-cardiac derived cardiomyocytes demonstrated that 1) tissue of origin matters to generate a better-featured iPSC-CMs, 2) the sublocation within the cardiac tissue has marginal effects on the differentiation process.

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Allele-specific NKX2-5 binding underlies multiple genetic associations with human electrocardiographic traits

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