Identifying and characterizing causal genes in GWAS-identified loci for heart rate variability using high-throughput, image-based screens in zebrafish larvae

von der Heyde B, Emmanouilidou A, Klingström T, Vicenzi S, Brooke H, Allalou A, den Hoed M

ASHG 2017 Annual Meeting - The American Society of Human Genetics - (-) - [2017-10-17; online 2017-10-17]

Introduction: Heart rate variability (HRV) is associated with an increased risk for cardiovascular disease (CVD), the major cause of death worldwide. A recent meta-analysis of genome-wide association studies (GWAS) in data from 53,172 individuals has identified variants in eight loci that show robust associations with HRV. The causal genes and mechanisms through which these loci influence heart rate variability are still unknown. We aimed to characterize positional candidate genes for HRV using a zebrafish model system. Methods: We prioritized 18 candidate genes for functional follow-up based on results from five bioinformatic tools. Twelve of these genes had a total of 16 orthologous genes in zebrafish. We targeted these 16 orthologues in three pools of up to eight targeted genes using a multiplex CRISPR-Cas9 approach in fertilized eggs from fish that transgenically express green fluorescent protein on smooth muscle cells, which enables visualization of the beating heart. After three months, founder mutants were crossed, and heart rate and rhythm were visualized and quantified in vivo in the offspring by recording 30 sec videos of the atrium at 2 and 5 days post-fertilisation (dpf, repeated measures). Heart rate and two different measures for HRV were quantified in batch mode using a custom-written MatLab script. CRISPR-Cas9 induced mutations were quantified using next-generation sequencing and data were analyzed using mutually adjusted multiple linear/logistic regression models (additive model). Results: We have so far characterized one of three multiplex mutant lines and show that: 1) each additional disrupted allele in rgs6 increases the odds of cardiac arrests at 2dpf by 2.52 (95% CI 1.13-5.61, n=372); 2) each additional disrupted allele in gngt1 is associated with a 0.21±0.09 higher RMSSD (n=303), a 3.49 higher odds of a reduced contractility (95% CI 1.29-9.43, n=326) and an 8.88 higher odds of an abnormal morphology of the heart (95% CI 2.16-36.48, n=297) at 5dpf; and 3) each additional affected allele in kiaa1755 results in a 0.14±0.06 (SD) higher heart rate (n=289). Conclusion: Zebrafish larvae facilitate large-scale, objective, image-based genetic screens of heart rate and rhythm, and allowed us to identify previously unanticipated genes that influence heart rate variability and early cardiac development (gngt1); the risk of cardiac arrests (rgs6); and heart rate (kiaa1755).

BioImage Informatics [Collaborative]

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