In vivoidentification and validation of novel potential predictors for human cardiovascular diseases

Abstract

Genetics crucially contributes to cardiovascular diseases (CVDs), the global leading cause of death. Since the majority of CVDs can be prevented by early intervention there is a high demand for predictive markers. While genome wide association studies (GWAS) correlate genes and CVDs after diagnosis and provide a valuable resource for such markers, preferentially those with pre-assigned function are addressed further. To tackle the unaddressed blind spot of understudied genes, we particularly focused on the validation of heart GWAS candidates with little or no apparent connection to cardiac function. Building on the high conservation of basic heart function and underlying genetics from fish to human we combined CRISPR/Cas9 genome editing of the orthologs of human GWAS candidates in isogenic medaka with automated high-throughput heart rate analysis. Our functional analyses of understudied human candidates uncovered a prominent fraction of heart rate associated genes from adult human patients displaying a heart rate effect in embryonic medaka already in the injected generation. Following this pipeline, we identified 16 GWAS candidates with potential diagnostic and predictive power for human CVDs.

Precise in vivo functional analysis of DNA variants with base editing using ACEofBASEs target prediction

“…The fish facility is under the supervision of the local representative of the animal welfare agency. The following medaka lines: Cab as wild-type (Loosli et al, 2000), Cab (myl7::EGFP) (Gierten et al, 2020), HdrR (myl7::EGFP myl7::H2A-mCherry) (Hammouda et al, 2021). The zebrafish line AB was used in this study as wild-type.…”

“…External development, rapid growth, and transparency of medaka (Oryzias latipes) and zebrafish (Danio rerio) embryos make these established vertebrate human disease models ideal for studying genetic variants' consequences in vivo (Gut et al, 2017;Hammouda et al, 2021;Meyer et al, 2020). Both ABEs and CBEs have been employed in zebrafish (Carrington et al, 2020;Qin et al, 2018;Rosello et al, 2021b;Zhang et al, 2017;Zhao et al, 2020), reaching up to 40% and 91% editing efficiencies, respectively.…”

“…We assayed the loss of GFP fluorescence in a medaka EGFP and mCherry double transgenic line with heart-specific, nuclear fluorophore expression (myl7::EGFP, myl7::H2A-mCherry) (Hammouda et al, 2021). Co-injection of ABE8e mRNA with the GFP-C71 sgRNA into 1-cell stage embryos resulted in the complete loss of GFP fluorescence in all hearts assayed at 4 dpf (n=41), indicating quantitative editing as also reflected by the absence of mosaicism.…”