An In Vivo Cardiac Assay to Determine the Functional Consequences of Putative Long QT Syndrome Mutations

Abstract

Rationale Genetic testing for Long QT Syndrome (LQTS) is now a standard and integral component of clinical cardiology. A major obstacle to the interpretation of genetic findings is the lack of robust functional assays to determine the pathogenicity of identified gene variants in a high throughput manner. Objective The goal of this study was to design and test a high throughput in vivo cardiac assay to distinguish between disease-causing and benign KCNH2 (hERG1) variants, using the zebrafish as a model organism. Methods and Results We tested the ability of previously characterized LQTS hERG1 mutations and polymorphisms to restore normal repolarization in the kcnh2-knockdown embryonic zebrafish. The cardiac assay correctly identified a benign variant in 9 of 10 cases (negative predictive value 90%) while correctly identifying a disease-causing variant in 39/39 cases (positive predictive value 100%). Conclusion The in vivo zebrafish cardiac assay approaches the accuracy of the current benchmark in vitro assay for the detection of disease-causing mutations and is far superior in terms of throughput rate. Together with emerging algorithms for interpreting a positive LQTS genetic test, the zebrafish cardiac assay provides an additional tool for the final determination of pathogenicity of gene variants identified in LQTS genetic screening.

A Functional Assay for Sick Sinus Syndrome Genetic Variants

“…First, despite being an in vivo cardiac system, some would argue that zebrafish is not an ideal model for human arrhythmogenic disease since it is a non-mammalian organism with a 2-chamber heart. However, the electrophysiological properties of zebrafish myocardium are more similar to human myocardium than other mammalian systems, such as mice [7, 14]. Second, while the assay is not particularly quantitative, it does provide a moderate-throughput approach to characterize whether an HCN4 variant is pathogenic, benign or hypomorphic.…”

“…Zebrafish display many similar cardiac electrophysiological characteristics as humans and are more amenable to high-throughput functional experiments [6]. We have previously developed an in vivo zebrafish assay to characterize the functional consequences of human Long QT Syndrome genetic variants [7]. We now present another in vivo cardiac assay to functionally characterize potential SSS genetic variants with the goal to provide meaningful clinical interpretation to HCN4 variants detected by genetic testing.…”

Anti-Inflammatory Effect of Plantago Sp Ethanolic Extract in Murine RAW264.7 Macrophage Cells

“…However, increase in activated macrophages may lead to acute or chronic inflammation that will cause dysfunction of tissues. Thus, inhibition of activated macrophage proliferation can prevent prolonged exposure to inflammation and encourage healthy tissue restoration [11]. Inflammation promotes wound healing and tissue repair but prolonged inflammation can cause tissue dysfunction that lead to inflammatory diseases.…”

Precise Editing of the Zebrafish Genome Made Simple and Efficient

“…The near transparency of the embryo has even permitted visualization and analysis of the dynamics of signaling molecules in intact tissues (Yu et al 2009; Muller et al 2012). Furthermore, the extent of functional conservation among vertebrates makes the zebrafish an outstanding system for examining the mechanistic basis of disease (Jurynec et al 2008), assaying human gene activity (Bamford et al 2000; Jou et al 2013), or discovering compounds that augment or inhibit developmental processes or disease-like conditions (Peterson et al 2004; North et al 2007; Hagedorn et al 2014). The sophistication with which biological processes can be probed in the zebrafish make it poised to exploit tools of genome manipulation, which could be used to generate models of human disease, study gene function in specific tissue or temporal contexts, or generate alleles that allow study of tagged protein activities under physiological conditions.…”