Development and validation of high-resolution cardiovascular phenotyping strategies in zebrafish reveals the clinical relevance of ROBO4 to congenital heart defects

Zebrafish;Bicuspid Aortic Valve;Echocardiography;Electrocardiography;Phenotype Methods;Genetics;Human Genetics and Molecular Biology

Woods, Courtney ElizabethDietz, Harry C ;

Johns Hopkins University

关键词: Zebrafish;    Bicuspid Aortic Valve;    Echocardiography;    Electrocardiography;    Phenotype Methods;    Genetics;    Human Genetics and Molecular Biology;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/60184/WOODS-DISSERTATION-2017.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

Bicuspid aortic valve (BAV) is a congenital heart defect, affecting 1-2% of the general population. Approximately 30% of individuals with BAV develop ascending aortic aneurysm (AscAA). Although the etiology of BAV/AscAA is unknown, previous family-based studies suggest that BAV is highly heritable. Using whole-exome sequencing, we have systematically screened nine patient families, 286 probands, and 193 unrelated controls to identify causative variants. ROBO4 is a gene in which we have detected multiple independent predicted deleterious alleles showing appropriate familial segregation. Targeted sequencing of an additional 441 unrelated probands and 183 controls detected six additional rare alleles predicted deleterious. Using zebrafish, we have established a robust pipeline to functionally evaluate the biological relevance of genes/variants implicated in BAV/AscAA. It provides an unprecedented capacity to interrogate the cardiovascular structural and functional correlates of BAV/AscAA in genetically defined developing and adult zebrafish. First, we establish mutant fish lines for selected genes using CRISPR/Cas9 or identify functionally equivalent mutants in public resources. For robo4, we generated a 7 base pair deletion in exon six of 19. As predicted, this mutation results in nonsense mediated mRNA decay, significantly reducing mRNA levels as quantified by qRT-PCR. In order to measure complete cardiac function in adult zebrafish, we have optimized two different methods – echocardiography and electrocardiography (ECG). We have completed a longitudinal study of wild-type zebrafish, establishing normative values for three to eleven months of age for both echocardiography and ECG – a critical resource to document genetic predisposition and progression of disease. These data enabled development of a linear model, adjusting for the influence of specific parameters such as age, sex, and body size on cardiovascular measurements in zebrafish. Furthermore, we were able to apply the newly developed echocardiography and ECG methods as well as this linear model to two well-characterized zebrafish models of arrhythmia as proof-of-principle. Echocardiography of robo4 mutants at approximately four to six months reveals extreme outflow turbulence and regurgitation (11/41), compared to wild-type fish (4/45). Importantly, our echocardiograms from mice homozygous for a Robo4 loss-of-function mutation display a similar phenotype. These data establish ROBO4 as a gene critical for the normal development and homeostasis of the left ventricular outflow tract and launch a vigorous pipeline and protocols for functional validation of other candidate genes of BAV/AscAA.