Introduction: Bicuspid aortic valve (BAV) is the most common adult congenital heart defect and is 30 times more prevalent in Turner Syndrome (TS). The genetic causes of BAV are mostly unknown. We previously identified ten large and recurrent rare genomic copy number variant regions (CNVRs) in patients with sporadic BAV and TS.
Hypothesis: BAV-associated recurrent rare CNVs contain dosage-sensitive genes that may cause valve and outflow tract defects when mutated in animal models.
Methods: Candidate CNV genes were prioritized using bioinformatics tools for knockdown (deletions) or overexpression (duplications) in zebrafish embryos. One to four cell stage Tg(acta2:EGFP/gata1:dsRED) and Tg(flk1:EGFP/gata1:dsRED) embryos were microinjected with in vitro transcribed mRNA (SLC2A3) or translation-blocking morpholinos (other genes). Embryos were visualized with fluorescence confocal video microscopy at 5-6 dpf, and Doppler echocardiography was performed at 28 dpf. Morphants were scored for bulbar phenotypes based on the severity of pericardial effusions, cardiomegaly, dilation of the bulbus arteriosus and bulboventricular regurgitation.
Results: Knockdown of kdm6a or bgnb or overexpression of human SLC2A3 produced highly penetrant bulbar phenotypes, characterized by cardiomegaly and bulboventricular regurgitation (Fig). The regurgitant fraction of SLC2A3 embryos was eight times that of control embryos. Thsd7aa and hmgxb4aa morphants demonstrated similar, but less penetrant defects. In contrast, knockdown of ddx3 and usp9, two control genes without known cardiovascular functions, caused non-specific growth retardation.
Conclusions: Knockdown of kdm6a or overexpression of SLC2A3, two candidate genes for BAV, caused cardiac defects in zebrafish that are orthologous to human BAV-associated aortopathy. Additional studies to quantify bulboventricular morphology and function in older morphants with developmentally mature valves are in progress.