Using Drosophila to model a variant of unknown significance in the human cardiogenic geneNkx2.5

Abstract

AbstractSequencing of human genome samples has unearthed genetic variants for which functional testing is necessary to validate their clinical significance. We used the Drosophila system to analyze a variant of unknown significance in the human congenital heart disease gene,Nkx2.5. We generated an R321N allele of theNkx2.5orthologtinman(tin) to model a human K158N variant and tested its function in vitro and in vivo. The R321N Tin isoform bound poorly to DNA in vitro and was deficient in activating a Tin-dependent enhancer in tissue culture. Mutant Tin also showed a significantly reduced interaction with a Drosophila Tbox cardiac factor named Dorsocross1. We generated atinR321Nallele using CRISPR/Cas9, for which homozygotes were viable and had normal heart specification, but showed defects in the differentiation of the adult heart that were exacerbated by further loss oftinfunction. We conclude that the human K158N mutation is likely pathogenic through causing both a deficiency in DNA binding and a reduced ability to interact with a cardiac cofactor, and that cardiac defects might arise later in development or adult life.