Uncovering functional insights into human pathogenic variants in CDK19 using Drosophila models

Heterozygous missense variants in CDK19 have been found in patients diagnosed with Developmental and epileptic encephalopathy-87 (DEE87) who present with global developmental delay, intellectual disability and other muscular and neurological deficiencies. Two missense variants in CDK19, Y32H and T196A , were first proposed to be dominant negative in nature based on experiments in a Drosophila model. Subsequently, another group proposed that Y32H is a gain of function based on elevated kinase activity. We present a detailed evaluation of the activity and functionality of these dominant variants in several contexts in fly models of DEE-87 in which endogenous cdk8 , the fly ortholog of human CDK8 and CDK19 , is knocked down while we overexpressed the human genes. Depletion of Drosophila cdk8 causes thicker muscle myofibrils, fused mitochondria, and climbing defects. The expression of human CDK19 ^WT in a fly cdk8-RNAi background rescues these defects, highlighting functional conservation. To investigate functional differences between the variants, we compared effects of variants to expression of wildtype CDK19. Ubiquitous expression of Y32H can rescue the cdk8 knockdown phenotype through a gain of function compensatory effect, while T196A is unable to do so through a possible reduction in kinase activity. We found that supplementation of the antioxidant drug, N-acetylcysteine amide (NACA), rescues phenotypes of only the T196A adult flies, illustrating a divergence in variant functionality. Our studies in flies allowed us to assay these variants in numerous contexts to gain further insight into their mechanism and obtain translational knowledge to apply back to human health.