Loss-of-function variants in MARK2 cause neurodevelopmental disorder.

Microtubule-affinity regulating kinase 2 (MARK2) is a conserved serine/threonine kinase that plays a critical role in microtubule dynamics and neuronal polarity. Rare MARK2 variants have recently been reported in individuals with autism spectrum disorder (ASD) and neurodevelopmental disorder (NDD), and here we expand the number of affected individuals, including a familial case. Despite these clinical findings, the functional significance and underlying mechanisms of patient-derived variants remain poorly understood. To address this gap, we established a humanized Drosophila model and systematically evaluated eight NDD-linked MARK2 variants using tissue-specific expression assays. Functional assessments in Drosophila indicate that MARK2 variants span a range of functional consequences, primarily exhibiting hypomorphic or loss-of-function effects. Truncating variants consistently showed reduced protein levels and rescued lethality, a loss-of-function mechanism. Most missense variants (e.g., p.Val231Met, p.Gly135Arg, p.Ala80Val, and p.His167Pro) maintained protein expression and induced mild to moderate phenotypes, consistent with partial loss of function. The p.Lys224Glu variant behaved similarly to the wild type in both viability and wing patterning assays and exhibited elevated protein levels but did not display overt pathogenicity in flies. Based on these findings, we propose a functional classification of MARK2 variants into three categories: not pathogenic (p.Lys224Glu), hypomorphic (missense variants), and loss of function (truncating variants). These results highlight the value of Drosophila as a scalable in vivo platform for variant interpretation and provide insight into how reduced MARK2 activity may contribute to neurodevelopmental disorders.