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 delay (NDD) [1-3], 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, p.His167Pro) maintained protein expression and induced mild to moderate phenotypes, consistent with partial loss-of-function. The p.Lys224Glu variant behaved similarly to 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.