Deep phenotyping of EHMT1 ankyrin repeat domain missense variants in Kleefstra syndrome by multi-tiered structural genomics analyses at atomic resolution.

EHMT1 is a lysine methyltransferase that writes and reads histone methylation marks to repress transcription. Deleterious mutations in EHMT1 cause Kleefstra syndrome (KLEFS1; OMIM #610253), a congenital neurodevelopmental disorder. Many of these affect the ankyrin repeat (ANKR) domain, which recognizes histone H3 lysine-9 methylation and mediates interactions with other epigenetic regulators. However, the functional consequences of EHMT1 missense variants within the ANKR domain remain poorly characterized, as nearly 50% are currently classified as variants of unknown significance (VUS). To address this gap, we performed a comprehensive structural genomics analysis to predict how 56 Kleefstra syndrome-associated missense variants alter EHMT1 ANKR domain structure and function. We integrated sequence-based pathogenicity algorithms with protein structural and dynamics metrics to generate variant molecular fitness scores. From these predictions, we selected 16 representative variants for experimental validation. Using differential scanning fluorimetry and circular dichroism, we quantified variant-specific effects on ANKR domain stability and secondary structure. We further assessed methyl-lysine binding using fluorescence polarization assays. By combining computational and biochemical analyses, we re-classified 80% of VUS as tolerated or damaging based on their effects on ANKR domain structural integrity and methyl-lysine binding. The resulting molecular fitness scores reveal mechanisms by which damaging variants disrupt EHMT1 ANKR domain stability, ligand binding, or both. Together, our integrated approach refines EHMT1 variant annotation and provides mechanistic insights that enhance current tools for variant interpretation in Kleefstra syndrome, which can be translated to missense variants in other epigenetic proteins and rare diseases.