本研究调查了常染色质组蛋白赖氨酸甲基转移酶 1 (EHMT1) 中各种不确定意义 (VUS) 的功能意义，这对于早期发育和正常生理机能至关重要。 EHMT1 突变会导致 Kleefstra 综合征，并与多种人类癌症有关。然而，尚未对这些变异进行准确的功能解释，这限制了诊断和未来的研究。为了克服这个问题，我们将传统的变异调用工具与计算生物物理学和生物化学相结合，对 EHMT1 的 SET 催化结构域进行多层机制分析，这对于该蛋白质功能至关重要。我们使用基于分子力学和分子动力学 (MD) 的指标来分析 SET 结构域结构和由该结构域内 97 个 Kleefstra 综合征错义变异产生的功能运动。我们的方法允许我们以机械的方式将变体分类为 SV（结构变体）、DV（动态变体）、SDV（结构和动态变体）和 VUS（意义不确定的变体）。我们的研究结果表明，破坏性变异主要分布在活性位点、底物结合位点和预设置区域周围。总的来说，我们报告了这种方法相对于传统变异解释工具的改进，同时提供了变异功能障碍的分子机制。© 2023 作者。

This study investigates the functional significance of assorted variants of uncertain significance (VUS) in euchromatic histone lysine methyltransferase 1 (EHMT1), which is critical for early development and normal physiology. EHMT1 mutations cause Kleefstra syndrome and are linked to various human cancers. However, accurate functional interpretations of these variants are yet to be made, limiting diagnoses and future research. To overcome this, we integrate conventional tools for variant calling with computational biophysics and biochemistry to conduct multi-layered mechanistic analyses of the SET catalytic domain of EHMT1, which is critical for this protein function. We use molecular mechanics and molecular dynamics (MD)-based metrics to analyze the SET domain structure and functional motions resulting from 97 Kleefstra syndrome missense variants within the domain. Our approach allows us to classify the variants in a mechanistic manner into SV (Structural Variant), DV (Dynamic Variant), SDV (Structural and Dynamic Variant), and VUS (Variant of Uncertain Significance). Our findings reveal that the damaging variants are mostly mapped around the active site, substrate binding site, and pre-SET regions. Overall, we report an improvement for this method over conventional tools for variant interpretation and simultaneously provide a molecular mechanism for variant dysfunction.© 2023 The Authors.