Abstract
Protein sequences primarily determine their stability and functions. Mutations may occur at one, two, or three positions at the same time (low-order variants) or at multiple positions simultaneously (high-order variants), which affect protein functions. So far, low-order variants, such as single variants, double variants, and triple variants, have been well-studied through high-throughput experimental scanning techniques and computational prediction methods. However, research on high-order variants remains limited because of the difficulty of scanning an exponentially large number of potential variant combinations. Nonetheless, studying higher-order variants is crucial for understanding the pathogenesis of complex diseases, advancing protein engineering, and driving precision medicine. In this work, we introduce a novel deep learning model, namely MultiMut Recursive Tree (MMRT), to address this challenge of predicting the functional effects of high-order variants. MMRT integrates deep learning with a recursive tree framework to leverage the information from low-order variants to predict functional effects of high-order variants. We evaluated MMRT on datasets comprising 685,593 high-order variants. Our results (mean Spearman's correlation coefficient 0.55) demonstrated that MMRT outperformed three existing state-of-the-art methods: ESM (evolutionary scale modeling), DeepSequence, and ECNet (evolutionary context-integrated neural network). MMRT thus provides more accurate prediction of the functional effects of high-order protein variants, offering great potential for aiding the interpretation of variants in human disease studies.