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<div><p>(1) Background</p><p>The Mixed lineage leukemia 1 <i>(MLL1)</i> gene, located on chromosome 11q23, plays a pivotal role in histone lysine-specific methylation and is consistently associated with various types of leukemia. Non-synonymous Single Nucleotide Polymorphisms (nsSNPs) have been tied to numerous diseases, including cancers, and have become valuable cancer biomarkers. There’s a notable gap in studies probing the influence of SNPs on <i>MLL1</i> protein structure, function, and subsequent modifications;</p><p>(2) Methods</p><p>We utilized an array of bioinformatics tools, including PredictSNP, InterPro, ConSurf, I-Mutant2.0, MUpro, Musitedeep, Project HOPE, RegulomeDB, Mutpred2, and both CScape and CScape Somatic, to meticulously analyze the consequences of nsSNPs in the <i>MLL1</i> gene;</p><p>(3) Results</p><p>Out of 2,097 nsSNPs analyzed, 62 were determined to be significantly pathogenic by the PredictSNP tool, with ten crucial <i>MLL1</i> functional domains identified using InterPro. Additionally, 50 of these nsSNPs had high conservation scores, hinting at potential effects on protein structure and function, while 32 were found to undermine <i>MLL1</i> protein stability. Notably, four nsSNPs were deemed oncogenic, with two identified as cancer drivers. The nsSNP, D2724G, between the <i>MLL1</i> protein’s FY-rich domains, could disrupt proteolytic cleavage, altering gene expression patterns and potentially promoting cancer;</p><p>(4) Conclusions</p><p>Our research provides a comprehensive assessment of nsSNPs’ impact in the <i>MLL1</i> protein structure and function and consequently on leukemia development, suggesting potential avenues for personalized treatment, early detection, improved prognosis, and a deeper understanding of hematological malignancy genesis.</p></div>