BackgroundThe Alternative Lengthening of Telomeres (ALT) pathway represents a non-canonical mechanism of telomere maintenance that operates independently of the conventional telomerase activity. The three biologically significant proteins, designated as SMARCAL1 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A-like protein 1), DAXX (Death domain-associated protein 6) and ATRX (alpha-thalassemia/mental retardation, X-linked) are associated with ALT in certain cancer types. The purpose of this study was to identify the most high-risk nsSNPs (non-synonymous Single Nucleotide Polymorphisms) within these three genes and assess their impacts on the structure and function of the proteins they encode. MethodsThe reported genetic polymorphisms of SMARCAL1, DAXX and ATRX genes were retrieved from the Ensembl database. Later, various computational tools like PROVEAN, PolyPhen2, SNPs and GO, SNAP2, Predict-SNP, Panther and PMut were used to predict the most deleterious nsSNPs. MutPred was used to understand the underlying molecular reasons of those nsSNPs being deleterious, followed by prediction of Post Translational Modification Sites (PTMs) using ModPred. I-Mutant and MUpro were used to predict the effect of SNP on energy stability. Later, 3D clustering analysis was done using Mutation 3D server. Moreover, ConSurf was utilized to identify the conservation scores of wild-type amino acids. Additionally, the NCBI conserved domain search tool was employed to pinpoint conserved domains within these three proteins. Project-Hope helped for biophysical validation, followed by prediction of these genes’ interaction and function by using GeneMANIA. ResultAnalysis on SMARCAL1 protein revealed that among 665 nsSNPs, four were identified as the most deleterious: L578S, T581S, P582A, and P582S. Similarly, within the DAXX protein, among a pool of 480 nsSNPs, P284S, R230C, and R230S were found out to be the most deleterious variants. In case of ATRX protein, V178D, R246C, and V277G, from the total of 1009 nsSNPs, were predicted to be the most deleterious. All these nsSNPs were found to occur at residue positions that are 100 % conserved within protein domains and were predicted to be most damaging from both structural and functional perspectives and highly destabilizing to their corresponding proteins. ConclusionComputational investigation on the 3 proteins-SMARCAL1, DAXX and ATRX through different bioinformatics analysis tools concludes that the identified high risk nsSNPs of these proteins are pathogenic SNPs. These variants potentially exert functional and structural influences, thus making them valuable candidates for future genetic studies.