In silico identification and verification of deleterious gene mutations in B-cell acute lymphoblastic leukemia

Abstract

The gene is a well-known regulator of the development of lymphocytes, and genetic variation in this gene has been found repeatedly to be associated with leukemogenesis and poor prognosis in B-cell ALL. Determining the neutral and functionally deleterious single nucleotide substitutions within this gene is an important step towards the use of variants in this gene as diagnostic and/or prognosis markers. This was a non-experimental, descriptive, and analytical in silico study; it did not involve wet-laboratory experiments or clinical trials. Transcript variants of the gene were retrieved from the NCBI Nucleotide database using the Homo sapiens and RefSeq filters. FASTA sequences were analyzed using BLASTx to identify amino acid substitutions. Mutations were designated based on position and residue change (e.g., R83P). Each identified mutation was analyzed with the following tools PolyPhen-2: Predicts potential impact on protein structure/function. SIFT: Predicts whether amino acid changes affect protein function. MuPRO and I-Mutant: Predict stability changes post-mutation. PhD-SNP: Assesses disease association. Study was a computational/databased study and not performed at a clinical site. The study was conducted without any human or animal subjects, and only reference gene sequences were used that are publicly available. Predicted functional effect (benign/neutral versus damaging/deleterious) and predicted stability change of each identified amino acid substitution, output by PolyPhen-2, SIFT, MuPro, I-Mutant, and PhD-SNP. Eight amino acid substitutions were detected and confirmed: V20L, E5D, I33V, P19T, R83P, D22N, Q11H and K75E. The majority of substitutions (V20L, E5D, I33V, and P19T) were predicted to be neutral, benign, and tolerated with little effect on protein function, though several predicted a decrease in protein stability. R83P, Q11H, K75E, and D22N were identified by several tools as potentially or probably damaging and have been flagged multiple times. The majority of the identified single-nucleotide polymorphisms in were predicted to be benign despite being associated with the presence of changes in the protein's stability, while the four polymorphisms (R83P, D22N, Q11H and K75E) were consistently predicted as deleterious and could affect protein function and disease progression in B-cell acute lymphoblastic leukemia. These results emphasize the relevance of alterations for leukemogenesis and resistance to therapy, and suggest that these variants are potential diagnostic and/or prognostic markers which should be further validated in experimental studies. This was an in silico bioinformatics analysis, not a clinical trial, and was not eligible for trial registration.