In Silico Analysis of Missense Mutations as a First Step in Functional Studies: Examples from Two Sphingolipidoses

Abstract

In order to delineate a better approach to functional studies, we have selected 23 missense mutations distributed in different domains of two lysosomal enzymes, to be studied by in silico analysis. In silico analysis of mutations relies on computational modeling to predict their effects. Various computational platforms are currently available to check the probable causality of mutations encountered in patients at the protein and at the RNA levels. In this work we used four different platforms freely available online (Protein Variation Effect Analyzer- PROVEAN, PolyPhen-2, Swiss-model Expert Protein Analysis System—ExPASy, and SNAP2) to check amino acid substitutions and their effect at the protein level. The existence of functional studies, regarding the amino acid substitutions, led to the selection of the distinct protein mutants. Functional data were used to compare the results obtained with different bioinformatics tools. With the advent of next-generation sequencing, it is not feasible to carry out functional tests in all the variants detected. In silico analysis seems to be useful for the delineation of which mutants are worth studying through functional studies. Therefore, prediction of the mutation impact at the protein level, applying computational analysis, confers the means to rapidly provide a prognosis value to genotyping results, making it potentially valuable for patient care as well as research purposes. The present work points to the need to carry out functional studies in mutations that might look neutral. Moreover, it should be noted that single nucleotide polymorphisms (SNPs), occurring in coding and non-coding regions, may lead to RNA alterations and should be systematically verified. Functional studies can gain from a preliminary multi-step approach, such as the one proposed here.

Is the alpha‐galactosidase A variant p.Asp313Tyr (p.D313Y) pathogenic for Fabry disease? A systematic review

“…Later, the same patient was also identified as carrying a second missense disease causing variant, the p.Cys172Gly (c.514T>G) variant, questioning the pathogenetic role of p.Asp313Tyr 9 . On the other hand, the p.Asp313Tyr silico analysis identified the variant as deleterious or probably damaging, adding to the controversy 10‐13 . The identification of pathogenic variants plays a central role in the establishment of a definite FD diagnosis.…”

Assessing Lysosomal Disorders in the NGS Era: Identification of Novel Rare Variants

“…The effect of this variant has been addressed by a number of teams and it has already been demonstrated in vitro that this particular amino acidic substitution leads to an unstable protein, with 5.7–9% of wt GCase activity (reviewed in Duarte A.J. et al [ 34 ]).…”

“…Importantly, this sort of studies, while being a valuable option for immediate guidance regarding patient care and counseling, are relatively fast and inexpensive. In fact, attentive in silico analysis may confer an indication of prognosis value to the genotyping results [ 34 ].…”

The Impact of Mutations on the Pathogenic and Antigenic Activity of SARS-CoV-2 during the First Wave of the COVID-19 Pandemic: A Comprehensive Immunoinformatics Analysis

“…The effect of such mutations on the structure and function of the respective viral proteins is important to predict the evolutionary potential of the viral proteins. However, in silico prediction of the impact of amino acid variants on proteins' structure and function may, sometimes, be considered as an alternative to or a pre-study indicator of in vitro expression level studies [22]. In addition, the interpretation of the proteomic variants in light of their phenotypic effects is one of the emerging crucial tasks we have to perform in order to advance our understanding of how these variants affect SARS-CoV-2 proteins structural and functional behavior.…”