The Role of CYBB Gene Mutations in Chronic Granulomatous Disease: Exploring Immunogenicity and Immune Deficiencies.

This study aims to characterise the structural impacts of missense mutations in the CYBB gene associated with Chronic Granulomatous Disease (CGD), by comparing them to wild-type CYBB and to assess their effects on protein stability and dynamics and thus on NADPH oxidase function. The goal is to elucidate the pathogenesis of CGD and inform personalised therapeutic strategies. The study used a computational approach combining data from the ClinVar database to identify and characterise CYBB missense variants. It included sequence conservation analysis, using multiple sequence alignments to identify conserved regions of the CYBB protein. DDMut, a deep-learning based stability assessment tool, was used to predict changes in Gibbs Free Energy (ΔΔG) upon mutation. Furthermore, molecular dynamics (MD) simulations were performed to investigate the stability and dynamics of CYBB and selected mutants. Root Mean Square Deviation (RMSD) and Root Mean Square Fluctuation (RMSF) data were analyzed to assess structural stability and flexibility, respectively. K-means clustering was used to group simulations. Analysis revealed numerous missense variants with varying pathogenicity classifications. Many variants classified as "likely benign" were predicted to destabilize the CYBB protein, while some "likely pathogenic" variants were predicted to increase stability. Mutations in conserved regions, including those classified as "likely benign," were found to be destabilizing. MD simulations revealed that some variants, despite maintaining overall structural similarity to the wild-type protein, induced substantial local dynamic alterations. Specific mutations within functional domains such as the transmembrane domain (e.g., S142P, R54G, A156D, I15V) and FAD and NAD-binding domains (e.g., P390L, W361R, G359R, E462A, C537F, K438R, Y476C, M465I) were predicted to be destabilizing. Some mutations outside the defined domains (C257R, F262C, I273V) were also found to be destabilizing. There are inconsistencies between ClinVar classifications and computational predictions regarding the impact of CYBB missense mutations on protein stability and dynamics. Many "likely benign" variants may have functional impacts and some "likely pathogenic" variants might not destabilize the protein. The study highlights the importance of using multiple computational approaches, including structural and dynamic analysis, to assess the impact of mutations in the CYBB gene. These findings contribute to a better understanding of the molecular mechanisms of CGD and will help in the development of more accurate diagnostic and therapeutic strategies. Experimental validation of these predictions is needed to further confirm these findings.