The objective of this study was to provide the first genomic diagnosis of chronic granulomatous disease (CGD) in a referral hospital in Southern Brazil, a rare and underdiagnosed disease with limited data in the country. Six male patients (P1-P6) were clinically and genetically analyzed, through medical reports review and massively parallel sequencing by a panel for the CYBB, CYBA, NCF1, NCF2, and NCF4 genes and whole genome sequencing. The gene-scan technique was used to detect the Tyr26HisfsTer variant (ΔGT) in NCF1 and to distinguish it of its pseudogenes (ΨNCF1), which naturally have ΔGT. Variants were classified according to the American College of Medical Genetics and Genomics guidelines. Structural modelling was performed for missense variants using PyMOL and I-TASSER to verify their potential impact on the NADPH oxidase complex, which is defective in CGD. Among the clinical manifestations, the most commonly affected organs were the lungs, skin, and lymph nodes, with all patients presenting with recurrent infections and pneumonia. Adverse reactions to BCG vaccination were observed in two patients. Four patients carried variants in CYBB: (P1) p.Cys257Ser, a novel variant, and (P2) p.Cys257Arg, both classified as likely pathogenic and predicted to significantly affect the structure of NADPH oxidase, with Gibbs free energy values of 6.66 and 6.23 kcal/mol, respectively (reference value: >1.6 kcal/mol); p.Arg157Ter (P3), and p.Trp483Ter (P4), both classified as pathogenic and predicted to undergo nonsense-mediated mRNA decay. Gene-scan analysis revealed the ΔGT in two siblings: P5 (homozygous) and P6 (heterozygous). It was hypothesized that P6 may have an NCF1-related pseudogene lacking the ΔGT originated by unequal recombination with NCF1, resulting in the absence of functional alleles in P6. This study underscores the importance of genetic characterization for accurate diagnosis, reducing diagnosis odysseys and supporting the indication of bone marrow transplantation to prevent fatal outcomes, and reinforcing the contraindication of Bacillus Calmette–Guérin vaccination in patients with CGD.
