Identification and functional characterization of two novel mutations in the α-helical loop (residues 484-503) of CYBB/gp91(phox) resulting in the rare X91(+) variant of chronic granulomatous disease.

Chronic granulomatous disease (CGD) is mainly caused by mutations in X-linked CYBB that encodes gp91. We have identified two novel mutations in CYBB resulting in the rare X91(+)-CGD variant, c.1500T>G (p.Asp500Glu) in two male siblings and c.1463C>A (p.Ala488Asp) in an unrelated male. Zymosan and/or PMA (Phorbol 12-myristate 13-acetate)-induced recruitment of p47(phox) and p67(phox) to the membrane fraction was normal for both mutants. Cell-free assays using recombinant wild-type and the mutant proteins revealed that these mutants were not activated by NADPH (nicotinamide adenine dinucleotide phosphate). Interestingly, the Ala488Asp mutant was activated by NADPH in the presence of glutathione. These data suggest that the mutations prevented NADPH from binding to gp91(phox) and the requirement of a negative charge at residue 500 in gp91(phox) for NADPH oxidase assembly, in contrast to a previously described Asp500Gly change. These mutations and the effect of glutathione provide a unique insight into disease pathogenesis and potential therapy in variant X91(+)-CGD.