Abstract
Genetic analysis of a patient with the variant cytochrome b-245-positive form of chronic granulomatous disease revealed a missense mutation resulting in a Arg54-->Ser substitution in the gp91phox subunit of cytochrome b-245. As a consequence, although no O2- is made, NADPH oxidase-associated FAD accepts electrons from NADPH in the cell-free activation system and becomes reduced. The reduced flavin exhibits normal levels of iodonitrotetrazolium violet diaphorase activity, and the patient's neutrophils exhibit high levels of intracellular oxidant production and show a low level of NBT staining in the NBT slide test. Thus, this mutation appears to render the heme center of NADPH oxidase present but nonfunctional, while leaving the flavin center fully functional.
Highlights
Genetic analysis of a patient with the variant cytochrome b_ 2415·positive form of chronic granulomatous disease revealed a missense mutation resulting in a Arg!54 - Ser substitution in the gp9]phOX subunit of cytochrome b-2415' As a consequence, no 0; is made, NADPH oxidase-associated FAD accepts electrons from
Patient A had been diagnosed with CGD at 7 years of age following a history of recurrent infections and an abnormal NBT slide test
Neutrophils and monocytes from patient A displayed a uniform population (98 -100%) of weakly staining cells. This staining pattern is similar to that observed in a very few other variant X-linked CGD patients (X91- CGD), with diminished levels (3-25%) of a functional cytochrome b_ 24 5 3 Unlike patient A, these X91CGD patients have neutrophils that are capable of diminished levels ofO; production that broadly correlate with their degree of cytochrome expression
Summary
0i, superoxide anion radical; eGO, chronic granulomatous disease; INT, p-iodonitrotetrazolium violet; NBT,nitro blue tetrazolium; OG,octyl-J3-D-glucopyranoside; DCFH-DA, 2',7' -dichlorofluorescein diacetate (2',7' -dichlorodihydrofluorescein diacetate); PVDF, polyvinylidene difluoride; PMN, polymorphonuclear neutrophil; PMA, phorbol 12-myristate 13-acetate. In addition to the membrane-bound redox centers of NADPH oxidase, there are at least three other components required for the fully active enzyme that are present in the cytosolic fraction of disrupted neutrophils. Subsequent studies using wholly or partially recombinant systems have shown that Rac (either form) is absolutely required for NADPH oxidase activity. We describe a new patient (patient A) with neutrophils that express normal levels of a cytochrome that is non functional in terms of O2 production, but retains normal dyereductase activity. By comparison with another patient (patient B) previously described by Dinauer et at. Ref. 11) whose neutrophils express a cytochrome b -245 in which there is a Pro4 15 ~ His substitution in gp p h ox , we can infer that patient A has a mutation affecting electron transfer processes subsequent to flavin reduction, whereas patient B has a mutation affecting the initial electron transfer processes preventing enzyme flavin reduction
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