Persistence of the bacterial pathogen Granulibacter bethesdensis in chronic granulomatous disease monocytes and macrophages lacking a functional NADPH oxidase.
We, for the first time, detected somatic mosaicism in two unrelated male patients with X-CGD caused by de novo nonsense mutations (p.Gly223X and p.Glu462X) in the CYBB gene.
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.
Among the seven foetuses whose mothers were X-CGD carriers, four foetuses (Family 2, 4, 5 and 7) had CYBB gene mutations and showed very low hydrogen peroxide generation (stimulation index < 10) and no gp91(phox) expression.
High-performance liquid chromatography under partially denaturing conditions (dHPLC) is a fast and cost-effective method for screening molecular defects: four novel mutations found in X-linked chronic granulomatous disease.
Germline mutations in CYBB, the human gene encoding the gp91(phox) subunit of the phagocyte NADPH oxidase, impair the respiratory burst of all types of phagocytes and result in X-linked chronic granulomatous disease (CGD).
Zinc finger nuclease-mediated gene targeting of a single-copy gp91(phox) therapeutic minigene into one allele of the "safe harbor" AAVS1 locus in X-CGD iPSCs without off-target inserts resulted in sustained expression of gp91(phox) and substantially restored neutrophil ROS production.
We further demonstrate that additive gene transfer using lentiviral vectors encoding gp91(phox) is capable of restoring NADPH-oxidase activity in mature neutrophils derived from X-CGD iPSC.
These findings were recapitulated in human neutrophils derived from transduced X-CGD CD34(+) cells in vivo, and suggest that the chimeric promoter will have utility for gene therapy of myeloid lineage disorders such as CGD.
Chronic granulomatous disease is caused by missense, nonsense, frameshift, splice, or deletion mutations in the genes for p22(phox), p40(phox), p47(phox), p67(phox) (autosomal chronic granulomatous disease), or gp91(phox) (X-linked chronic granulomatous disease), which result in variable production of neutrophil-derived ROIs.
For each step in the protocol, a general overview of principles is provided, followed by an in depth analysis of one example, scanning of CYBB, the gene that is mutated in X-linked chronic granulomatous disease.
Superoxide release and CYBB gene expression by EDA-ID cells were significantly decreased compared with healthy cells and similar to cells from patients with X-linked chronic granulomatous disease (X91(0) CGD).