Clinical Information

Chronic granulomatous disease (CGD) is caused by genetic defects in the gene components that encode the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme complex. These defects result in an inability to produce superoxide anions required for killing of bacterial and fungal organisms. Other clinical features include a predisposition to systemic granulomatous complications and autoimmunity.(1) There are 5 known genetic defects associated with the clinical phenotype of CGD.(2) The gene defects include mutations in the CYBB gene, encoding the gp91phox protein, which is X-linked and accounts for approximately 70% of CGD cases. Other gene defects are autosomal recessive: NCF1 (p47phox), NCF2 (p67phox), CYBA (p22phox), and NCF4 (p40phox). Typically, patients with X-linked CGD have the most severe disease, while patients with p47phox defects tend to have the best outcomes. Mutations in NCF4 encoding the p40phox protein have been the most recently described (3) and appears to be associated with more gastrointestinal disease with fewer infections. There is significant clinical variability even among individuals with similar mutations, in terms of NADPH oxidase function, indicating that there can be several modulating factors including the genetic defect, infection history, and granulomatous and autoimmune complications. There appears to be a correlation between very low NADPH superoxide production and worse outcomes. CGD can be treated with hematopoietic cell transplantation (HCT), which can be effective for the inflammatory and autoimmune manifestations.

It has been shown that survival of patients with CGD was strongly associated with residual reactive oxygen intermediate (ROI) production, independent of the specific gene defect.(4) Measurement of NADPH oxidase activity through the dihydrorhodamine (DHR) flow cytometry assay contributed to the assessment of ROI. The diagnostic laboratory assessment for CGD includes evaluation of NADPH oxidase function in neutrophils, using either the nitroblue tetrazolium test (NBT) or the more analytically sensitive DHR test, as described here. Activation of neutrophils with phorbol myristate acetate (PMA) results in oxidation of DHR to a fluorescent compound, rhodamine 123, which can be measured by flow cytometry. Flow cytometry can distinguish between the different genetic forms of CGD.(5, 6) Complete myeloperoxidase (MPO) deficiency can cause a false-positive result for CGD in the DHR flow cytometric assay (7); however, there is a difference between the % DHR+ neutrophils and the mean fluorescence intensity (MFI) after PMA stimulation that allows discrimination between true X-linked CGD and complete MPO deficiency. Further, the addition of recombinant human MPO enhances the DHR signal in MPO-deficient neutrophils but not in CGD neutrophils.(7)

It is important to have quantitative measures in the DHR flow cytometry assay to effectively use the test for diagnosis of the different forms of CGD as well as for monitoring chimerism and NADPH oxidase activity post-HCT. These quantitative measures include assessment of the relative proportion (%) of neutrophils that are positive for DHR fluorescence after PMA stimulation and the relative fluorescence intensity of DHR (MFI) on neutrophils after activation. This assay can also be used for the diagnostic evaluation of Rac2 deficiency, which is a neutrophil defect that causes profound neutrophil dysfunction with decreased chemotaxis, polarization, superoxide anion production, azurophilic granule secretion. This disease is caused by inhibitory mutations in the RAC2 gene, which encodes a Rho family GTPase essential to neutrophil activation and NADPH oxidase function.(8) Patients with Rac2 deficiency have been shown to have normal neutrophil oxidative burst when stimulated with PMA, indicating normal NADPH oxidase activity, but abnormal neutrophil responses to N-formyl-methionyl-leucyl-phenylalanine (fMLP), which is a physiological activator of neutrophils. The defective oxidative burst to fMLP, but not to PMA, indicates a signaling defect in Rac2 deficiency.(9)

Female carriers of X-linked CGD can become symptomatic for CGD due to skewed lyonization (X chromosome inactivation).(10) Age-related acquired skewing of lyonization can also cause increased susceptibility to infections in carriers of X-linked CGD.(11) While germline mutations are more common in CGD, there have been reports of de novo, sporadic mutations in the CYBB gene, causing X-linked CGD in male patients whose mothers are not carriers for the affected allele. Additionally, somatic mosaicism has been reported in patients with X-linked CGD who have small populations of normal cells.(12) There are also reports of triple somatic mosaicism in female carriers (13,14) as well as late-onset disease in an adult female who was a somatic mosaic for a novel mutation in the CYBB gene.(15)

Therefore, the clinical, genetic, and age spectrum of CGD is varied and laboratory assessment of NADPH oxidase activity after neutrophil stimulation, coupled with appropriate interpretation, is critical to achieving an accurate diagnosis or for monitoring patients posttransplant.