Debra A. Long Priel

Residual NADPH Oxidase and Survival in Chronic Granulomatous Disease

BACKGROUND Failure to generate phagocyte-derived superoxide and related reactive oxygen intermediates (ROIs) is the major defect in chronic granulomatous disease, causing recurrent infections and granulomatous complications. 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. We hypothesized that residual ROI production might be linked to survival in patients with chronic granulomatous disease. METHODS We assessed the risks of illness and death among 287 patients with chronic granulomatous disease from 244 kindreds. Residual ROI production was measured with the use of superoxide-dependent ferricytochrome c reduction and flow cytometry with dihydrorhodamine oxidation assays. Expression of NADPH oxidase component protein was detected by means of immunoblotting, and the affected genes were sequenced to identify causal mutations. RESULTS Survival of patients with chronic granulomatous disease was strongly associated with residual ROI production as a continuous variable, independently of the specific gene affected. Patients with mutations in p47(phox) and most missense mutations in gp91(phox) (with the exception of missense mutations in the nucleotide-binding and heme-binding domains) had more residual ROI production than patients with nonsense, frameshift, splice, or deletion mutations in gp91(phox). After adolescence, mortality curves diverged according to the extent of residual ROI production. CONCLUSIONS Patients with chronic granulomatous disease and modest residual production of ROI have significantly less severe illness and a greater likelihood of long-term survival than patients with little residual ROI production. The production of residual ROI is predicted by the specific NADPH oxidase mutation, regardless of the specific gene affected, and it is a predictor of survival in patients with chronic granulomatous disease. (Funded by the National Institutes of Health.).

Retrovirus gene therapy for X-linked chronic granulomatous disease can achieve stable long-term correction of oxidase activity in peripheral blood neutrophils.

Chronic granulomatous disease (CGD) is associated with significant morbidity and mortality from infection. The first CGD gene therapy trial resulted in only short-term marking of 0.01% to 0.1% of neutrophils. A recent study, using busulfan conditioning and an SFFV retrovirus vector, achieved more than 20% marking in 2 patients with X-linked CGD. However, oxidase correction per marked neutrophil was less than normal and not sustained. Despite this, patients clearly benefited in that severe infections resolved. As such, we initiated a gene therapy trial for X-CGD to treat severe infections unresponsive to conventional therapy. We treated 3 adult patients using busulfan conditioning and an MFGS retroviral vector encoding gp91(phox), achieving early marking of 26%, 5%, and 4% of neutrophils, respectively, with sustained long-term marking of 1.1% and 0.03% of neutrophils in 2 of the patients. Gene-marked neutrophils have sustained full correction of oxidase activity for 34 and 11 months, respectively, with full or partial resolution of infection in those 2 patients. Gene marking is polyclonal with no clonal dominance. We conclude that busulfan conditioning together with an MFGS vector is capable of achieving long-term correction of neutrophil oxidase function sufficient to provide benefit in management of severe infection. This study was registered at www.clinicaltrials.gov as #NCT00394316.

Signal transducer and activator of transcription 1 (STAT1) gain-of-function mutations and disseminated coccidioidomycosis and histoplasmosis.

BACKGROUND Impaired signaling in the IFN-γ/IL-12 pathway causes susceptibility to severe disseminated infections with mycobacteria and dimorphic yeasts. Dominant gain-of-function mutations in signal transducer and activator of transcription 1 (STAT1) have been associated with chronic mucocutaneous candidiasis. OBJECTIVE We sought to identify the molecular defect in patients with disseminated dimorphic yeast infections. METHODS PBMCs, EBV-transformed B cells, and transfected U3A cell lines were studied for IFN-γ/IL-12 pathway function. STAT1 was sequenced in probands and available relatives. Interferon-induced STAT1 phosphorylation, transcriptional responses, protein-protein interactions, target gene activation, and function were investigated. RESULTS We identified 5 patients with disseminated Coccidioides immitis or Histoplasma capsulatum with heterozygous missense mutations in the STAT1 coiled-coil or DNA-binding domains. These are dominant gain-of-function mutations causing enhanced STAT1 phosphorylation, delayed dephosphorylation, enhanced DNA binding and transactivation, and enhanced interaction with protein inhibitor of activated STAT1. The mutations caused enhanced IFN-γ-induced gene expression, but we found impaired responses to IFN-γ restimulation. CONCLUSION Gain-of-function mutations in STAT1 predispose to invasive, severe, disseminated dimorphic yeast infections, likely through aberrant regulation of IFN-γ-mediated inflammation.

The CXCR4 antagonist plerixafor corrects panleukopenia in patients with WHIM syndrome

WHIM syndrome is a rare congenital immunodeficiency disorder characterized by warts, hypogammaglobulinemia, infections, and myelokathexis (neutropenia because of impaired egress from the BM); most patients also have severe panleukopenia. Because WHIM syndrome is caused by mutations in the chemokine receptor CXCR4 that result in increased agonist-dependent signaling, we hypothesized that the CXCR4 antagonist plerixafor (Mozobil [Genyzme Corporation], AMD3100), might be an effective treatment. To test this, we enrolled 3 unrelated adult patients with the most common WHIM mutation, CXCR4(R334X), in a phase 1 dose-escalation study. Plerixafor increased absolute lymphocyte, monocyte, and neutrophil counts in blood to normal without significant side effects in all 3 patients. Peak responses occurred at 3-12 hours after injection and waned by 24 hours after injection which tracked the drugs pharmacokinetics. All 3 cell types increased in a dose-dependent manner with the rank order of responsiveness absolute lymphocyte > monocyte > neutrophil. These data provide the first pharmacologic evidence that panleukopenia in WHIM syndrome is caused by CXCL12-CXCR4 signaling-dependent leukocyte sequestration, and support continued study of plerixafor as mechanism-based therapy in this disease. This study is registered at http://www.clinicaltrials.gov as NCT00967785.

Loss-of-function of the protein kinase C δ (PKCδ) causes a B-cell lymphoproliferative syndrome in humans

Defective lymphocyte apoptosis results in chronic lymphadenopathy and/or splenomegaly associated with autoimmune phenomena. The prototype for human apoptosis disorders is the autoimmune lymphoproliferative syndrome (ALPS), which is caused by mutations in the FAS apoptotic pathway. Recently, patients with an ALPS-like disease called RAS-associated autoimmune leukoproliferative disorder, in which somatic mutations in NRAS or KRAS are found, also were described. Despite this progress, many patients with ALPS-like disease remain undefined genetically. We identified a homozygous, loss-of-function mutation in PRKCD (PKCδ) in a patient who presented with chronic lymphadenopathy, splenomegaly, autoantibodies, elevated immunoglobulins and natural killer dysfunction associated with chronic, low-grade Epstein-Barr virus infection. This mutation markedly decreased protein expression and resulted in ex vivo B-cell hyperproliferation, a phenotype similar to that of the PKCδ knockout mouse. Lymph nodes showed intense follicular hyperplasia, also mirroring the mouse model. Immunophenotyping of circulating lymphocytes demonstrated expansion of CD5+CD20+ B cells. Knockdown of PKCδ in normal mononuclear cells recapitulated the B-cell hyperproliferative phenotype in vitro. Reconstitution of PKCδ in patient-derived EBV-transformed B-cell lines partially restored phorbol-12-myristate-13-acetate-induced cell death. In summary, homozygous PRKCD mutation results in B-cell hyperproliferation and defective apoptosis with consequent lymphocyte accumulation and autoantibody production in humans, and disrupts natural killer cell function.

Anti-CD20 (rituximab) therapy for anti-IFN-γ autoantibody-associated nontuberculous mycobacterial infection

Patients with anti-IFN-γ autoantibodies have impaired IFN-γ signaling, leading to severe disseminated infections with intracellular pathogens, especially nontuberculous mycobacteria. Disease may be severe and progressive, despite aggressive treatment. To address the underlying pathogenic IFN-γ autoantibodies we used the therapeutic monoclonal rituximab (anti-CD20) to target patient B cells. All subjects received between 8 and 12 doses of rituximab within the first year to maintain disease remission. Subsequent doses were given for relapsed infection. We report 4 patients with refractory disease treated with rituximab who had clinical and laboratory evidence of therapeutic response as determined by clearance of infection, resolution of inflammation, reduction of anti-IFN-γ autoantibody levels, and improved IFN-γ signaling.

Severe congenital neutropenia resulting from G6PC3 deficiency with increased neutrophil CXCR4 expression and myelokathexis

Mutations in more than 15 genes are now known to cause severe congenital neutropenia (SCN); however, the pathologic mechanisms of most genetic defects are not fully defined. Deficiency of G6PC3, a glucose-6-phosphatase, causes a rare multisystem syndrome with SCN first described in 2009. We identified a family with 2 children with homozygous G6PC3 G260R mutations, a loss of enzymatic function, and typical syndrome features with the exception that their bone marrow biopsy pathology revealed abundant neutrophils consistent with myelokathexis. This pathologic finding is a hallmark of another type of SCN, WHIM syndrome, which is caused by gain-of-function mutations in CXCR4, a chemokine receptor and known neutrophil bone marrow retention factor. We found markedly increased CXCR4 expression on neutrophils from both our G6PC3-deficient patients and G6pc3(-/-) mice. In both patients, granulocyte colony-stimulating factor treatment normalized CXCR4 expression and neutrophil counts. In G6pc3(-/-) mice, the specific CXCR4 antagonist AMD3100 rapidly reversed neutropenia. Thus, myelokathexis associated with abnormally high neutrophil CXCR4 expression may contribute to neutropenia in G6PC3 deficiency and responds well to granulocyte colony-stimulating factor.

Glycosylation, Hypogammaglobulinemia, and Resistance to Viral Infections

Genetic defects in MOGS, the gene encoding mannosyl-oligosaccharide glucosidase (the first enzyme in the processing pathway of N-linked oligosaccharide), cause the rare congenital disorder of glycosylation type IIb (CDG-IIb), also known as MOGS-CDG. MOGS is expressed in the endoplasmic reticulum and is involved in the trimming of N-glycans. We evaluated two siblings with CDG-IIb who presented with multiple neurologic complications and a paradoxical immunologic phenotype characterized by severe hypogammaglobulinemia but limited clinical evidence of an infectious diathesis. A shortened immunoglobulin half-life was determined to be the mechanism underlying the hypogammaglobulinemia. Impaired viral replication and cellular entry may explain a decreased susceptibility to infections.

An AAVS1-Targeted Minigene Platform for Correction of iPSCs From All Five Types of Chronic Granulomatous Disease

There are five genetic forms of chronic granulomatous disease (CGD), resulting from mutations in any of five subunits of phagocyte oxidase, an enzyme complex in neutrophils, monocytes, and macrophages that produces microbicidal reactive oxygen species. We generated induced pluripotent stem cells (iPSCs) from peripheral blood CD34(+) hematopoietic stem cells of patients with each of five CGD genotypes. We used zinc finger nuclease (ZFN) targeting the AAVS1 safe harbor site together with CGD genotype-specific minigene plasmids with flanking AAVS1 sequence to target correction of iPSC representing each form of CGD. We achieved targeted insertion with constitutive expression of desired oxidase subunit in 70-80% of selected iPSC clones. Neutrophils and macrophages differentiated from corrected CGD iPSCs demonstrated restored oxidase activity and antimicrobial function against CGD bacterial pathogens Staphylococcus aureus and Granulibacter bethesdensis. Using a standard platform that combines iPSC generation from peripheral blood CD34(+) cells and ZFN mediated AAVS1 safe harbor minigene targeting, we demonstrate efficient generation of genetically corrected iPSCs using an identical approach for all five genetic forms of CGD. This safe harbor minigene targeting platform is broadly applicable to a wide range of inherited single gene metabolic disorders.

Assessment of Atherosclerosis in Chronic Granulomatous Disease

Background— Patients with chronic granulomatous disease (CGD) experience immunodeficiency because of defects in the phagocyte NADPH oxidase and the concomitant reduction in reactive oxygen intermediates. This may result in a reduction in atherosclerotic injury. Methods and Results— We prospectively assessed the prevalence of cardiovascular risk factors, biomarkers of inflammation and neutrophil activation, and the presence of magnetic resonance imaging and computed tomography quantified subclinical atherosclerosis in the carotid and coronary arteries of 41 patients with CGD and 25 healthy controls in the same age range. Univariable and multivariable associations among risk factors, inflammatory markers, and atherosclerosis burden were assessed. Patients with CGD had significant elevations in traditional risk factors and inflammatory markers compared with control subjects, including hypertension, high-sensitivity C-reactive protein, oxidized low-density lipoprotein, and low high-density lipoprotein. Despite this, patients with CGD had a 22% lower internal carotid artery wall volume compared with control subjects (361.3±76.4 mm3 versus 463.5±104.7 mm3; P<0.001). This difference was comparable in p47phox- and gp91phox-deficient subtypes of CGD and independent of risk factors in multivariate regression analysis. In contrast, the prevalence of coronary arterial calcification was similar between patients with CGD and control subjects (14.6%, CGD; 6.3%, controls; P=0.39). Conclusions— The observation by magnetic resonance imaging and computerized tomography of reduced carotid but not coronary artery atherosclerosis in patients with CGD despite the high prevalence of traditional risk factors raises questions about the role of NADPH oxidase in the pathogenesis of clinically significant atherosclerosis. Additional high-resolution studies in multiple vascular beds are required to address the therapeutic potential of NADPH oxidase inhibition in cardiovascular diseases. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: NCT01063309.