Suk See DeRavin

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.).

Common Severe Infections in Chronic Granulomatous Disease

BACKGROUND Chronic granulomatous disease (CGD) is due to defective nicotinamide adenine dinucleotide phosphate oxidase activity and characterized by recurrent infections with a limited spectrum of bacteria and fungi as well as inflammatory complications. To understand the impact of common severe infections in CGD, we examined the records of 268 patients followed at a single center over 4 decades. METHODS All patients had confirmed diagnoses of CGD, and genotype was determined where possible. Medical records were excerpted into a standard format. Microbiologic analyses were restricted to Staphylococcus, Burkholderia, Serratia, Nocardia, and Aspergillus. RESULTS Aspergillus incidence was estimated at 2.6 cases per 100 patient-years; Burkholderia, 1.06 per 100 patient-years; Nocardia, 0.81 per 100 patient-years; Serratia, 0.98 per 100 patient-years, and severe Staphylococcus infection, 1.44 per 100 patient-years. Lung infection occurred in 87% of patients, whereas liver abscess occurred in 32%. Aspergillus incidence was 55% in the lower superoxide-producing quartiles (quartiles 1 and 2) but only 41% in the higher quartiles (rate ratio, <0.0001). Aspergillus and Serratia were somewhat more common in lower superoxide producing gp91phox deficiency. The median age at death has increased from 15.53 years before 1990 to 28.12 years in the last decade. Fungal infection carried a higher risk of mortality than bacterial infection and was the most common cause of death (55%). Gastrointestinal complications were not associated with either infection or mortality. CONCLUSIONS Fungal infections remain a major determinant of survival in CGD. X-linked patients generally had more severe disease, and this was generally in those with lower residual superoxide production. Survival in CGD has increased over the years, but infections are still major causes of morbidity and mortality.

Expansion of immunoglobulin-secreting cells and defects in B cell tolerance in Rag-dependent immunodeficiency

The contribution of B cells to the pathology of Omenn syndrome and leaky severe combined immunodeficiency (SCID) has not been previously investigated. We have studied a mut/mut mouse model of leaky SCID with a homozygous Rag1 S723C mutation that impairs, but does not abrogate, V(D)J recombination activity. In spite of a severe block at the pro–B cell stage and profound B cell lymphopenia, significant serum levels of immunoglobulin (Ig) G, IgM, IgA, and IgE and a high proportion of Ig-secreting cells were detected in mut/mut mice. Antibody responses to trinitrophenyl (TNP)-Ficoll and production of high-affinity antibodies to TNP–keyhole limpet hemocyanin were severely impaired, even after adoptive transfer of wild-type CD4+ T cells. Mut/mut mice produced high amounts of low-affinity self-reactive antibodies and showed significant lymphocytic infiltrates in peripheral tissues. Autoantibody production was associated with impaired receptor editing and increased serum B cell–activating factor (BAFF) concentrations. Autoantibodies and elevated BAFF levels were also identified in patients with Omenn syndrome and leaky SCID as a result of hypomorphic RAG mutations. These data indicate that the stochastic generation of an autoreactive B cell repertoire, which is associated with defects in central and peripheral checkpoints of B cell tolerance, is an important, previously unrecognized, aspect of immunodeficiencies associated with hypomorphic RAG mutations.

Human syndromes of immunodeficiency and dysregulation are characterized by distinct defects in T-cell receptor repertoire development

BACKGROUND Human immunodeficiencies characterized by hypomorphic mutations in critical developmental and signaling pathway genes allow for the dissection of the role of these genes in the development of the T-cell receptor (TCR) repertoire and the correlation of alterations of the TCR repertoire with diverse clinical phenotypes. OBJECTIVE The presence of T cells in patients with Omenn syndrome (OS) and patients with atypical presentations of severe combined immunodeficiency gene mutations presents an opportunity to study the effects of the causal genes on TCR repertoires and provides a window into the clinical heterogeneity observed. METHODS We performed deep sequencing of TCRβ complementarity-determining region 3 (CDR3) regions in subjects with a series of immune dysregulatory conditions caused by mutations in recombination activating gene 1/2 (RAG 1/2), IL-2 receptor γ (IL2RG), and ζ chain-associated protein kinase 70 (ZAP70); a patient with atypical DiGeorge syndrome; and healthy control subjects. RESULTS We found that patients with OS had marked reductions in TCRβ diversity compared with control subjects, as expected. Patients with atypical presentations of RAG or IL2RG mutations associated with autoimmunity and granulomatous disease did not have altered overall diversity but instead had skewed V-J pairing and skewed CDR3 amino acid use. Although germline TCRs were more abundant and clonally expanded in patients with OS, nongermline sequences were expanded as well. TCRβ from patients with RAG mutations had less junctional diversity and smaller CDR3s than patients with OS caused by other gene mutations and healthy control subjects but relatively similar CDR3 amino acid use. CONCLUSIONS High-throughput TCR sequencing of rare immune disorders has demonstrated that quantitative TCR diversity can appear normal despite qualitative changes in repertoire and strongly suggests that in human subjects RAG enzymatic function might be necessary for normal CDR3 junctional diversity.

X-linked carriers of chronic granulomatous disease: Illness, lyonization, and stability

Background: Chronic granulomatous disease (CGD) is characterized by recurrent life‐threatening bacterial and fungal infections and aberrant inflammation. Mutations in CYBB cause X‐linked CGD and account for 65% to 70% of cases in Western countries. Objective: We sought to understand the clinical manifestations associated with the X‐linked CGD carrier state. Methods: We undertook a comprehensive retrospective study of 162 affected female subjects. We examined dihydrorhodamine 123 (DHR) oxidation data for percentage of X‐chromosome inactivation. We correlated lyonization (%DHR+) with clinical features. Where possible, we followed %DHR+ values over time. Results: Clinical data were available for 93 female subjects: %DHR+ values were 46% (mean) and 47% (median; SD, 24). Using the %DHR+ value as the criterion for X inactivation, 78% of patients had levels of inactivation of 20% to 80%, suggesting random inactivation that was independent of age. In contrast, carriers with CGD‐type infections had median %DHR+ values of 8% (n = 14; range, 0.06% to 48%), and those with only autoimmune or inflammatory manifestations had median %DHR+ values of 39% (n = 31; range, 7.4% to 74%). Those with both infections and autoimmunity had low %DHR+ values (n = 6; range, 3% to 14%). A %DHR+ value of less than 10% was strongly associated with infections (odds ratio, 99). Strong association persisted when %DHR+ values were less than 20% (odds ratio, 12). Autoimmunity was not associated with %DHR+ values. In 2 sets of identical twins, the %DHR+ populations tracked closely over time. Although the %DHR+ populations were very similar between sisters, those between mothers and daughters were unrelated. Conclusions: A low %DHR+ value strongly predicts infection risk in X‐linked CGD carriers, and the carrier state itself is associated with autoimmunity.

Gastrointestinal Features of Chronic Granulomatous Disease Found During Endoscopy

BACKGROUND & AIMS Chronic granulomatous disease (CGD) is an inherited disorder of the reduced nicotinamide adenine dinucleotide phosphate oxidase complex within phagocytic cells that predisposes people to bacterial and fungal infections. Approximately 40% of patients with CGD have gastrointestinal involvement. We aimed to characterize the endoscopic features of gastrointestinal CGD and define the role of endoscopy in patients. METHODS We created a database of all patients with CGD seen at the National Institutes of Health from 1990 through 2010. We identified patients who had an endoscopy, and collected information from those with CGD-associated inflammatory bowel disease. We analyzed clinical data (demographic information and symptoms), endoscopic data (indication, preparation quality, degree of inflammation, mucosal findings, and complications), and pathologic data. RESULTS A total of 211 endoscopies (96 esophagogastroduodenoscopies, 82 colonoscopies, and 33 flexible sigmoidoscopies) were performed at the National Institutes of Health on 78 patients with CGD. Esophageal, gastric, and duodenal inflammation were detected in 21%, 74%, and 37% of patients, respectively. Esophageal dysmotility and structural abnormalities were noted in 26%. Of the patients who had colonic CGD-inflammatory bowel disease, 74% had skip lesions and 93% had anorectal disease. Enteric fistulae were found in 18% of patients; 73% of these were perianal. Colonic strictures were observed in 24% of patients; 80% were in the anorectal area. CONCLUSIONS Based on an analysis of clinical and endoscopic data from 78 patients, CGD-inflammatory bowel disease is a distinct entity, primarily involving the anus and rectum, with skip lesions in the remaining bowel. Bowel strictures and fistulae are present in a significant number of patients. Upper gastrointestinal tract inflammatory disease is common, although typically not as severe as colonic disease. Upper and lower endoscopies are important in characterizing the gastrointestinal features of CGD.

The Changing Paradigm of Management of Liver Abscesses in Chronic Granulomatous Disease

Background Chronic granulomatous disease (CGD) is a rare genetic disorder causing recurrent infections. More than one-quarter of patients develop hepatic abscesses and liver dysfunction. Recent reports suggest that disease-modifying treatment with corticosteroids is effective for these abscesses. Comparison of corticosteroid therapy to traditional invasive treatments has not been performed. Methods Records of 268 patients with CGD treated at the National Institutes of Health from 1980 to 2014 were reviewed. Patients with liver involvement and complete records were included. We recorded residual reactive oxygen intermediate (ROI) production by neutrophils, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase germline mutation status, laboratory values, imaging characteristics, time to repeat hepatic interventions, and overall survival among 3 treatment cohorts: open liver surgery (OS), percutaneous liver-directed interventional radiology therapy (IR), and high-dose corticosteroid management (CM). Results Eighty-eight of 268 patients with CGD suffered liver involvement. Twenty-six patients with a median follow-up of 15.5 years (8.5-32.9 years of follow-up) had complete records and underwent 100 standard interventions (42 IR and 58 OS). Eight patients received a treatment with high-dose corticosteroids only. There were no differences in NADPH genotype, size, or number of abscesses between patients treated with OS, IR, or CM. Time to repeat intervention was extended in OS compared with IR (18.8 vs 9.5 months, P = .04) and further increased in CM alone (median time to recurrence not met). Impaired macrophage and neutrophil function measured by ROI production correlated with shorter time to repeat intervention (r = 0.6, P = .0019). Conclusions Treatment of CGD-associated liver abscesses with corticosteroids was associated with fewer subsequent hepatic interventions and improved outcome compared to invasive treatments.

Interleukin-27 Enhances the Potential of Reactive Oxygen Species Generation from Monocyte-derived Macrophages and Dendritic cells by Induction of p47 phox

Interleukin (IL)-27, a member of the IL-12 cytokine family, plays an important and diverse role in the function of the immune system. We have previously demonstrated that IL-27 is an anti-viral cytokine which inhibits HIV-1, HIV-2, Influenza virus and herpes simplex virus infection, and enhances the potential of reactive oxygen species (ROS) generating activity during differentiation of monocytes to macrophages. In this study, we further investigated the mechanism of the enhanced potential for ROS generation by IL-27. Real time PCR, western blot and knock down assays demonstrate that IL-27 is able to enhance the potential of superoxide production not only during differentiation but also in terminally differentiated-macrophages and immature dendritic cells (iDC) in association with the induction of p47phox, a cytosolic component of the ROS producing enzyme, NADPH oxidase, and the increase in amounts of phosphorylated p47phox upon stimulation. We also demonstrate that IL-27 is able to induce extracellular superoxide dismutase during differentiation of monocytes but not in terminal differentiated macrophages. Since ROS plays an important role in a variety of inflammation, our data demonstrate that IL-27 is a potent regulator of ROS induction and may be a novel therapeutic target.

Gene Correction for SCID-X1 in Long-Term Hematopoietic Stem Cells

Gene correction in human long-term hematopoietic stem cells (LT-HSCs) could be an effective therapy for monogenic diseases of the blood and immune system. High frequencies of reproducible targeted integration of a wild-type cDNA into the endogenous start codon of a gene in LT-HSCs could provide a robust genome editing approach to cure genetic diseases in which patients have different mutations throughout the gene. We describe a clinically relevant method for correcting X-linked severe combined immunodeficiency (SCID-X1). By using a highly specific and active CRISPR/Cas9-AAV6 based strategy and selection-free approach, we achieve up to 20% genome integration frequencies in LT-HSCs of a full-length IL2RG cDNA at the endogenous start site as demonstrated by serial transplantation and analysis of genome edited human cells eight months following initial transplantation. In addition to high frequencies of functional gene correction in LT-HSCs we observed no evidence of abnormal hematopoiesis following transplantation, a functional measure of the lack of genotoxicity. Deep analysis of potential off-target activity detected two sites with low frequency (<0.3%) of off-target mutations. The level of off-target mutations was reduced to below the limit of detection using a high fidelity Cas9. Moreover, karyotype evaluation identified no genomic instability events. We achieved high levels of genome targeting frequencies (median 45%) in CD34+ HSPCs from six SCID-X1 patients and demonstrate rescue of lymphopoietic defect of patient derived cells both in vitro and in vivo. In sum, our study provides specificity, toxicity and efficacy data supportive of clinical development of genome editing to treat SCID-Xl.

57. Seamless Targeted Correction of CYBB Exon 5 Mutations Restores Granulocyte Function in X-Linked Chronic Granulomatous Disease iPSCs

X-linked chronic granulomatous disease (X-CGD) is an immune deficiency resulting from lack of production of microbicidal reactive oxygen species (ROS) by phagocytic cells. Mutations causing X-CGD can occur throughout the >30-kb CYBB gene encoding gp91phox, with the majority of patients exhibiting a single causative mutation within one of the 13 exons or adjoining intronic splice sites, resulting in a loss of gp91phox protein expression. We previously demonstrated targeted “safe-harbor” gene therapy of X-CGD in iPSCs through insertion of a codon-optimized CYBB minigene into the AAVS1 locus, resulting in constitutive gp91phox expression from a CAG promoter. Another approach targeting insertion of the codon-optimized minigene or normal CYBB cDNA to the start site of endogenous CYBB resulted in little or no gp91phox expression or ROS activity in iPSC-derived granulocytes, suggesting that regulatory elements downstream of the CYBB promoter may be required for efficient expression at this locus. In order to maintain normal regulation of gp91phox expression with minimal alterations in corrected cells, we now demonstrate a strategy for seamless targeted repair of CYBB exon 5 mutations using an exon 5-specific TALEN pair or CRISPR, along with a donor plasmid containing the corrected 146-bp exon 5 interrupted by a piggyBac transposon cassette containing a PGK promoter and puroDtk gene for positive/negative selection, and flanked by ~900-bp upstream and downstream intron sequences for homologous recombination. iPSCs from X-CGD patients with exon 5 458T>G mutation or 461A deletion were nucleofected with donor and TALEN or CRISPR expression plasmids. Both TALENs and CRISPR enabled efficient targeted insertion in 82-98% of puromycin-resistant clones, 25-65% of which contained off-target inserts, for an overall efficiency of 29-75% of selected clones containing only the targeted insert. Targeted iPSC clones lacking off-target inserts were nucleofected with “excision-only” piggyBac transposase expression plasmid and selected with ganciclovir for removal of the PGK-puroDtk cassette, leaving only a silent codon change to accommodate the TTAA sequence left behind after piggyBac excision, without additional alterations detected beyond correction of the CYBB mutation. Corrected iPSCs maintained pluripotency and upon in vitro granulocyte differentiation exhibited restoration of gp91phox expression and ROS production comparable to normal blood neutrophils (3100% of normal gp91phox expression and 85-99% of normal ROS activity by mean fluorescence intensity in DHR assay). Our findings demonstrate efficient and seamless targeted repair of exon 5 mutations in X-CGD iPSCs with an exon replacement strategy, resulting in normal regulation of gp91phox expression and functional correction of the disease defect in iPSC-derived granulocytes. The development of similar approaches for the other exons of CYBB will enable seamless repair of the majority of causative mutations found in X-CGD patients.