Autosomal Recessive Chronic Granulomatous Disease Research Articles

Clinical and molecular significance of flow cytometric analysis for reactive oxygen species production and residual p67phox expression in p67phox-deficient chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency disease caused by molecular defects in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. p67phox-CGD is an autosomal recessive CGD, which is caused by a defect in the cytosolic components of NADPH oxidase, p67phox, encoded by NCF2. We previously established a flow cytometric analysis for p67phox expression, which allows accurate assessment of residual protein expression in p67phox-CGD. We evaluated the correlation between oxidase function and p67phox expression, and assessed the relevancy to genotypes and clinical phenotypes in 11 patients with p67phox-CGD. Reactive oxygen species (ROS) production by granulocytes was evaluated using dihydrorhodamine-1,2,3 (DHR) assays. p67phox expression was evaluated in the monocyte population. DHR activity and p67phox expression were significantly correlated (r = 0.718, p < 0.0162). Additionally, DHR activity and p67phox expression were significantly higher in patients carrying one missense variant in combination with one nonsense or frameshift variant in the NCF2 gene than in patients with only null variants. The available clinical parameters of our patients (i.e., age at disease onset, number of infectious episodes, and each infection complication) were not linked with DHR activity or p67phox expression levels. In summary, our flow cytometric analysis revealed a significant correlation between residual ROS production and p67phox expression. More deleterious NCF2 genotypes were associated with lower levels of DHR activity and p67phox expression. DHR assays and protein expression analysis by using flow cytometry may be relevant strategies for predicting the genotypes of p67phox-CGD.

Prime Editing Efficiently and Precisely Corrects Causative Mutation in Chronic Granulomatous Disease, Restoring Myeloid Function: Toward Development of a Prime Edited Autologous Hematopoietic Stem Cell Therapy

Chronic granulomatous disease (CGD) is an inherited primary immunodeficiency characterized by a lack of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in phagocytic myeloid cells such as neutrophils. NADPH oxidase produces reactive oxygen species (ROS) that directly kill bacteria and fungi to control infection. CGD patients lack this ability to control infection, which results in chronic and severe infections, inflammation, autoimmunity, poor quality of life, and reduced lifespan. Current standard care for CGD is antimicrobial prophylaxis and aggressive management of infection. For patients with a suitable human leukocyte antigen (HLA) matched donor, an allogeneic hematopoietic stem cell (HSC) transplant (allo-HSCT) offers a curative approach. However, many patients do not have access to HLA matched donors and allo-HSCT also carries a risk of GvHD and graft rejection. P47phox-deficient CGD, the most common autosomal recessive CGD, is caused by mutations in the NCF1 gene, which encodes the p47phox protein, a subunit of the NADPH oxidase complex. Approximately 80% of p47phox-deficient CGD patients carry the same homozygous mutation, a 2 nucleotide GT deletion (delGT) in exon 2 of NCF1 which is correctable using Prime Editing. Transplantation of Prime Edited patient autologous CD34 + cells, in which the delGT mutation has been corrected with Prime Editing, may provide a curative treatment for p47phox-deficient CGD patients. To evaluate whether correction of the delGT NCF1 mutation in CGD patient CD34 + cells restores p47phox protein expression and NADPH oxidase activity in myeloid progeny, Prime Editors were designed to correct this mutation precisely and efficiently. The Prime Editors were delivered to CD34 + cells from two p47phox-deficient CGD patients homozygous for delGT NCF1. In both studies, ≧75% of Prime Edited patient CD34 + cells carried at least 1 corrected allele as determined by ddPCR and DNA sequencing analyses. Myeloid progeny differentiated from Prime Edited patient CD34 + cells had restored p47phox protein expression and NADPH oxidase activity to ~80% of normal healthy donors as determined by multiple ROS detection assays. To evaluate reproducibility, Prime Editors were delivered to CD34 + cells from 6 different healthy donors. On average, greater than 90% of CD34 + cells were Prime Edited across 6 donors in independent experiments. To confirm that Prime Edited CD34 + cells retain stem cell multipotency, engraftment, and hematopoietic reconstitution properties, Prime Edited and mock treated CD34 + cells from three different donors were transplanted into immunodeficient mice. Sixteen weeks after CD34 + cell transplantation, &amp;gt;87% of long-term hematopoietic stem cells (LT-HSC) that repopulated the bone marrow had at least 1 allele corrected. There were no significant differences between ‘electroporation only’ mock treated and Prime Editor electroporated human CD34 + cells with respect to long-term CD34 + cell engraftment, human CD45 + blood cell chimerism, hematopoietic multilineage blood cell reconstitution and biodistribution, or LT-HSC cell potency. Across these studies, &amp;gt;95% human CD45 + cell chimerism was achieved with Prime Edited CD34 + cells. Despite high on-target correction of the delGT mutation in NCF1, off-target editing, unintended editing, and chromosomal rearrangements (larger deletions or translocation) events were not detected using a robust suite of genome-wide assays. In summary, Prime Editing corrects a mutation that causes the most common autosomal recessive from of CGD; correction of this delGT mutation at NCF1 restores NADPH oxidase activity and function in myeloid progeny of Prime Edited CGD CD34 + cells; and Prime Edited long-term HSCs retain multipotency and functionality in vivo. These results support the development of a Prime Edited CGD patient autologous CD34 + cell therapy as a potential curative approach for p47phox-deficient CGD patients.

Chronic granulomatous disease in the United Arab Emirates: clinical and molecular characteristics in a single center.

Chronic granulomatous disease (CGD) is a genetic disorder caused by defective oxidative burst within phagocytes, manifesting as recurrent, severe infections as well as hyperinflammation. This is the first report from the United Arab Emirates (UAE) to describe the demographic, clinical, laboratory, radiological, and genetic characteristics of patients with CGD. This is a retrospective study that was conducted at Tawam Hospital in the UAE on patients with confirmed CGD between 2017 and 2022. A total of 14 patients were diagnosed with CGD, of whom 13 patients had autosomal recessive (AR) CGD due to NCF1 deficiency. Consanguinity was noted in all patients with AR CGD, whereas positive family history was identified in 50% of cases. The median age of onset of symptoms was 24 months, while the median age at diagnosis was 72 months. Lymphadenitis was the most common clinical feature identified in 71% of patients. Other common infectious manifestations included abscess formation (57%), pneumonia (50%), invasive aspergillosis (21%), oral thrush (14%), and sepsis (14%). Disseminated trichosporonosis was reported in one patient. Autoimmune and inflammatory manifestations included celiac disease in two patients, diabetes mellitus and asymptomatic colitis in one patient each. Genetic analysis was performed in all patients; NCF1 deficiency was diagnosed in 13 (93%) patients, with c.579G>A being the most prevalent pathogenic variant identified. The treatment modalities, as well as treatment of acute infections, treatment modalities included antimicrobial prophylaxis in 12 (86%) patients and hematopoietic stem cell transplant in six patients (42%). This is the first report from the UAE describing the clinical and molecular characteristics of patients with CGD. The homozygous variant c.579G>A causing NCF1 deficiency can be considered as a founder mutation for AR CGD in the UAE.

Discoid lupus erythematosus associated with chronic granulomatous disease

To the Editor, Chronic granulomatous disease (CGD) is a primary immunodeficiency disorder in which a reduced activity of nicotinamide dinucleotide phosphate (NAPDH) oxidase leads to defective reactive oxygen intermediates (ROS) and impairs intracellular killing of microorganisms. Patients have recurrent bacterial and fungal infections as well as granuloma formations, and have a higher risk of autoimmune and inflammatory disorders like lupus erythematosus.1 A 20-year-old caucasian male with a diagnosis of autosomal recessive CGD was referred to our hospital because of a pruritic photosensitive rash on the face and hands, present for more than a year. [...]

Detection of CYBA Gene Expression by Reverse Transcription PCR as a Diagnostic Tool for Autosomal Recessive Chronic Granulomatous Disease in a Sample of Egyptian Children

Abstract Chronic granulomatous disease (CGD) is an inherited primary immunodeficiency disease caused by functional impairment of the nicotinamide dinucleotide phosphate (NADPH) oxidase complex in neutrophilic granulocytes and monocytes, characterized by recurrent and severe infections, dysregulated inflammation, and autoimmunity. Approximately two-thirds of patients with CGD have defects in Cytochrome Bβ (CYBB), the X-linked gene encoding glycoprotein 91 phagocytic oxidase (gp91phox). Rarer autosomal recessive (AR) forms of CGD are caused by autosomal recessive mutations in CYBA genes encoding protein 22 phagocytic oxidase (p22phox). However, the incidence of autosomal recessive CGD can be higher than X-linked CGD in countries with high rates of consanguineous marriage. In the present study we aimed to diagnose the commonest AR-type of CGD by detection of CYBA gene expression using real time RT-PCR as a cheaper diagnostic method for CGD subtypes categorization among immunodeficient children. This case-control study was conducted on 15 children provisionally diagnosed as CGD patients with DHR stimulation index using phorbol myristate acetate (PMA) &amp;lt; 30% (Group I). The study also included 12 mothers and 8 fathers of the studied patients to detect the genetic mutations in carriers, if any (Group IIa and IIb respectively), and 14 apparently healthy children as a control group (Group III). We found that cases with a fold change of CYBA gene expression less than 0.6 were considered under-expressed. At this cut-off value, from our molecularly studied subjects 1 case and 2 mothers showed under-expression of CYBA gene. In conclusion, we could establish the diagnosis of AR-form of CGD, derives from defects in the CYBA gene, which encodes p22phox of the NADPH oxidase using real time RT-PCR, without the need to use complex and expensive methodologies such as northern blot, slot blot, or genomic DNA sequencing.

Detection of NCF-1 Gene Expression as a Diagnostic Tool for Autosomal Recessive Chronic Granulomatous Disease among Egyptian Children with Phagocytic Dysfunction

Abstract Background Chronic granulomatous disease (CGD) is an inherited primary immunodeficiency disorder caused by functional impairment of the nicotinamide dinucleotide phosphate (NADPH) oxidase complex in neutrophilic granulocytes and monocytes and characterized by recurrent and severe infections, dysregulated inflammation and autoimmunity. Aim of the Work To diagnose the autosomal recessive (AR) type of CGD among a group of Egyptian pediatric patients with phagocytic dysfunction by detection of Neutrophil Cytosolic Factor-1 (NCF-1) gene expression using reverse transcription polymerase chain reaction (RT-PCR) and to correlate it with other diagnostic tests. Subjects and Methods A case-control study was conducted on 15 provisionally diagnosed CGD pediatric patients (Group I) by the use of DHR test, with the stimulation index using PMA &amp;lt; 30%. They were recruited from different university hospitals in Egypt. The study also included 12 mothers of the studied patients to detect the genetic mutations in carriers, if any, (Group II) and 14 apparently healthy children as a control group (Group III). Full medical history was taken and thorough clinical examination was done. The selected groups underwent testing for phagocytic and lytic indices, measurement of NCF-1 gene expression by real time RT-PCR. Results The comparative statistics between group I and group III as regards the phagocytic index (PI) revealed a high statistical significant difference between both groups. In addition, a statistically significant difference between both groups as regards lytic index (LI) was also revealed. Correlation studies were done between fold change of NCF-1 gene expression and each of PI, LI and DHR and showed no significant statistical correlation between these parameters in the 3 studied groups. Cases with a fold change of NCF-1 gene expression less than 0.48 (cut-off value calculated by the 25th percentile fold change of the control group), are considered having defective NCF-1 gene expression. At this cut-off value 2 out of 15 CGD cases and 3 mothers out of 12 showed NCF-1 gene under-expression. Conclusion Assessment of NCF-1 gene expression which encodes p47phox by real time RT- PCR in the present study could detect defective gene expression only among two CGD children and their mother as well as two other mothers with no correlation to other studied parameters. Since CGD is a genotypically heterogeneous disease; the findings of the present study highlight the importance of molecular testing for diagnosis after screening for NADPH oxidase protein deficiency by flow cytometry.

Non-infectious skin manifestations in patients with Chronic Granulomatous Disease: A report of 4 cases

BackgroundChronic granulomatous disease (CGD) is a monogenic inborn error of immunity caused by defects in the phagocytic NADPH oxidase complex. These defects lead to an increased risk of severe infection, autoimmunity, widespread granuloma formation and dysregulated inflammation. In individuals with CGD, immune dysregulation and autoimmune manifestations may impact almost every organ system with atypical dermatologic findings frequently reported. Case PresentationsHere we describe four cases of inflammatory skin lesions in patients with genetically confirmed CGD. Patient A is a 4-year-old male who presented with diffuse alopecia totalis preceded by a widespread skin eruption composed of polycyclic, annular, erythematous plaques and prominent facial erythema and edema. Whole exome sequencing was obtained due to concern for ectodermal dysplasia, which identified a variant in CYBB (CYBB c.901G>T; pVal301Phe). Patient B is a 4-year-old male with X-CGD (CYBB c.1016 C > A, p.Pro339His) who developed annular to polycyclic indurated red plaques on his face and arms. Patient C is a 19-year-old female with autosomal recessive CGD (NCF1 c.75_76delGT) and persistent hidradenitis suppurativa-like lesions in the axilla, inguinal areas, and popliteal fossa. Patient D is a 12-year-old female with autosomal recessive CGD (p47phox deficiency) and psoriasiform dermatitis of the scalp.Patient A had persistence of lesions that required chronic systemic corticosteroids and methotrexate. He underwent hematopoietic stem cell transplantation using a matched sibling donor with improvement in his dermatitis. Skin lesions in patient B resolved with the use of topical corticosteroids. Patient C improved with a combination of isotretinoin therapy and both intralesional and systemic corticosteroid treatment. Patient D had improvement in her psoriasiform dermatitis with topical corticosteroid solution and salicylic acid shampoo. ConclusionsThis case series expands our understanding of cutaneous non-infectious eruptions that can occur in patients with both X-linked and autosomal recessive forms of CGD. Each of these cases illustrates a unique clinical history, histologic findings and treatment plan in patients who developed cutaneous lesions secondary to immune dysregulation in the context of CGD. This series provides additional insights that may assist providers in identifying and managing non-infectious dermatologic manifestations of CGD.

Abnormal Dihydrorhodamine Result in a Patient with Diabetes Mellitus, Recurrent Candida Liver Abscesses: Diagnostic Possibilities

A 17-year-old female with type I diabetes mellitus (DM) and a history of recurrent candidal vaginal infections and liver abscesses was assessed fora potential neutrophil oxidative burst defect by DHR flow cytometry. The DHR result was abnormal with significantly decreased MFI (MFI = 9; ref. cutoff: >/ = 60) after stimulation with PMA though all of her neutrophils (99%; ref. cutoff >/ = 95%) showed respiratory burst. This DHR pattern has been previously observed in patients with complete myeloperoxidase deficiency (cMPO), p40phox deficiency and hypomorphic X-linked chronic granulomatous disease (CGD). Genetic evaluation of the genes commonly associated with CGD (CYBB, CYBA, NCF1, NCF2 and NCF4) was negative. It must be noted that many genetic laboratories either do not sequence the NCF1 gene or have limited sequencing to the most commonly observed pathogenic variant in exon 2. Most targeted genetic panels currently do not include MPO (myeloperoxidase) or CYBC1 (Eros). The clinical phenotype of Candida infection in the context of diabetes has been previously reported in 5% of cMPO deficiency. The patient underwent a partial hepatectomy and was initiated on an 8-month course of fluconazole. Three months later, there was a recurrence of hepatic abscesses (Figure 1), requiring treatment with a 12-week course of IV micafungin. Complete MPO deficiency can cause a false-positive result on the DHR assay but the pattern is different from that observed in typical XL- and autosomal recessive CGD. There are two ways of confirming cMPO deficiency, besides genetic testing: 1) staining for MPO in neutrophils in a peripheral smear, or 2) repeating the DHR assay and gating on eosinophils as eosinophil peroxidase can overcome the lack of MPO deficiency and demonstrate a normal oxidative burst. These studies are currently under investigation. In summary, cMPO deficiency should be included in the differential diagnosis of patients with diabetes and invasive Candida infections. Communication with the clinical laboratory is essential to identify the DHR pattern and obtain additional confirmatory testing. From a clinical perspective, cMPO-deficient patients do not require prophylactic antibiotics but they have a high rate of recurrence of fungal infections and require aggressive management with antifungal therapies. [Display omitted]

Lupus nephritis with clinical and laboratory features of humoral immune deficiency in a 29-year-old female with a novel NCF1 variant consistent with autosomal recessive chronic granulomatous disease

Chronic Granulomatous Disease (CGD) is a rare, inherited disorder of the innate immune system characterized by defects in the phagocyte NADPH oxidase complex. This results in poor phagocyte function causing frequent infections (bacterial and fungal) and chronic inflammation. Here we discuss a rare presentation of lupus nephritis and antibody deficiency in an adult female patient with a novel homozygous nonsense mutation in the NCF-1 gene causing autosomal recessive CGD.Our patient is a 29-year-old female with history of ITP, biopsy-confirmed lupus nephritis, systemic lupus erythematosus (SLE), idiopathic avascular necrosis of the hip, and previous diagnosis of common variable immune deficiency (CVID) treated with Immunoglobulin replacement therapy (IVIG). She presented to our clinic to establish care in the setting of persistent skin abscesses/cellulitis, poor wound healing, necrotic lymphadenitis, and recurrent urinary tract infections and pneumonia despite years of IVIG therapy. She had near-normal total IgG, absent IgA and IgE, and protective vaccine antibody titers (pneumococcus, diphtheria, and tetanus) prior to the start of IVIG. Immunophenotyping demonstrated quantitatively normal number of total B cells, absent CD27+ memory B cells, and normal T cell subsets. She had reassuring lymphocyte proliferation with mitogen stimulation and normal TLR function. Dihydrorhodamine (DHR) flow cytometric assay demonstrated abnormally low neutrophil oxidative burst activity. Gene sequencing revealed homozygous nonsense mutation in the NCF-1 gene (c.75-76del; pY26Hfs*26) and a pathogenic heterozygous variant in FANCM (c.3732-3735dup; p. Leu1246Asnfs*8). She was started on daily prophylactic trimethoprim-sulfamethoxazole and itraconazole. She has marked decrease in infections/hospitalizations and her lupus nephritis is well controlled with hydroxy-chloroquine. She is being referred to the NIH for HSCT evaluation. The majority of CGD cases in the United States are due to X-linked CGD (70%) and AR variants in NCF1 (20%). While rare, discoid lupus erythematosus and SLE have been documented with X-linked forms of CGD. Similarly, there are few case reports of antibody deficiency in patients with CGD. This is the first documented case of AR CGD with SLE/lupus nephritis and humoral immune defects. Additionally, we report a disease-causing, novel variant in the NCF1 gene. This case report adds to the existing literature on CGD and associated diseases.

CGDilemma: Fitting Together the Puzzle Pieces for Definitive Diagnosis

IntroductionChronic granulomatous disease (CGD) is a rare inherited primary immunodeficiency. ΔGT mutations in NCF1, which encodes phagocyte nicotinamide adenine phosphate (NADPH) p47-phox protein, accounts for 23% of CGD. Oftentimes testing focuses on ΔGT defects. However, 20% of p47 CGD patients have mutations at other sites in NCF1 which can be missed. Further, NCF1 has 2 pseudogenes near chromosomal locus 7q11.23 which may undergo reciprocal crossover between functional genes exceeding expected frequency, complicating diagnosis, especially in atypical presentations. Case PresentationA 4-year-old male from Saudi Arabia presented with a necrotic lesion on his left cheek, unresponsive to antibiotics. Two years prior he had a Rhizopus infection on his right leg necessitating debridement. Biopsy revealed recurrent Rhizopus infection. He received debridement, linezolid, posaconazole and amphotericin. He was discharged on posaconazole and trimethoprim/sulfamethoxazole, after a protracted hospital stay. His wound improved, however margins remained positive.Immune workup revealed an oxidative burst assay with a broad abnormal peak of intermediate granulocyte dihydrorhodamine fluorescence suggesting autosomal recessive CGD. Genetic panels identified no variants in CGD genes. Western blot showed absent p47phox protein, confirming NCF1-deficient CGD. Genetic testing did not show the common ΔGT deletion at the start of exon 2 in 6 copies of NCF1/NCF1 pseudogenes. Sanger sequencing revealed a single nucleotide change distinct to the patient. This coded for NCF1 c.579 G > A, p. Trp193X, a previously reported mutation in NCF1. ConclusionDinucleotide deletion at the beginning of exon 2 of NCF1 pseudogenes leads to a frameshift and stop. When a pseudogene replaces the authentic NCF1, the overall ratio of functional genes to pseudogenes is altered. This potentially masks CGD carriers who possess pseudogene to functional gene ratios that renders them indistinguishable from healthy individuals with most testing. Non-ΔGT mutations occur in ~20% of p47phox patients. Because of the extensive sequence homology of NCF1 with the pseudogenes, Sanger sequencing may be insufficient to determine the underlying defect in NCF1. Therefore, gene panels and even whole genome sequencing may miss p47phox deficiency. This multimodality approach allowed us to identify p47phox-deficient autosomal recessive CGD.

The Risk of the Next Child Getting Affected by Chronic Granulomatous Disease in Families with at Least One Autosomal Recessive CGD Child.

Chronic granulomatous disease (CGD) is a rare primary immunodeficiency disorder more common in autosomal recessive (AR) than X-linked in Iran. This study aimed to assess whether having a child with AR-CGD would increase the likelihood of the next child being affected by CGD. Ninety-one families with at least one child affected by AR-CGD entered this study. Out of the 270 children, 128 were affected by AR-CGD. We used a cross tab for the odds ratio (OR) calculation, in which exposure to a previously affected child and the next child's status were evaluated. This study illustrated that the chances of having another child afflicted with AR-CGD are significantly increased if the previous child had AR-CGD (OR=2.77, 95% CI=1.35-5.69).Althoug h AR disorders affect 25% of each pregnancy, we showed that the chance that the next child would be affected by CGD, given that the previous child was affected, is 2.77 times greater than in families with a normal child. It is recommended to warn families with one or more affected children to evaluate the risk of CGD in their subsequent pregnancies with prenatal diagnosis.

Clinical features and outcomes of patients with chronic granulomatous disease in Taiwan.

Chronic granulomatous disease (CGD) is a rare primary immunodeficiency disease characterized by defective neutrophil killing of microbial pathogens and recurrent infections. We aimed to investigate the clinical, genetic features, treatment, and outcomes in patients with CGD. Pediatric patients diagnosed with CGD from a medical center in Taiwan were enrolled from January 1999 to Oct 2021. Nine pediatric patients with CGD were enrolled: six X-linked (XL) CGD with CYBB gene mutations, three autosomal recessive (AR) CGD with two NCF1 and one CYBA gene mutations.The median age of onset and age of diagnosis was 0.92 and 2.64 years, respectively. Patients with XL-CGD had a younger age of onset (4.6 months vs. 1.83 years, P=0.06) and age of diagnosis (1.71 vs. 8.86 years, P=0.024) than AR-CGD patients. The most common sites of infections were skin and soft tissue abscesses. The most common pathogens were Staphylococcus, Serratia, and Salmonella spp. Prophylactic antibiotics, anti-fungal agents, and interferon-gamma (IFN-γ) were given in 9 (100%), 7 (77.8%), and 8(88.9%) patients, respectively. The mean duration of IFN-γ usage was 5.15 years. One male patient with XL-CGD was successfully treated with hematopoietic stem cell transplantation at 2.2 years. The mortality rate was 11.1%, and the estimated overall survival at 20 years was 66.7%. Staphylococcus aureus, Serratia marcescens, and Salmonella infections are important in Taiwanese CGD patients. Patients with XL-CGD have early disease onset. IFN-γ prophylaxis and prophylactic anti-microbial agents might have an effect on alleviating the infection episodes in CGD patients.

Clinical, Immunological, and Molecular Profile of Chronic Granulomatous Disease: A Multi-Centric Study of 236 Patients From India.

BackgroundChronic granulomatous disease (CGD) is an inherited defect in phagocytic respiratory burst that results in severe and life-threatening infections in affected children. Single center studies from India have shown that proportion of autosomal recessive (AR) CGD is more than that reported from the West. Further, affected patients have high mortality rates due to late referrals and difficulties in accessing appropriate treatment. However, there is lack of multicentric collaborative data on CGD from India.ObjectiveTo describe infection patterns, immunological, and molecular features of CGD from multiple centers in India.MethodsA detailed proforma that included clinical and laboratory details was prepared and sent to multiple centers in India that are involved in the care and management of patients with inborn errors of immunity. Twelve centers have provided data which were later pooled together and analyzed.ResultsOf the 236 patients analyzed in our study, X-linked and AR-CGD was seen in 77 and 97, respectively. Male female ratio was 172:64. Median age at onset of symptoms and diagnosis was 8 and 24 months, respectively. Common infections documented include pneumonia (71.6%), lymphadenitis (31.6%), skin and subcutaneous abscess (23.7%), blood-stream infection (13.6%), osteomyelitis (8.6%), liver abscess (7.2%), lung abscess (2.9%), meningoencephalitis (2.5%), splenic abscess (1.7%), and brain abscess (0.9%). Forty-four patients (18.6%) had evidence of mycobacterial infection. Results of molecular assay were available for 141 patients (59.7%)—CYBB (44.7%) gene defect was most common, followed by NCF1 (31.9%), NCF2 (14.9%), and CYBA (8.5%). While CYBA variants were documented only in Southern and Western parts of India, a common dinucleotide deletion in NCF2 (c.835_836delAC) was noted only in North Indian population. Of the 174 patients with available outcome data, 67 (38.5%) had expired. Hematopoietic stem cell transplantation was carried out in 23 patients, and 12 are doing well on follow-up.ConclusionsIn India, proportion of patients with AR-CGD is higher as compared to Western cohorts, though regional differences in types of AR-CGD exist. Clinical profile and mortality rates are similar in both X-linked and AR-CGD. However, this may be a reflection of the fact that milder forms of AR-CGD are probably being missed.

Hemophagocytic Lymphohistiocytosis in Children with Chronic Granulomatous Disease—Single-Center Experience from North India

Chronic granulomatous disease (CGD) is an inherited defect in components of the nicotinamide adenine dinucleotide phosphate oxidase complex that results in potential life-threatening infective and noninfective complications. Hemophagocytic lymphohistiocytosis (HLH) is an unusual but important inflammatory complication of CGD. Optimal management strategies have not yet been identified in children with CGD who develop HLH. To analyze clinical and laboratory features of HLH in CGD from a tertiary-care center in North India. A retrospective review of medical records of children with CGD diagnosed in the last 20 years was performed. Clinical and laboratory features of children with CGD who developed HLH were analyzed. Of 80 patients diagnosed with CGD, 5 (6.25%) had evidence of HLH. All 5 were males; 4 had X-linked CGD and 1 had autosomal recessive CGD (NCF2 defect). Two children with CGD had HLH as the predominant presenting manifestation mimicking the clinical presentation of congenital HLH. Infectious triggers identified were bloodstream infections (n= 3) (Candida albicans, Burkholderia cenocepacia, Francisella noatuensis), pneumonia (n= 4), and splenic abscess (n= 1). We document the first human infection with a fish pathogen, F.noatuensis, in a child with X-linked CGD. Although mortality was seen in 3 children who received only intravenous (IV) immunoglobulin therapy, the other 2 who received IV methylprednisolone pulse therapy survived. HLH can be a presenting manifestation of CGD, and workup for CGD must be considered in children with HLH. Early recognition with optimal management of both infectious trigger and HLH is very important to prevent mortality.

Bacillus Calmette-Guérin (BCG) Infections at High Frequency in Both AR-CGD and X-CGD Patients Following BCG Vaccination.

Patients with chronic granulomatous disease (CGD) develop severe infections, including Bacillus Calmette-Guérin (BCG). Although the autosomal recessive CGD (AR-CGD) patients should hypothetically develop relatively fewer infections compared to the X-linked CGD (X-CGD) patients due to more residual reactive oxygen intermediates, the impacts of BCG vaccination on AR-CGD and X-CGD patients are unclear. Herein, we demonstrated the clinical features of BCG infections, treatments, and genetic factors in CGD patients after BCG vaccination under the Japanese immunization program. We collected data retrospectively from 43 patients with CGD and assessed their history of initial infection, age at diagnosis of CGD, BCG vaccination history, clinical course, treatment for BCG infections, and genetic mutations associated with CGD. Fourteen CGD patients avoided BCG vaccination because of other preceding infections and family history. Of 29 patients with CGD who received BCG vaccination, 20 patients developed BCG infections. Although the age at onset of initial infection in X-CGD patients was significantly younger than that in AR-CGD patients (P < .01), the onset and frequency of BCG infections were similar in X-CGD and AR-CGD patients. In X-CGD patients, BCG infections equally developed in the patients carrying missense, insertion, deletion, nonsense, and splice mutations of CYBB. All CGD patients with BCG infections were successfully treated with anti-tuberculous drugs. Although X-CGD patients develop severe infections at a younger age than AR-CGD patients, our data suggested that BCG infections develop at high frequency in both AR-CGD and X-CGD patients, regardless of genotype and mutant forms.

Hematopoietic Cell Transplantation for Chronic Granulomatous Disease in Japan.

Hematopoietic cell transplantation (HCT) is established as a curative treatment for severe chronic granulomatous disease (CGD). However, outcomes of HCT for CGD in Japan had not been precisely reported. We evaluated the outcome of HCT for CGD in Japan by means of a nationwide survey. A total of 91 patients (86 males and 5 females) with CGD who received HCT between 1992 and 2013 was investigated. Their median age at HCT was 11 years (0–39). Sixty-four patients had X-linked CGD caused by CYBB gene mutations, 13 had autosomal recessive CGD (7 CYBA and 6 NCF2), and 14 were genetically undetermined. Seventy patients are still alive at a median follow-up of 38.9 (3.7–230) months. Three-year OS and EFS was 73.7 and 67.6%, respectively. Twenty-one patients died mainly from transplant-related mortality. The cumulative incidence of grade II to IV acute GVHD and extensive chronic GVHD was 27.2 and 17.9%, respectively. Risk factors for EFS after HCT for CGD were age >30 years (P < 0.01), non-CYBB gene mutations (P < 0.01) and CBT (P < 0.01). Regarding the reduced intensity conditioning (RIC) regimen, risk factors for EFS included anti-thymocyte globulin (P = 0.048) and not using low-dose irradiation therapy (P < 0.01), in addition to the preceding risk factors. We report outcomes of HCT for CGD in Japan. Future studies are needed to improve such outcomes, especially for patients harboring non-CYBB gene mutations and suffering from adult CGD. A RIC regimen including low-dose irradiation may be a good option to explore further.

Diagnostic Modalities Based on Flow Cytometry for Chronic Granulomatous Disease: A Multicenter Study in a Well-Defined Cohort

Chronic granulomatous disease (CGD) is characterized by defective microbial killing due to mutations affecting subunits of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. Definitive genetic identification of disease subtypes may be delayed or not readily available. Sought to investigate the role of intracellular staining of NADPH oxidase enzyme subunits in predicting the respective genetic defects in patients with CGD and carriers. Thirty-four patients with genetically inherited CGD, including 12 patients with X-linked CGD (gp91phagocyte oxidase (phox) deficiency due to cytochrome b-245, beta polypeptide [CYBB] mutations) and 22 patients with autosomal-recessive CGD (p22phox, p47phox, and p67phox deficiency due to cytochrome b-245, alpha polypeptide [CYBA], neutrophil cytosolic factor 1 [NCF1] and NCF2 mutations, respectively) were recruited from different immunology centers and followed up prospectively. Dihydrorhodamine testing and NADPH oxidase subunit expression in white blood cells were determined by flow cytometry. gp91phox and p22phox defects, which result in simultaneous loss of both proteins due to their complex formation, were differentiated only by comparative analysis of patients' and mothers' intracellular staining. p47phox and p67phox protein expression was almost undetectable in patients compared with carrier mothers and healthy controls. The expression values of the respective subunits were found to be significantly higher in all controls as compared with carrier mothers, which in turn were higher than those of patients. Analysis of NADPH oxidase enzyme subunits by flow cytometry in patients and carriers is useful in the rapid prediction of the genetic defect of patients with CGD, thus guiding targeted sequencing and aiding in their early diagnosis.

Novel NCF2 Mutation Causing Chronic Granulomatous Disease.

Chronic granulomatous disease (CGD) is a rare primary immunodeficiency disorder caused by defects in the NADPH oxidase complex. Mutations in NCF2 encoding the cytosolic factor p67phox result in autosomal recessive CGD. We describe three patients with a novel c.855G>C NCF2 mutation presenting with diverse clinical phenotype. Two siblings were heterozygous for the novel mutation and for a previously described exon 8-9 duplication, while a third unrelated patient was homozygous for the novel mutation. Mutation pathogenicity was confirmed by abnormal DHR123 assay and absent p67phox production and by sequencing of cDNA which showed abnormal RNA splicing. Clinically, the homozygous patient presented with suspected early onset interstitial lung disease and NCF2 mutation was found on genetic testing performed in search for surfactant-related defects. The two siblings also had variable presentation with one having history of severe pneumonia, lymphadenitis, and recurrent skin abscesses and the other presenting in his 30s with discoid lupus erythematosus and without significant infectious history. We therefore identified a novel pathogenic NCF2 mutation causing diverse and unusual clinical phenotype.

Radiographic features of patients with chronic granulomatous disease

Introduction: Chronic granulomatous disease (CGD) is one of the most common primary immunodeficiencies of childhood, and is caused by defects in the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. Alongside neutrophil dysfunction, dysregulation of the immune system predisposes patients to recurrent life-threatening infections as well as granuloma formation, hyperinflammation, and autoimmunity. Examination by imaging (radiography, ultrasound, computed tomography, magnetic resonance) in conjunction with biopsy and tissue or fluid cultures are essential to identify the extent and severity of infections as well as the microorganisms responsible. These modalities also help to guide the management of inflammatory complications.Aim: We highlight the common radiographic findings in 10 pediatric CGD patients followed at our centre over a period of 10 years.Methods: Medical records of patients with confirmed CGD diagnosis were reviewed retrospectively. All had low neutrophil oxidative burst index (NOBI) and pathogenic mutation in 1 of the 5 subunits of the NADPH oxidase. Three patients had autosomal recessive CGD and 7 had X-linked recessive CGD. All but 1 are male.Results: The most common radiographic presentation was hilar lymphadenopathy and pulmonary nodules. Other lung complications include cavitating lesions, lung abscess, pulmonary nodule, and pleuritic nodules. Lymphatic tissue and lymph nodes were involved in 50% of our cohort of patients, while gastrointestinal manifestations were noted in approximately 35% of our patients. These include the presence of pigmented macrophages, multiple granulomas, liver abscess, or detection of Aspergillus in tissue or fluid culture.Discussion: It is essential for clinicians to keep primary immunodeficiency as one of the differential diagnoses in patients who present with severe infection or inflammation. We encourage physicians to consider CGD in patients with above described findings and consider measuring NOBI in patients with early onset infection, inflammation, or granuloma formation.Statement of novelty: We describe the radiographic findings of a pediatric cohort of patients with CGD.

Genetic Characteristics, Infectious, and Noninfectious Manifestations of 32 Patients with Chronic Granulomatous Disease

Background: Chronic granulomatous disease (CGD) is a rare genetic disorder characterized by failure of phagocytic leukocytes to destroy certain microbes. We present a study on CGD patients enrolled at a single medical center concerning the infectious and noninfectious complications and genetic properties of the disease. Methods: Icotinamide adenine dinucleotide phosphate oxidase activity and the expression of flavocytochrome b₅₅₈ were measured by flow cytometry, and clinical outcomes of the patients were listed in relation to the genetic results. Results: The clinical and genetic findings of 32 pediatric cases with CGD from 23 families were enrolled. Pneumonia and anemia were the most common infectious and noninfectious symptoms. Genetic analysis showed that 10 families (43.5%) carried CYBB variants and 13 families (56.5%) have autosomal recessive (AR) CGD, in which 6 families (26%) carried NCF1 variants, 4 (17.4%) carried CYBA variants, and 3 (13%) carried NCF2 variants. The median age of clinical onset was 3.3 and 48 months for patients with X-linked CGD (X-CGD) and AR-CGD, respectively. The onset of symptoms before age 1 year was 94% in X-CGD, 28.5% in AR-CGD, and 12.5% in patients with oxidase residual activity. Moreover, a de novo germline mutation at c.1415delG in CYBB (OMIM#300481) and a novel c.251_263del13bp in CYBA (OMIM#608508) were also investigated. Conclusions: Ihydrorhodamine-1,2,3 assay could not detect carrier mother in de novo case with CYBB variant. Most X-CGD patients have the onset of symptoms before age 1 year. Additionally, residual oxidase activity in AR-CGD causes a delay in onset, diagnosis, and prophylaxis. The protective role of residual activity is limited while the infection is ongoing and becoming serious.