Molecular diagnosis of chronic granulomatous disease

Patients with chronic granulomatous disease (CGD) suffer from recurrent, life‐threatening bacterial and fungal infections of the skin, the airways, the lymph nodes, liver, brain and bones. Frequently found pathogens are Staphylococcus aureus, Aspergillus species, Klebsiella species, Burkholderia cepacia and Salmonella species. CGD is a rare (～1:250 000 births) disease caused by mutations in any one of the five components of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in phagocytes. This enzyme generates superoxide and is essential for intracellular killing of pathogens by phagocytes. Molecular diagnosis of CGD involves measuring NADPH oxidase activity in phagocytes, measuring protein expression of NADPH oxidase components and mutation analysis of genes encoding these components. Residual oxidase activity is important to know for estimation of the clinical course and the chance of survival of the patient. Mutation analysis is mandatory for genetic counselling and prenatal diagnosis. This review summarizes the different assays available for the diagnosis of CGD, the precautions to be taken for correct measurements, the flow diagram to be followed, the assays for confirmation of the diagnosis and the determinations for carrier detection and prenatal diagnosis.     

Two X-Linked Chronic Granulomatous Disease Patients with Unusual NADPH Oxidase Properties

Background

Chronic granulomatous disease (CGD) is an immune deficiency syndrome caused by defects in the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, the enzyme that generates reactive oxygen species (ROS) in phagocytizing leukocytes. This study evaluates the NADPH oxidase capacity in two X-linked CGD patients with mutations in gp91^phox that alter the regions in this membrane-bound NADPH oxidase component involved in docking of the cytosolic component p47^phox.

Materials and Methods

Hydrogen peroxide and superoxide generation, bactericidal activity, and NADPH oxidase protein expression by the patients?? neutrophils were measured, and genetic analysis was performed.

Results

We report two patients, each with a novel missense mutation in CYBB, the gene that encodes gp91^phox. Surprisingly, neutrophils from these patients showed total absence of superoxide production, although they retained 13?C30% of the hydrogen peroxide production capability. We speculate that this is due to direct electron transfer from flavin adenine dinucleotide (FAD) in gp91^phox to oxygen, leading to inefficient hydrogen peroxide formation instead of efficient superoxide production.

Conclusions

X-linked CGD patients with mutations that alter the gp91^phox protein in regions involved in docking of the cytosolic NADPH oxidase component p47^phox may have higher than expected hydrogen peroxide generation capability.     

Clinical Manifestations of Disease in X-Linked Carriers of Chronic Granulomatous Disease

Chronic Granulomatous Disease (CGD) is a rare primary immunodeficiency due to a defect in one of the NADPH oxidase complex subunits; 70 % of cases are X-linked, due to a CYBB mutation, resulting in defective production of gp91^PHOX. Female carriers of X-linked CGD have previously been considered to be unaffected. It is increasingly recognized that they may suffer from similar problems to CGD patients. This review will examine the literature about clinical manifestations of disease in X-linked carriers of CGD.     

Clinical and molecular findings of chronic granulomatous disease in Oman: family studies

Chronic granulomatous disease (CGD), a rare inherited disorder of the innate immune system, results from mutations in any one of the five genes encoding the subunits of the nicotinamide adenine dinucleotide phosphate‐oxidase (NADPH) oxidase enzyme, and is characterized by recurrent life‐threatening bacterial and fungal infections. Molecular analysis of 14 Omani CGD patients from 10 families, diagnosed to have CGD on clinical (recurrent infections) and biochemical grounds (positive for both the nitroblue tetrazolium (NBT) test and the dihydrorhodamine (DHR‐1,2,3 assay), revealed that only one patient had X‐linked CGD, with a large deletion involving both the gp91‐phox gene (CYBB) and the McLeod gene (XK). The remaining 13 patients were all homozygotes from a previously described c.579G>A (p.Trp193X) mutation in the NCF1 gene on chromosome 7, responsible for autosomal recessive CGD (AR‐CGD). Although X‐linked CGD is the most common type of CGD disorder in most population groups, AR‐CGD is the most prevalent type in Oman.     

X-linked chronic granulomatous disease secondary to skewed X chromosome inactivation in a female with a novel CYBB mutation and late presentation

Chronic granulomatous disease (CGD) is characterized by defects in the superoxide producing enzyme NADPH oxidase causing phagocytes to improperly clear invading pathogens. Here we report findings of a late presenting 16-year-old female with X-linked CGD. The patient presented with community-acquired pneumonia, but symptoms persisted for 2 weeks during triple antimicrobial coverage. Cultures revealed Aspergillus fumigatus which was resolved through aggressive voriconazole treatment. Neutrophil studies revealed NADPH oxidase activity and flavocytochrome b(558) levels that were 4-8% of controls and suggested carrier status of the mother. We found a null mutation in the CYBB gene (c.252insAG) predicting an aberrant gp91(phox) protein (p.Cys85fsX23) in the heterozygous state. Methylation analysis demonstrated extremely skewed X chromosome inactivation favoring the maternally inherited defective gene. In conclusion, a novel mutation in the CYBB gene and an extremely skewed X-inactivation event resulted in the rare expression of the CGD phenotype in a carrier female.     

Diabetes, Renal and Cardiovascular Disease in p47 phox−/− Chronic Granulomatous Disease

Chronic granulomatous disease is a rare immunodeficiency due to defects in the phagocyte NADPH oxidase. The X-linked form (gp91 ^phox deficiency) accounts for about 70 % of cases; autosomal recessive p47 ^phox deficiency accounts for about 25 % of cases. We identified a 10 % incidence of diabetes in p47 ^phox deficient CGD, but none in X-linked CGD. Renal and cardiovascular diseases were also higher in p47 ^phox deficiency. p47 ^phox deficient CGD has non-infectious morbidities distinct from those in X-linked CGD.     

Chronic Granulomatous Disease: A 25-Year Patient Registry Based on a Multistep Diagnostic Procedure, from the Referral Center for Primary Immunodeficiencies in Greece

Chronic Granulomatous Disease (CGD) is an uncommon primary immunodeficiency caused by the absence or dysfunction of one of NADPH oxidase subunits, with heterogeneous genetic aetiologies. The aim of this study was the CGD patient registry in Greece, the identification of the responsible genotype and the potential correlation with the patient’s clinical phenotype. Medical charts of 24 CGD patients, investigated by NBT test or DHR for NADPH oxidase activity, Western blot analysis for NADPH oxidase component expression and DNA sequencing (pyro- and cycle sequencing) for mutation analysis, were reviewed. All patients, but one, were classified into the different types of CGD. Sixteen from 14 unrelated families had X-linked CGD (66.7 %), four patients had mutations in the NCF1 gene (16.7 %), and three, from two unrelated families, had mutations in NCF2 (12.5 %). Fifteen mutations were detected in the CYBB gene, including nonsense (53.8 %), splice site (30.8 %) and missense mutations (7.7 %), and deletions (7.7 %). Two novel mutations were identified; one in CYBB and one in NCF1. Carrier detection for X-CGD revealed that the de novo mutation rate was about 7 %. Prenatal diagnosis identified one affected male in three male fetuses tested. In both the X-linked and the autosomal recessive (AR-CGD) group, the gastrointestinal and respiratory manifestations were more common, followed by lympadenopathy in X-CGD and skin infections in the AR-CGD group. The patients with a mutation in CYBB had a wider variability of clinical manifestations and earlier diagnosis (4.6 years) compared to the AR-CGD group (12.9 years). The incidence of CGD in Greece is estimated at 0.90 (95 % CI 0.89–0.91) per 100,000 live births for the last decade.     

Utility of Immunohistochemistry and Immunofluorescence in Determining the Pathogenic Variants of Chronic Granulomatous Disease

Background

Chronic granulomatous disease (CGD) is a primary immunodeficiency disorder of phagocytes due to defects in any of the five subunits of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. An initial diagnosis of CGD is made by flow cytometry-based dihydrorhodamine assay or nitro blue tetrazolium test, which is further confirmed by molecular assays. Expression of five subunits of NADPH oxidase components by either flow cytometric or western blot analysis provides clues toward the potential gene targets which are subsequently confirmed by various genetic assays. Immunohistochemistry (IHC) and immunofluorescence (IF) have never been earlier used to determine the expression of different subunits of NADPH oxidase system. We evaluated the utility of IHC and IF in determining the underlying pathogenic variants of CGD.

Materials and Methods

Twelve genetically confirmed cases of CGD, comprising of biopsy specimens (n?=?6), tissue blocks from autopsy cases (n?=?3), and cellblocks of cell pellet prepared from peripheral blood (n?=?4) were included. IHC for p67phox and p47phox subunits and IF for cytochrome b558 were performed.

Results

All 4 cases with pathogenic variation of NCF2 gene showed loss of expression for p67phox subunit. Two cases with pathogenic variation of NCF1 gene showed loss of expression for p47phox subunit. Five cases, except a single case with CYBB gene pathogenic variation, showed loss of expression for cytochrome b558 on IF. Thus, loss of expression consistently matched with the underlying genetic defects assessed by sequencing.

Conclusions

Our results confirm our hypothesis that IHC and IF are two rapid, economical, pathologist-friendly techniques providing pertinent information regarding the underlying pathogenic variants and such immuno-analysis can be easily performed on the tissue.

     

Clinical and Immunological Characteristics of 63 Patients with Chronic Granulomatous Disease: Hacettepe Experience

Background

Chronic granulomatous disease (CGD), one of the phagocytic system defects, is the primary immunodeficiency caused by dysfunction of the NADPH oxidase complex which generates reactive oxygen species (ROS), which are essential for killing pathogenic microorganisms, especially catalase-positive bacteria and fungi.

Objective

The objective of our study was to assess the clinical and laboratory characteristics, treatment modalities, and prognosis of patients with CGD.

Methods

We retrospectively reviewed 63 patients with CGD who have been diagnosed, treated, and/or followed-up between 1984 and 2018 in Hacettepe University, Ankara, in Turkey, as a developing country.

Results

The number of female and male patients was 26/37. The median age at diagnosis was 3.8 (IQR: 1.0–9.6) years. The rate of consanguinity was 63.5%. The most common physical examination finding was lymphadenopathy (44/63), growth retardation (33/63), and hepatomegaly (27/63). One adult patient had squamous cell carcinoma of the lung. The most common infections were lung infection (53/63), skin abscess (43/63), and lymphadenitis (19/63). Of the 63 patients with CGD, 6 patients had inflammatory bowel disease (IBD). Twelve of the 63 patients died during follow-up. CYBA, NCF1, CYBB, and NCF2 mutations were detected in 35%, 27.5%, 25%, and 12.5% of the patients, respectively.

Conclusion

We identified 63 patients with CGD from a single center in Turkey. Unlike other cohort studies in Turkey, due to the high consanguineous marriage rate in our study group, AR form of CGD was more frequent, and gastrointestinal involvement were found at relatively lower rates. The rate of patients who treated with HSCT was lower in our research than in the literature. A majority of the patients in this study received conventional prophylactic therapies, which highlight on the outcome of individuals who have not undergone HSCT.

     

Chronic granulomatous disease in two sisters

Two sisters with chronic granulomatous disease (CGD) have been studied. The diagnosis was suggested by the histopathological findings from the spleen and lymph nodes of the proband and confirmed by the low values obtained in the following tests performed on polymorphonuclear leukocytes (PMN): chemiluminescence, nitroblue tetrazolium (NBT) reduction, killing ofStaphylococcus aureus, and O ₂ ^– production. NADPH oxidase activity was not detected in the homogenates of the patients' PMN but cytochromeb was normally present. In addition, PMN depolarization induced by phorbol-myristate acetate was absent, thus suggesting a defect of the activation mechanism of the respiratory enzyme. The normal depolarization induced by ouabain indicated that the membrane polarity regulated by the Na/K pump in the patients' cells was not affected. The low, but not completely absent, respiratory activity of the patients' PMN could suggest an X-linked mode of inheritance with incomplete Lyonization. From a clinical point of view, one sister had mild symptoms whereas the other was almost symptomless, thus confirming once more the heterogeneity of CGD syndrome.     

Impaired host defence against Mycobacterium avium in mice with chronic granulomatous disease

Patients with chronic granulomatous disease (CGD), an inherited disorder of phagocytic cells, often contract recurrent life‐threatening bacterial and fungal infections. CGD is considered to arise from a functional defect of the O₂‐generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in phagocytes. To determine whether or not NADPH oxidase is crucial to the host defence against Mycobacterium avium, we investigated the response against M. avium using CGD model mice (gp91‐phox^‐) of C57BL/6 strain. A tracheal injection of 1 × 10⁷ colony‐forming units (CFU)/head of M. avium strain FN into the CGD mice resulted in a pulmonary infection, while also increasing the mortality rate. In contrast, normal C57BL/6 mice injected with same dose of the organisms did not develop severe pulmonary infection and were able to survive through 2 months of observation. The macrophages obtained from the CGD mice were observed to have a higher burden of the bacterial growth than macrophages from normal C57BL/6 mice. These results suggest that the defect of the NADPH oxidase function impairs the host defence against M. avium infection.     

Hyper‐responsive Toll‐like receptor 7 and 9 activation in NADPH oxidase‐deficient B lymphoblasts

Chronic granulomatous disease (CGD) is an inherited immunodeficiency linked with mutations in the multi-subunit leucocyte NADPH oxidase. Myeloid-derived phagocytic cells deficient in NADPH oxidase fail to produce sufficient levels of reactive oxygen species to clear engulfed pathogens. In this study we show that oxidase also influences B-cell functions, including responses to single-stranded RNA or unmethylated DNA by endosomal Toll-like receptors (TLRs) 7 and 9. In response to TLR7/9 ligands, B-cell lines derived from patients with CGD with mutations in either the NADPH oxidase p40^phox or p47^phox subunits produced only low levels of reactive oxygen species. Remarkably, cytokine secretion and p38 mitogen-activated protein kinase activation by these oxidase-deficient B cells was significantly increased upon TLR7/9 activation when compared with oxidase-sufficient B cells. Increased TLR responsiveness was also detected in B cells from oxidase-deficient mice. NADPH oxidase-deficient patient-derived B cells also expressed enhanced levels of TLR7 and TLR9 mRNA and protein compared with the same cells reconstituted to restore oxidase activity. These data demonstrate that the loss of oxidase function associated with CGD can significantly impact B-cell TLR signalling in response to nucleic acids with potential repercussions for auto-reactivity in patients.     

Impaired macrophage function following bacterial stimulation in chronic granulomatous disease

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is critical for phagocyte anti-microbial activity and plays a major role in innate immunity. Defects in genes coding for components of the NADPH oxidase enzyme system are responsible for chronic granulomatous disease (CGD), a rare primary neutrophil immunodeficiency associated with recurrent, life-threatening bacterial and fungal infections. Microbial killing and digestion within the neutrophil phagosomal compartment are defective in these patients. NADPH oxidase activity is also crucial for optimal macrophage and dendritic cell function and has recently been implicated in both cross-presentation and T-cell priming. We present evidence of impaired macrophage function in CGD, with attenuated pro-inflammatory cytokine and increased interleukin-10 secretion following bacterial stimulation. These results highlight additional abnormalities in macrophage function associated with CGD and the importance of NADPH oxidase activity in immunity.     

Rapid whole-blood flow cytometry assay for diagnosis of chronic granulomatous disease.

Chronic granulomatous disease (CGD) is characterized by defective killing of intracellular microorganisms due to mutations in one of the four known components of the NADPH oxidase system. This system is responsible for the generation of superoxide and related antimicrobial oxidants. Diagnosis of CGD requires the demonstration of an abnormal oxidase system in the leukocytes of affected patients. Recently, several flow cytometry-based procedures which measure various reactive oxygen intermediates generated by the NADPH oxidase system have been developed. Most of the procedures developed to date require time-consuming granulocyte isolation, washing, and counting procedures, or they lack sensitivity. We have modified an existing procedure such that cell labelling and stimulation are performed directly in whole blood. Optimization of this procedure and its use in the diagnosis of patients with CGD or X-linked carriers are presented.     

Chronic Granulomatous Disease: Two Decades of Experience From a Tertiary Care Centre in North West India

Chronic granulomatous disease (CGD) results from an inherited defect in the phagocytic cells of the immune system. It is a genetically heterogenous disease caused by defects in one of the five major subunits of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. There is a paucity of data from India on CGD. We herein describe the clinical features in 17 children with CGD from a single tertiary referral center in India. A detailed analysis of the clinical features, laboratory investigations and outcome of 17 children 7 with X-linked (XL) and 10 with autosomal recessive (AR) form was performed. Diagnosis of CGD was based on an abnormal granulocyte oxidative burst evaluated by either Nitroblue Tetrazolium (NBT) test or flow cytometry based Dihyrorhodamine 123 assay or both. The molecular diagnosis was confirmed by genetic mutation analysis in 13 cases. The mean age at diagnosis and the age at onset of symptoms was significantly lower in children diagnosed with XL- CGD compared those with AR disease. Mutations were detected in CYBB gene in 6 patients with XL-CGD and NCF-1 gene mutations were observed in 7 cases of AR- CGD. The course and outcome of the disease was much worse in children diagnosed with X-linked form of disease compared to AR forms of the disease; 4/7 (57 %) children with X-CGD were dead at the time of data analysis. This is one of the largest series on chronic granulomatous disease from any developing country.     

The search for a genetic defect in Polish patients with chronic granulomatous disease

INTRODUCTION: Chronic granulomatous disease (CGD)is a rare inherited disorder in which phagocytic cells are unable to generate superoxide anions.Patients with CGD are predisposed to recurrent bacterial and fungal infections because the superoxide-generating NADPH oxidase activity is needed for efficient killing of microbes.Among the at least 5 subunits cre-ating a functional NADPH oxidase, a molecular defect located in any of the gp91phox, p22phox, p47phox, or p67phox subunits may cause CGD. MATERIAL/METHODS: In this study,8 patients were diagnosed with CGD on the basis of clinical findings and absence of nitroblue tetrazolium reduction in phagocytes. Southern blot analysis, GeneScan, and direct sequencing were performed to define particular DNA mutations. RESULTS: Among 6 X-linked CGD (X-CGD)patients, 4 different mutations were identified in the X-linked CYBB gene (encoding gp91phox)by direct sequencing. A novel missense mutation, located in the NADPH-binding region of gp91phox,was found in 2 brothers. One frameshift 1578delA, one splicing 252G->A mutation, and one partial gene deletion were also identified. The molecular defect in the NCF1 gene (encoding p47phox)was established in 2 patients. One was DeltaGT/DeltaGT homozygote, the other carried, besides this GT deletion on one allele, a unique Phe118stop mutation on the other. CONCLUSIONS: In general, the X-CGD patients within the group followed a more severe clinical course than the patients with an NCF1 defect. However, the lack of a straightforward genotype phenotype correlation indicates that the clinical severity of CGD depends also on other antimicrobial host-defense systems.     

Three novel mutations in CYBA among 22 Iranians with Chronic granulomatous disease

Chronic granulomatous disease (CGD) is a rare primary immunodeficiency caused by defect in one of the components of nicotinamide adenine dinucleotide phosphate (NADPH)‐oxidase enzyme. The enzyme is at least composed of membrane‐bound subunits gp91‐phox and p22‐phox (also named cytochrome b₅₅₈), and cytosolic ones p40‐phox, p47‐phox and p67‐phox. A defect in the enzyme activity leads to impaired intracellular killing of phagocytic cells. The CYBA gene encoding p22‐phox is located on chromosome 16q24. In this study, new genetic changes of CYBA gene in 22 Iranian patients with autosomal recessive‐CGD (AR‐CGD) were identified. Twenty‐two patients with CGD were referred to Immunology, Asthma and Allergy Research Institute (IAARI) and enrolled in this study based on defect in NADPH oxidase activity, demographic data and clinical histories. All patients had p22‐phox deficiency based on Western blotting. Genomic DNA was extracted from peripheral blood mononuclear cells (PBMCs), and PCR followed by direct sequencing was performed to find p22‐phox mutations. Mutation analysis of CYBA revealed 12 different mutations, including three novel mutations: one was deletion of exon 1, and two were point mutations in exon 3 (c.136G>A (p.Gly46Ser)), and exon 6 (c.388C>T (p.Gln130X)). Three new mutations of CYBA gene in four of 22 Iranian patients with AR‐CGD were found. These three novel mutations can partly complete the database of Human Gene Mutation Database (HGMD) and other related ones. It can also be helpful for further prenatal diagnosis in the affected families. Given that currently bone marrow transplantation is considered to be the curative treatment for patients with CGD, finding mutations will also be useful for timely decision‐making in bone marrow transplantation.     

Genetical Analysis of All Danish Patients Diagnosed with Chronic Granulomatous Disease

Chronic granulomatous disease (CGD) is a rare inherited disorder of the innate immune system caused by a defect in NADPH oxidase, leaving the granulocytes unable to kill invading microorganisms. CGD is caused by mutation in one of the five components gp91phox, p22phox, p47phox, p67phox and p40phox, encoded by the X‐linked CYBB gene and the autosomal CYBA, NCF1, NCF2 and NCF4 genes respectively. We have collected samples from all Danish patients with known CGD followed in the clinic or newly diagnosed during a 5‐year period, a cohort of 27 patients, and characterized them genetically. The cohort includes 10 male patients with X‐linked CGD and one female with extremely lyonized expression of a defective CYBB allele. Six patients had mutation in CYBA. Seven of 10 patients with a defect in NCF1 were homozygous for the common GT deletion, one was compound heterozygous for the GT deletion and a splice‐site mutation, and two patients were homozygous for a nonsense mutation in exon 7. Three novel mutations were detected, a deletion of exon 6 in CYBA, a duplication of exon 8–13 in CYBB and a splice site mutation in intron 7 of NCF1.     

Molecular analysis of X-linked chronic granulomatous disease in five unrelated Korean patients

Chronic granulomatous disease (CGD) is a fatal genetic disorder in which phagocytes fail to produce antimicrobial superoxide because of NADPH oxidase deficiency. Molecular defects in CYBB gene causing X-linked CGD are responsible for about 70% of all cases. This study was done to confirm genetic defects of CYBB gene in five Korean patients who were highly suggestive of having CGD by clinical history. We performed initial screening for five unrelated Korean patients using single strand conformation polymorphism (SSCP) and then selective sequencing for the regions involving the abnormal bands. Activated NBT tests revealed that all patients were X-linked. SSCP analysis for CYBB gene showed abnormal bands in all patients. The molecular defects of five patients were as follows: c.1663insT, c.1111-1G>T, c.39_40insG, c.927delC and c.434T>C mutation. This result will help the families with prenatal diagnosis or genetic counseling.     

Genetic and mutational heterogeneity of autosomal recessive chronic granulomatous disease in Tunisia

NADPH oxidase, a multi-subunit protein consisting of cytosolic components and the membrane-bound heterodimer, plays an instrumental role in host defence mechanisms of phagocytes. Genetic deficiency of the enzymatic complex results in an inherited disorder, chronic granulomatous disease (CGD), which is characterized by an impaired phagocyte microbicidal activity. X-Linked (XL) CGD results from a mutation in the CYBB gene encoding the gp91phox subunit, while autosomal recessive (AR) CGD is associated with mutations in one of the NCF1, NCF2 and CYBA genes that encode the p47phox, p67phox and p22phox subunits, respectively. In the study reported here, we investigated genetic defects underlying CGD in 15 Tunisian patients from 14 unrelated families. Haplotype analyses and homozygosity mapping with microsatellite markers around known CGD genes assigned the genetic defect to NCF1 in four patients, to NCF2 in four patients and to CYBA in two patients. However, one family with two CGD patients seemed not to link the genetic defect to any known AR-CGD genes. Mutation screening identified two novel mutations in NCF2 and CYBA in addition to the recurrent mutation, ΔGT, in NCF1 and a splice site mutation previously reported in a North African patient. Our results revealed the genetic and mutational heterogeneity of the AR recessive form of CGD in Tunisia.