Immune Thrombocytopenic Purpura (ITP) and Chorioretinopathy in Chronic Granulomatous Disease: A Case Report.

BACKGROUND: Chronic Granulomatous Disease (CGD) is a rare immunodeficiency disorder characterized by impaired phagocytic function, leading to recurrent infections and granuloma formation. Genetic mutations in NADPH oxidase complex components, such as CYBB, NCF1, NCF2, and CYBA genes, contribute to the pathogenesis. This case report explores the possible ocular and hematologic complications associated with CGD. CASE PRESENTATION: A 6-year-old girl with a history of vitrectomy, membranotomy, and laser therapy due to congenital blindness (diagnosed with chorioretinopathy) was referred to the hospital with generalized ecchymosis and thrombocytopenia. Diagnostic workup initially suggested chronic immune thrombocytopenic purpura (ITP). Subsequent admissions revealed necrotic wounds, urinary tract infections, and recurrent thrombocytopenia. Suspecting immunodeficiency, tests for CGD, Nitroblue tetrazolium (NBT) and dihydrorhodamine (DHR) were performed. She had a low DHR (6.7), and her NBT test was negative (0.0%). Her whole exome sequencing results confirmed autosomal recessive CGD with a homozygous NCF1 mutation. CONCLUSION: This case underscores the diverse clinical manifestations of CGD, including recurrent thrombocytopenia and possible early-onset ocular involvement. The diagnostic challenges highlight the importance of a multidisciplinary approach involving hematologists, immunologists, and ophthalmologists for accurate diagnosis and management. The rare coexistence of ITP in CGD emphasizes the intricate link between immunodeficiency and autoimmunity, requiring tailored therapeutic strategies.

The association of APOH and NCF1 polymorphisms on susceptibility to recurrent pregnancy loss in women with antiphospholipid syndrome.

BACKGROUND: Recurrent pregnancy loss (RPL) is the main manifestation of pathological pregnancy in antiphospholipid syndrome (APS) women. The immune state plays a significant role in the occurrence/development of APS and RPL susceptibility, but there is little research on genetic factors. METHOD: Previous studies have described the important role of APOH and NCF1 in APS and pregnancy. To explore the association of APOH and NCF1 gene variants with RPL susceptibility in APS patients, we collected and analyzed 871 controls, 182 APS and RPL, and 231 RPL patients. Four single nucleotide polymorphisms (SNPs) (rs1801690, rs52797880, and rs8178847 of APOH and rs201802880 of NCF1) were selected and genotyped. RESULTS: We found rs1801690 (p = 0.001, p = 0.003), rs52797880 (p = 8.73e-04, p = 0.001), and rs8178847 (p = 0.001, p = 0.001) of APOH and rs201802880 (p = 3.77e-26, p = 1.31e-26) of NCF1 showed significant differences between APS and RPL patients and controls in allelic and genotype frequencies respectively. Moreover, rs1801690, rs52797880, and rs8178847 showed strong linkage disequilibrium. Especially, our results revealed a complete linkage disequilibrium (D' = 1) between rs52797880 and rs8178847. Furthermore, higher serum TP (total protein) level was described in APOH rs1801690 CG/GG (p = 0.007), rs52797880 AG/GG (p = 0.033), and rs8178847 CT/TT (p = 0.033), while the higher frequency of positive serum ACA-IgM was found in NCF1 rs201802880 GA (p = 0.017) in APS and RPL patients. CONCLUSION: Rs1801690, rs52797880, and rs8178847 of APOH and rs201802880 of NCF1 were associated with RPL susceptibility in APS patients.

NAPDH Oxidase-Specific Flow Cytometry Allows for Rapid Genetic Triage and Classification of Novel Variants in Chronic Granulomatous Disease.

PURPOSE: Chronic granulomatous disease (CGD) is an innate immune deficiency, primarily affecting the phagocytic compartment, and presenting with a diverse phenotypic spectrum ranging from severe childhood infections to monogenic inflammatory bowel disease. Dihydrorhodamine (DHR) flow cytometry is the standard diagnostic test for CGD, and correlates with NADPH oxidase activity. While there may be genotype correlation with the DHR flow pattern in some patients, in several others, there is no correlation. In such patients, assessment by flow cytometric evaluation of NADPH oxidase-specific (NOX) proteins provides a convenient and rapid means of genetic triage, though immunoblotting has long been used for this purpose. METHODS AND RESULTS: We describe the clinical utility of the NOX flow cytometry assay through assessment of X-linked and autosomal recessive CGD patients and their first-degree relatives. The assessment of specific NOX proteins was correlated with overall NADPH oxidase function (DHR flow), clinical phenotype and genotype. NOX-specific protein assessment is a valuable adjunct to DHR assessment and genotyping to classify and characterize CGD patients. CONCLUSIONS: The atypical clinical presentation of some CGD patients can make genotype-phenotype correlation with DHR flow data challenging. Genetic testing, while useful for confirmation of diagnosis, can take several weeks, and in some patients does not provide a conclusive answer. However, NADPH-oxidase-specific protein flow assessment offers a rapid alternative to identification of the underlying genetic defect in cellular subsets, and can be utilized as a reflex test to an abnormal DHR flow. Further, it can provide insight into correlation between oxidative burst relative to protein expression in granulocytes and monocytes.

Independent and inter-dependent immunoregulatory effects of NCF1 and NOS2 in experimental autoimmune encephalomyelitis.

BACKGROUND: Increasing evidence has suggested that a single nucleotide polymorphism in the Ncf1 gene is associated with experimental autoimmune encephalomyelitis (EAE). However, the mechanisms of NCF1-induced immunoregulatory effects remain poorly understood. In this study, we focus on NCF1 deficiency-mediated effects on EAE in NOS2 dependent and independent ways. METHODS: To determine the effects of NCF1 and NOS2 during EAE development, we have established recombinant mouse strains deficient at NCF1 and/or NOS2 in a crossbreeding system. Different strains allow us to examine the entire course of the disease in the Nos2-null mice bearing a Ncf1 gene that encodes a mutated NCF1, deficient in triggering oxidative burst, after immunization with recombinant myelin oligodendrocyte glycoprotein (MOG)79-96 peptides. The peptide-induced innate and adaptive immune responses were analyzed by flow cytometry. RESULTS: NCF1-deficient mice developed a reduced susceptibility to EAE, whereas NCF1-NOS2 double-deficient mice developed an enhanced EAE, as compared with NOS2-deficient mice. Flow cytometry analyses show that double deficiencies resulted in an increase of neutrophils in the spleen, accompanied with higher release of interleukin-1ß in neutrophils prior to EAE onset. The additional deficiency in NCF1 had no added effect on either interleukin-17 or interferon-γ secretion of T cells during the priming phase. CONCLUSIONS: These studies show that NCF1 and NOS2 interact to regulate peptide-induced EAE.

Suppression of the Reactive Oxygen Response Alleviates Experimental Autoimmune Uveitis in Mice.

Reactive oxygen species (ROS) are produced by host phagocytes and play an important role in antimicrobial actions against various pathogens. Autoimmune uveitis causes blindness and severe visual impairment in humans at all ages worldwide. However, the role of ROS in autoimmune uveitis remains unclear. We used ROS-deficient (Ncf1-/-) mice to investigate the role of ROS in experimental autoimmune uveitis (EAU). Besides, we also used the antioxidant N-acetylcysteine (NAC) treatment to evaluate the effect of suppression of ROS on EAU in mice. The EAU disease scores of Ncf1-/- mice were significantly lower than those of wild-type mice. EAU induction increased the levels of cytokines (interleukin (IL)-1α, IL-1ß, IL-4, IL-6, IL-12, IL-17, and tumor necrosis factor (TNF)-α) and chemokines (monocyte chemoattractant protein (MCP)-1) in the retinas of wild-type mice but not in those of Ncf1-/- mice. EAU induction enhanced the level of NF-κB activity in wild-type mice. However, the level of NF-κB activity in Ncf1-/- mice with EAU induction was low. Treatment with the antioxidant NAC also decreased the severity of EAU in mice with reduced levels of oxidative stress, inflammatory mediators, and NF-κB activation in the retina. We successfully revealed a novel role of ROS in the pathogenesis of EAU and suggest a potential antioxidant role for the treatment of autoimmune uveitis in the future.

Ncf1 polymorphism reveals oxidative regulation of autoimmune chronic inflammation.

The current review on the function of neutrophil cytosolic factor 1 (NCF1) and induced reactive oxygen species (ROS) is based on a genetic search for the major genes controlling autoimmune inflammatory disorders. Surprisingly, the disease-promoting allele determined a lower ROS response and was therefore in complete contrast to the prevailing dogma. Once cloned, it opened the possibility to dissect this complex field from a new angle and with the possibilities to study the role of ROS in vivo. We found that NCF1 and NADPH oxidase 2 (NOX2) complex-derived ROS is an important regulator of several chronic inflammatory disorders by using models for rheumatoid arthritis, multiple sclerosis, psoriasis and psoriasis arthritis, gout, and lupus. ROS could therefore affect many different types of diseases and the common denominator seems to be that ROS regulate macrophages, which prevents inflammation from going chronic. The role of ROS is currently changing from being seen as toxic agents that will promote inflammation toward a more complex view with ROS as crucial regulators of immune and inflammatory pathways.

S-Glutathionylation of p47phox sustains superoxide generation in activated neutrophils.

Post-translational modifications (PTMs) induced conformational changes of proteins can cause their activation or inactivation. Neutrophils clear pathogen through phagocytosis and oxidative burst generation, while participate in inflammation through sustained and uncontrolled generation of ROS. In activated PMNs, cytosolic NOX-2 subunit p47phox following phosphorylation interacts with p67phox, p40phox and along with Rac2 translocate to the membrane. Phosphorylation of p47phox subunit occurs in both short spurts as well as sustained ROS generation, suggesting towards the unidentified molecular mechanism(s) driving these two diverse outcomes by various stimuli. The present study demonstrates that in PMA or NO treated neutrophils a subunit of NOX2, p47phox gets glutathionylated to sustain ROS generation along with a decrease in catalase, Grx-1 activity and change in GSH/GSSG ratio. Surprisingly, fMLP treated cells neither showed sustained ROS production nor glutathionylation of p47phox. S-Glutathionylation was always secondary to phosphorylation of p47phox and inhibition of glutathionylation did not alter phosphorylation but specifically impaired sustained ROS production. Interestingly, forced S-glutathionylation of p47phox converted the fMLP induced ROS generation into sustained release of ROS. We then identified the glutathionylation susceptible cysteine residues of p47phox by LC-MS/MS with IAM switch mapping. Site-directed mutagenesis of cysteine residues further mitigated p47phox S-glutathionylation. Thus, we demonstrate that p47phox S-glutathionylation plays an essential key role in the sustained ROS generation by human neutrophils.

Genetic and molecular findings of 38 Iranian patients with chronic granulomatous disease caused by p47-phox defect.

One of the components of NADPH oxidase is p47-phox, encoded by NCF1 gene. This study aims to find new genetic changes and clinical features in 38 Iranian patients with autosomal recessive chronic granulomatous disease (AR-CGD) caused by NCF1 gene defect. Patients who had abnormal NBT and DHR-1,2,3 assay with loss of p47-phox in Western blotting were included in this study. After recording demographic and clinical data, PCR amplification was performed followed by direct sequencing for all exons and exon-intron boundaries. The most common form of CGD in Iran was AR-CGD due to consanguinity marriages. Among patients with AR-CGD, NCF1 deficiency was found to be more common than other forms. Cutaneous involvements (53%), pulmonary infections (50%) and lymphadenopathy (29%) were more prevalent than other clinical manifestations of CGD. Mutation analysis of NCF1 gene identified five different mutations. Homozygous delta GT deletion (c.75_76delGT) was the most frequent mutation and was detected in more than 63% of families. Six families had a nonsense mutation in exon 7 (c.579G > A). Two novel mutations were found in exon 4 in two families, including a missense mutation (c.328C > T) and a nine-nucleotide deletion (c.331_339delTGTCCCCAC). Genetic detection of these mutations may result in early diagnosis and prevention of possible complications of the disease. This could be useful for timely decision-making for haematopoietic stem cell transplantation and for carrier detection as well as prenatal diagnosis of next children in the affected families. Our findings might help to predict outcomes, raise awareness and help effective treatment in these patients.

ATPR-induced differentiation and G0/G1 phase arrest in acute promyelocytic leukemia by repressing EBP50/NCF1 complex to promote the production of ROS.

Our previous study has demonstrated that 4-amino-2-trifluoromethyl-phenyl Retinate (ATPR) can induce human leukemia NB4 cells differentiation and G0/G1 phase arrest, but the underlying mechanism is still unclear. In this study, we used proteomics to screen differentially expressed protein profiles in NB4 cells before and after ATPR treatment in vitro. We analyzed the peptides digested from total cellular proteins by reverse phase LC-MS/MS and then performed label-free quantitative analysis. We found 27 significantly up-regulated proteins in the ATPR group compared to the control group. NCF1 was the most significantly changed protein. Immunoprecipitation and double immunofluorescent staining showed that EBP50 bind to NCF1. We further explored the potential molecular mechanism of EBP50/NCF1 complex in ATPR-induced differentiation and G0/G1 phase arrest. The results showed that ATPR remarkably reduced the expression of EBP50 in vivo and in vitro. Interestingly, the reduction of EBP50 contributed to ROS release by modulating the subcellular localization of NCF1. The reduction of EBP50 also contributed to G0/G1 phase arrest by inhibiting CyclinD1, CyclinA2 and CDK4, as well as promoting the differentiation of NB4 cells by increasing the expression of CD11b. Furthermore, we found that the overexpression of EBP50 restrained the effects of ATPR on differentiation and G0/G1 phase arrest in NB4 cells. These results suggest that ATPR-induced differentiation and G0/G1 phase arrest in acute promyelocytic leukemia (APL) by repressing EBP50/NCF1 complex to promote the production of ROS, and the results from in vivo experiments were consistent with those from in vitro studies. Therefore, our finding results suggest that EBP50 may be a new target for ATPR in the treatment of APL.

A false-carrier state for the c.579G>A mutation in the NCF1 gene in Ashkenazi Jews.

BACKGROUND: Mutations in the NCF1 gene that encodes p47phox, a subunit of the NADPH oxidase complex, cause chronic granulomatous disease (CGD). In Kavkazi Jews, a c.579G>A (p.Trp193Ter) mutation in NCF1 is frequently found, leading to CGD. The same mutation is found in about 1% of Ashkenazi Jews, although Ashkenazi CGD patients with this mutation have never been described. METHODS: We used Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), gene scan analysis and Ion Torrent Next Generation Sequencing for genetic analysis, and measured NADPH oxidase activity and p47phox expression. RESULTS: In an Ashkenazi couple expecting a baby, both parents were found to be heterozygotes for this mutation, as was the fetus. However, segregation analysis in the extended family was consistent with the fetus inheriting both carrier alleles from the parents. MLPA indicated four complete NCF1 genes in the fetus and three in each parent. Gene sequencing confirmed these results. Analysis of fetal leucocytes obtained by cordocentesis revealed substantial oxidase activity with three different assays, which was confirmed after birth. In six additional Ashkenazi carriers of the NCF1 c.579G>A mutation, we found five individuals with three complete NCF1 genes of which one was mutated (like the parents), and one individual with in addition a fusion gene of NCF1 with a pseudogene. CONCLUSION: These results point to the existence of a 'false-carrier' state in Ashkenazi Jews and have wide implications regarding pre-pregnancy screening in this and other population groups.

Respiratory Complications Lead to the Diagnosis of Chronic Granulomatous Disease in Two Adult Patients.

Chronic granulomatous disease (CGD) is a primary immunodeficiency associated to multiple life-threatening bacterial and fungal infections, beginning in childhood. There are rare cases of diagnosis in adulthood. We describe here two cases of late diagnosis in adults: a 45-year-old woman and 59-year-old-man. CGD diagnosis should be considered in adult patients with unexplained infectious diseases with tissue granuloma.

A single nucleotide polymorphism in the NCF1 gene leading to reduced oxidative burst is associated with systemic lupus erythematosus.

OBJECTIVES: Ncf1 polymorphisms leading to low production of reactive oxygen species (ROS) are strongly associated with autoimmune diseases in animal models. The human NCF1 gene is very complex with both functional and non-functional gene copies and genotyping requires assays specific for functional NCF1 genes. We aimed at investigating association and function of the missense single nucleotide polymorphism (SNP), rs201802880 (here denoted NCF1-339) in NCF1 with systemic lupus erythematosus (SLE). METHODS: We genotyped the NCF1-339 SNP in 973 Swedish patients with SLE and 1301 controls, using nested PCR and pyrosequencing. ROS production and gene expression of type 1 interferon-regulated genes were measured in isolated cells from subjects with different NCF1-339 genotypes. RESULTS: We found an increased frequency of the NCF1-339 T allele in patients with SLE, 11% compared with 4% in controls, OR 3.0, 95% CI 2.4 to 3.9, p=7.0×10-20. The NCF1-339 T allele reduced extracellular ROS production in neutrophils (p=0.004) and led to an increase expression of type 1 interferon-regulated genes. In addition, the NCF1-339 T allele was associated with a younger age at diagnosis of SLE; mean age 30.3 compared with 35.9, p=2.0×1-6. CONCLUSIONS: These results clearly demonstrate that a genetically controlled reduced production of ROS increases the risk of developing SLE and confirm the hypothesis that ROS regulate chronic autoimmune inflammatory diseases.

Mannan induces ROS-regulated, IL-17A-dependent psoriasis arthritis-like disease in mice.

Psoriasis (Ps) and psoriasis arthritis (PsA) are poorly understood common diseases, induced by unknown environmental factors, affecting skin and articular joints. A single i.p. exposure to mannan from Saccharomyces cerevisiae induced an acute inflammation in inbred mouse strains resembling human Ps and PsA-like disease, whereas multiple injections induced a relapsing disease. Exacerbation of disease severity was observed in mice deficient for generation of reactive oxygen species (ROS). Interestingly, restoration of ROS production, specifically in macrophages, ameliorated both skin and joint disease. Neutralization of IL-17A, mainly produced by γδ T cells, completely blocked disease symptoms. Furthermore, mice depleted of granulocytes were resistant to disease development. In contrast, certain acute inflammatory mediators (C5, Fcγ receptor III, mast cells, and histamine) and adaptive immune players (αß T and B cells) were redundant in disease induction. Hence, we propose that mannan-induced activation of macrophages leads to TNF-α secretion and stimulation of local γδ T cells secreting IL-17A. The combined action of activated macrophages and IL-17A produced in situ drives neutrophil infiltration in the epidermis and dermis of the skin, leading to disease manifestations. Thus, our finding suggests a new mechanism triggered by exposure to exogenous microbial components, such as mannan, that can induce and exacerbate Ps and PsA.

Role of Flow Cytometry in the Diagnosis of Chronic Granulomatous Disease: the Egyptian Experience.

INTRODUCTION: Chronic granulomatous disease (CGD) is an inherited mutational defect in any of the NADPH oxidase complex, CYBB (gp91-phox), NCF1 (p47-phox), CYBA (p22-phox), NCF2 (p67-phox), or NCF4 (p40-phox) leading to inability of phagocytes to perform effective respiratory burst and thus diminished killing of bacteria and fungi. The identification of defective proteins aids in establishing a diagnosis prior to genetic analysis, which is rather labor-intensive, expensive, and time-consuming. AIM: The present study aims at assessing the NADPH proteins by performing the intracellular staining with specific monoclonal antibodies and their assessment on flow cytometry. The use of flow cytometry is less laborious and faster to perform than western blot. It also confirms the diagnosis of CGD and detects the affected components allowing proper management of patients. MATERIALS AND METHODS: Twenty-eight patients from 25 different kindred, clinically suspected as CGD were recruited in Egypt. Dihydrorhodamine test was performed to confirm the diagnosis of the patients. Intracellular staining of NADPH components using specific monoclonal antibodies was performed followed by flow cytometric analysis. RESULTS: The present study revealed that the most common defective protein in our cohort is p22-phox, found in 13 patients (46.4 % of cases) followed by p47-phox in 8 patients (28.6 %), gp91-phox in 5 patients (17.9 %), and finally p67-phox in 2 patients (7.1 %). CONCLUSION: In countries with limited resources and yet large number of CGD patients, the analysis of the defective proteins by flow cytometry is an optimum solution for confirming the diagnosis and is a step for targeted sequencing in families seeking prenatal diagnosis.

Clinical, Immunological, and Molecular Findings of Patients with p47phox Defect Chronic Granulomatous Disease (CGD) in Indian Families.

Chronic granulomatous disease (CGD) is a group of inherited disorder of phagocytes, resulting from mutations in the components of the NADPH oxidase complex. Reduced or absent oxygen radical synthesis seen in these patients leads to impaired killing of intracellular bacteria and fungi. CGD clinically presents with recurrent and life-threatening infections as well as granulomatous inflammatory responses. p47phox encoded by the NCF1 gene is the most common autosomal recessive form of CGD which is often clinically milder. Here, we are presenting the data on clinical and immunological findings in 21 Indian patients with Del GT mutation in the NCF1 gene. Diagnosis of these patients was based on detailed clinical evaluation, measurement of respiratory burst activity by nitro blue tetrazolium and dihydrorhodamine-1,2,3 assay, expression of p47phox by flow cytometry, and molecular confirmation by GeneScan method. Seventeen male and four female patients with median age of onset of 1 year ranging from 1.5 months to 6 years were included in the study. Sixty-two percent (13 out of 21) of patients belonged to a consanguineous marriage with only one family having a history of a previous sibling death. Significant variability in clinical presentation was observed in spite of identical genetic defect ranging from asymptomatic to very severe presentation leading to early death or requiring transplantation. However, none of these patients showed difference in immunological parameters to account for this variability. Thus, this study highlights the phenotypic heterogeneity seen in these patients with Del GT mutation in the NCF1 gene and its implication in management of these patients.

Ncf1 affects osteoclast formation but is not critical for postmenopausal bone loss.

BACKGROUND: Increased reactive oxygen species and estrogen deficiency contribute to the pathophysiology of postmenopausal osteoporosis. Reactive oxygen species contribute to bone degradation and is necessary for RANKL-induced osteoclast differentiation. In postmenopausal bone loss, reactive oxygen species can also activate immune cells to further enhance bone resorption. Here, we investigated the role of reactive oxygen species in ovariectomy-induced osteoporosis in mice deficient in Ncf1, a subunit for the NADPH oxidase 2 and a well-known regulator of the immune system. METHODS: B10.Q wild-type (WT) mice and mice with a spontaneous point mutation in the Ncf1-gene (Ncf1*/*) were ovariectomized (ovx) or sham-operated. After 4 weeks, osteoclasts were generated ex vivo, and bone mineral density was measured using peripheral quantitative computed tomography. Lymphocyte populations, macrophages, pre-osteoclasts and intracellular reactive oxygen species were analyzed by flow cytometry. RESULTS: After ovx, Ncf1*/*-mice formed fewer osteoclasts ex vivo compared to WT mice. However, trabecular bone mineral density decreased similarly in both genotypes after ovx. Ncf1*/*-mice had a larger population of pre-osteoclasts, whereas lymphocytes were activated to the same extent in both genotypes. CONCLUSION: Ncf1*/*-mice develop fewer osteoclasts after ovx than WT mice. However, irrespective of genotype, bone mineral density decreases after ovx, indicating that a compensatory mechanism retains bone degradation after ovx.

A founder effect for p47(phox)Trp193Ter chronic granulomatous disease in Kavkazi Jews.

Chronic granulomatous disease (CGD) is a rare congenital immune deficiency caused by mutations in any of the five genes encoding NADPH oxidase subunits. One of these genes is NCF1, encoding the p47(phox) protein. A group of 39 patients, 14 of whom are of Kavkazi Jewish descent, was investigated for a founder effect for the mutation c.579G>A (p.Trp193Ter) in NCF1. We analyzed various genetic markers in the NCF1 region, including two single nucleotide polymorphisms (SNPs) in NCF1 and two short tandem repeats (STRs) located near NCF1. Most patients were homozygous for the c.579G>A mutation, but three patients were hemizygotes, with a deletion of NCF1 on the other allele, and three patients were compound heterozygotes with another mutation in NCF1. All Kavkazi Jewish patients had a c.295G_c.345T SNP combination in NCF1 and shared a common number of repeats in STR3. In addition, 90% of the Kavkazi Jewish patients shared a common number of repeats in STR1. This uniformity indicates that the c.579G>A mutation in NCF1 was introduced some 1200-2300 years ago in the Kavkazi Jewish population. Variation amongst the other investigated populations from the Middle East indicates that this mutation exists in these non-Kavkazi populations already for more than 5000 years.

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.

Different unequal cross-over events between NCF1 and its pseudogenes in autosomal p47(phox)-deficient chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a rare congenital disorder in which phagocytes cannot generate superoxide (O2(-)) and other microbicidal oxidants due to mutations in one of the five components of the O2(-)-generating NADPH oxidase complex. The most common autosomal subtype of CGD is caused by mutations in NCF1, encoding the NADPH subunit p47(phox). Usually, these mutations are the result of unequal exchange of chromatid between NCF1 and one of its two pseudogenes. We have now investigated in detail the breakpoints within or between these (pseudo) NCF1 genes in 43 families with p47(phox)-deficient CGD by means of multiplex ligase-dependent probe amplification (MLPA). In 24 families the patients totally lacked NCF1 sequences, indicating that in these families the cross-over points are located between NCF1 and its pseudogenes. Six other families were compound heterozygous for a total NCF1 deletion and another mutation in NCF1 on the other allele. In 8 families, the patients lacked NCF1 exons 1-4 but had retained NCF1 exons 6-10, indicating that a cross-over point is located within NCF1 between exons 4 and 6. Similarly, in 4 families a cross-over point was located within NCF1 between exons 2 and 4. Similar cross-overs, in heterozygous form, were observed in family members of the patients. Several patients were compound heterozygous for total and partial NCF1 deletions. Thus, at least three different cross-over points exist within the NCF1 gene cluster, indicating that autosomal p47(phox)-deficient CGD is genetically heterogeneous but can be dissected in detail by MLPA.

Clinical, functional, and genetic characterization of chronic granulomatous disease in 89 Turkish patients.

BACKGROUND: Chronic granulomatous disease (CGD) is a rare primary immunodeficiency disorder of phagocytes resulting in impaired killing of bacteria and fungi. A mutation in one of the 4 genes encoding the components p22(phox), p47(phox), p67(phox), and p40(phox) of the leukocyte nicotinamide dinucleotide phosphate reduced (NADPH) oxidase leads to autosomal recessive (AR) CGD. A mutation in the CYBB gene encoding gp91(phox) leads to X-linked recessive CGD. OBJECTIVE: The aim of this study is to show the correlation between clinical, functional, and genetic data of patients with CGD from Turkey. METHODS: We report here the results of 89 patients with CGD from 73 Turkish families in a multicenter study. RESULTS: Most of the families (55%) have an AR genotype, and 38% have an X-linked genotype; patients from 5 families with a suspected AR genotype (7%) were not fully characterized. We compared patients with CGD according to the severity of NADPH oxidase deficiency of neutrophils. Patients with A22(0), A67(0) or X91(0) phenotypes with a stimulation index of 1.5 or less have early clinical presentation and younger age at diagnosis (mean, 3.2 years). However, in p47(phox)-deficient cases and in 5 other AR cases with high residual oxidase activity (stimulation index ≥ 3), later and less severe clinical presentation and older age at diagnosis (mean, 7.1 years) were found. Pulmonary involvement was the most common clinical feature, followed by lymphadenitis and abscesses. CONCLUSION: Later and less severe clinical presentation and older age at diagnosis are related to the residual NADPH oxidase activity of neutrophils and not to the mode of inheritance. CGD caused by A22(0) and A67(0) subtypes manifests as severe as the X91(0) subtype.