Mutations In NCF2 Research Articles

A founder effect for p47phoxTrp193Ter 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 p47phox 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 1200–2300years 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 5000years.

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 patients from 14 unrelated families had X-linked CGD (66.7 %), four had mutations in the NCF1 gene (19 %), and three, from two unrelated families, had mutations in NCF2 (9.5 %) [Corrected]. 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.

A Glucose-6-Phosphate Isomerase Peptide Induces T and B Cell–Dependent Chronic Arthritis in C57BL/10 Mice: Arthritis without Reactive Oxygen Species and Complement

Immunization with human glucose-6-phosphate isomerase (hG6PI) protein or with several of its peptides induces arthritis in DBA/1 mice. We investigated G6PI peptide-induced arthritis in C57BL/10 mice and the effect of oxidative burst on disease. To study the arthritogenicity of G6PI peptides and its immune dependency, we used genetically modified and congenic mice on the C57BL/10 background and in vitro T- and B-cell assays. hG6PI(325-339) peptide induced arthritis in C57BL/10 mice. The disease was associated with major histocompatibility complex class II and was dependent on T cells, B cells, and complement C5. Th1 and Th17 cells primed with the hG6PI(325-339) peptide cross-reacted with the murine G6PI protein. The severity of the disease increased in mice carrying a mutation in Ncf1 (Ncf1*/*), which abolishes the NADPH oxidase 2 complex oxidative burst. Ncf1*/* mice developed arthritis also on immunization with the mouse G6PI325-339 peptide and in the absence of C5. The antibody responses to the G6PI protein and peptides were minimal in both Ncf1*/* and wild-type mice. Herein is described G6PI peptide as the first peptide to induce arthritis in C57BL/10 mice. The differences between the wild-type and Ncf1*/* mice suggest that an alternative complement-independent arthritogenic pathway could be operative in the absence of oxidative burst.

Different unequal cross-over events between NCF1 and its pseudogenes in autosomal p47phox-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 p47phox. 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 p47phox-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 p47phox-deficient CGD is genetically heterogeneous but can be dissected in detail by MLPA.

Abstract 129: Investigation of Atherosclerosis in Association with Arthritic Inflammation

Background and Aims It is well established that patients with Rheumatoid Arthritis (RA) have a higher risk of developing coronary artery disease (CAD). However, it is unclear how the inflammatory process affects atherosclerosis and what specific factors are involved. In this study, we investigated the effect of the pro-atherogenic factor hyperlipidaemia on arthritis incidence and severity. Simultaneously, we have investigated the mutation of the neutrophil cytosolic factor 1 (Ncf-1), a known genetic factor that promotes arthritis susceptibility, and MHCII H-2q on atherosclerosis development. Results We observed that diet-induced hyperlipidaemia prior to induction of collagen induced arthritis (CIA) protected mice against the disease while genetically hyperlipidemic without the use of diet, Ldlr-/- x human ApoB100 transgenic (Ldlr-/-hApoBtg), were equally susceptible to CIA compared to their heterozygous littermates. Next, we investigated Ncf-1 mutation on Ldlr-/-hApoBtg mice. Surprisingly, despite the increased inflammatory/arthritic phenotype induced by Ncf-1 mutation, no difference in the atherosclerotic lesion size was observed. Conclusions Our data shows that hyperlipidaemia-induced by diet have substantial effects on arthritis susceptibility, which in turn differs from hyperlipidaemia acquired on birth due to genetic alterations. By developing a model carrying pro-arthritogenic factors, MHCII H-2q and Ncf-1 mutation, as well as the pro-atherosclerotic factors, Ldlr-/- and hApoBtg, we have established a new model where both arthritis and atherosclerosis are present. These mice will be used to further characterize the impact of systemic inflammation in the form of arthritis in the atherosclerotic process and vice versa.

Functional and genetic characterization of two extremely rare cases of Williams–Beuren Syndrome associated with chronic granulomatous disease

Williams-Beuren syndrome (WBS) is a neurodevelopmental disorder with multi-systemic manifestations, caused by a heterozygous segmental deletion of 1.55-1.83 Mb at chromosomal band 7q11.23. The deletion can include the NCF1 gene that encodes the p47(phox) protein, a component of the leukocyte NADPH oxidase enzyme, which is essential for the defense against microbial pathogens. It has been postulated that WBS patients with two functional NCF1 genes are more susceptible to occurrence of hypertension than WBS patients with only one functional NCF1 gene. We now describe two extremely rare WBS patients without any functional NCF1 gene, because of a mutation in NCF1 on the allele not carrying the NCF1-removing WBS deletion. These two patients suffer from chronic granulomatous disease with increased microbial infections in addition to WBS. Interestingly, one of these patients did suffer from hypertension, indicating that other factors than NADPH oxidase in vascular tissue may be involved in causing hypertension.

Reactive Oxygen Species Produced by the NADPH Oxidase 2 Complex in Monocytes Protect Mice from Bacterial Infections

Chronic granulomatous disease (CGD) is an inherited disorder characterized by recurrent life-threatening bacterial and fungal infections. CGD results from defective production of reactive oxygen species by phagocytes caused by mutations in genes encoding the NADPH oxidase 2 (NOX2) complex subunits. Mice with a spontaneous mutation in Ncf1, which encodes the NCF1 (p47(phox)) subunit of NOX2, have defective phagocyte NOX2 activity. These mice occasionally develop local spontaneous infections by Staphylococcus xylosus or by the common CGD pathogen Staphylococcus aureus. Ncf1 mutant mice were more susceptible to systemic challenge with these bacteria than were wild-type mice. Transgenic Ncf1 mutant mice harboring the wild-type Ncf1 gene under the human CD68 promoter (MN(+) mice) gained the expression of NCF1 and functional NOX2 activity specifically in monocytes/macrophages, although minimal NOX2 activity was also detected in some CD11b(+)Ly6G(+) cells defined as neutrophils. MN(+) mice did not develop spontaneous infection and were more resistant to administered staphylococcal infections compared with MN(-) mice. Most strikingly, MN(+) mice survived after being administered Burkholderia cepacia, an opportunistic pathogen in CGD patients, whereas MN(-) mice died. Thus, monocyte/macrophage expression of functional NCF1 protected against spontaneous and administered bacterial infections.

A new model of arthritis induced by a glucose-6-phosphate isomerase peptide: immunological requirements and peptide characterisation

Background and objectivesImmune reactivity to the ubiquitous glucose-6-phosphate isomerase (GPI) protein leads to arthritis development. Immunisation with human GPI protein leads to severe arthritis on DBA/1 background, but not on...

Collagen Type II and a Thermo-Responsive Polymer of N-Isopropylacrylamide Induce Arthritis Independent of Toll-Like Receptors : A Strong Influence by Major Histocompatibility Complex Class II and Ncf1 Genes

We established and characterized an arthritis mouse model using collagen type II (CII) and a thermo-responsive polymer, poly(N-isopropylacrylamide) (PNiPAAm). The new PNiPAAm adjuvant is TLR-independent, as all immunized TLR including MyD88-deficient mice developed an anti-CII response. Unlike other adjuvants, PNiPPAm did not skew the cytokine response (IL-1β, IFN-γ, IL-4, and IL-17), as there was no immune deviation towards any one type of immune spectrum after immunization with CII/PNiPPAm. Hence, using PNiPAAm, we studied the actual immune response to the self-protein, CII. We observed arthritis and autoimmunity development in several murine strains having different major histocompatibility complex (MHC) haplotypes after CII/PNiPAAm immunization but with a clear MHC association pattern. Interestingly, C57Bl/6 mice did not develop CII-induced arthritis, with PNiPAAm demonstrating absolute requirement for a classical adjuvant. Presence of a gene (Ncf1) mutation in the NADPH oxidation complex has a profound influence in arthritis and using PNiPAAm we could show that the high CIA severity in Ncf1 mutated mice is independent of any classical adjuvant. Macrophages, neutrophils, eosinophils, and osteoclasts but not mast cells dominated the inflamed joints. Furthermore, arthritis induction in the adjuvant-free, eosinophil-dependent Vβ12 DBA/1 mice could be shown to develop arthritis independent of eosinophils using CII/PNiPAAm. Thus, biocompatible and biodegradable PNiPAAm offers unique opportunities to study actual autoimmunity independent of TLR and a particular cytokine phenotype profile.

CD68‐expressing cells can prime T cells and initiate autoimmune arthritis in the absence of reactive oxygen species

It is widely believed that DC, but not macrophages, prime naïve T cells in vivo. Here, we investigated the ability of CD68-expressing cells (commonly defined as macrophages) in priming autoreactive T cells and initiating collagen-induced arthritis (CIA) in the mouse. For this purpose, a transgenic mouse was developed (MBQ mouse) where macrophages exclusively expressed the MHC class II H2-A(q) (A(q)) on an H2-A(p) (A(p)) background. A(q), but not A(p) expression mediates susceptibility to CIA through presentation of type II collagen (CII) to T cells. CIA severity is enhanced by a mutation in the Ncf1 gene, impairing reactive oxygen species (ROS) production by the phagocyte NADPH oxidase (NOX2) complex. Expression of functional Ncf1 on macrophages was previously shown to protect from severe CIA. To study the effect of ROS on macrophage-mediated priming of T cells, the Ncf1 mutation was introduced in the MBQ mouse. Upon CII immunization, Ncf1-mutated MBQ mice, but not Ncf1 wild-type MBQ mice nor Ncf1-mutated A(p) mice, activated autoreactive T cells and developed CIA. These findings demonstrate for the first time that macrophages can initiate arthritis and that the process is negatively regulated by ROS produced via the NOX2 complex.

Four different NCF2 mutations in six families from Turkey and an overview of NCF2 gene mutations

One of the rarest forms of autosomal recessive chronic granulomatous disease (AR-CGD) is attributable to mutations in the NCF2 gene, which encodes the polypeptide p67(phox), a key cytoplasmic protein in the phagocyte NADPH oxidase system. NCF2 is localized on chromosome 1q25, encompasses 40 kb and contains 16 exons. We report here the clinical and molecular characterization of six patients with CGD from six consanguineous Turkish families. The ages of the five female patients were between 3 and 22 years and a male patient was 2 years old; all patients showed clear clinical symptoms of CGD. The mothers of the patients did not show a bimodal histogram pattern specific for X-CGD in the dihydrorhodamine-1,2,3 (DHR) assay. Moreover, p67(phox) protein expression was not detectable using flow cytometric analysis of the patients' neutrophils except in those from patient 6, which had a diminished expression. Mutation analysis of NCF2 revealed four different homozygous mutations: a novel nonsense mutation in exon 3 c.229C>T, p.Arg77X; a novel missense mutation in exon 4 c.279C>G, p.Asp93Glu; a nonsense mutation in exon 4 c.304C>T, p.Arg102X; and a novel missense mutation in exon 6 c.605C>T, p.Ala202Val. The parents were found to be heterozygotes for these mutations. The prevalence of NCF2 mutant families is approximately 15% in our series of 40 CGD families. This high incidence of A67 CGD in Turkey is undoubtedly caused by the high incidence of consanguineous marriages. We found three new mutations in NCF2 and one previously described. These are presented together with an overview of all NCF2 mutations now known.

Detection of Non-ΔGTNCF-1Mutations in Chronic Granulomatous Disease

Chronic granulomatous disease (CGD) is a rare inherited disorder caused by mutations in the subunits of the NADPH oxidase complex, leaving phagocytes unable to produce superoxide and thereby unable to kill invading microorganisms. A subgroup of CGD patients (approximately 20%) is reported to have mutations in NCF-1 encoding p47-phox, which is part of the cytosolic component of NADPH oxidase. More than 94% of these patients share the same mutation, a 2 bp GT deletion in the GTGT dinucleotide repeat in the start of exon 2. The presence of two pseudogenes more than 98% homologous to the functional NCF-1 has complicated the identification of other mutations in the gene. The aim of this study was to find a general technique for detection of non-GT deletion mutations in the coding region of NCF-1. A technique involving GeneScan analysis followed by amplification of cDNA with intact dinucleotide repeat was set up and used to identify a novel mutation in exon 7 of NCF-1 in a patient with autosomal recessive CGD, explaining the disease by changing a UGG codon to a premature UGA STOP codon. The method is generally applicable for the detection of NCF-1 mutations in patients with suspected CGD.

Ncf1-Associated Reduced Oxidative Burst Promotes IL-33R+ T Cell-Mediated Adjuvant-Free Arthritis in Mice

Reactive oxygen species (ROS) are important in the immune defense against invading pathogens, but they are also key molecules in the regulation of inflammatory reactions. Low levels of ROS production due to a polymorphism in the neutrophil cytosolic factor 1 (Ncf1) gene are associated with autoimmunity and arthritis severity in mouse models induced with adjuvant. We established an adjuvant-free arthritis model in which disease is induced by injection of the autoantigen collagen type II (CII) and depends on IL-5-producing T cells and eosinophils. In addition, the transgenic expression of mutated mouse CII allowed us to investigate an autoreactive immune response to an autologous Ag and by that natural tolerance mechanism. We show that a deficient ROS production, due to a spontaneous mutation in Ncf1, leads to increased autoantibody production and expansion of IL-33R-expressing T cells, impaired T cell tolerance toward tissue-specific CII, and severe arthritis in this unique model without disturbing adjuvant effects. These results demonstrate that the insufficient production of ROS promotes the breakdown of immune tolerance and development of autoimmune and adjuvant-free arthritis through an IL-5- and IL33R-dependent T cell activation pathway.

A novel mutation in NCF1 in an adult CGD patient with a liver abscess as first presentation

Chronic granulomatous disease (CGD) is an immunodeficiency caused by defects in the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase complex and is usually diagnosed in early childhood. CGD patients suffer from severe, recurrent infections with bacteria, fungi and yeasts. We report a 25-year-old female with protracted fever because of a Staphylococcus aureus liver abscess, which did not resolve until breakthrough into the stomach. Despite her age, CGD was considered on diagnosis on the basis of the clinical symptoms. Analysis of the NADPH-oxidase activity confirmed CGD as the underlying condition. Western blotting revealed the absence of p47(phox) and subsequent sequencing of the p47(phox)-encoding gene, neutrophil cytosolic factor (NCF1), identified a deletion of 837C in the maternal NCF1 allele. The paternal allele contained a stopcodon because of a conversion between NCF1 and one of its PsiNCF1 pseudogenes. The patient had one novel mutation, c.837delC, and one conversion in NCF1, resulting in the complete absence of the p47(phox) component of the NADPH-oxidase complex. This p47(phox)-deficient CGD patient had the highest age at diagnosis reported thus far.

Lack of Reactive Oxygen Species Breaks T Cell Tolerance to Collagen Type II and Allows Development of Arthritis in Mice

The view on reactive oxygen species (ROS) in inflammation is currently shifting from being considered damaging toward having a more complex role in regulating inflammatory reactions. We recently demonstrated a role of ROS in regulation of animal models for the autoimmune disease rheumatoid arthritis. Low levels of ROS production, due to a mutation in the Ncf1 gene coding for the Ncf1 (alias p47(phox)) subunit of the NADPH oxidase complex, was shown to be associated with increased autoimmunity and arthritis severity in both rats and mice. To further investigate the role of ROS in autoimmunity, we studied transgenic mice expressing collagen type II (CII) with a mutation (D266E) in the immunodominant epitope that mimics the rat and human CII (i.e., mutated mouse collagen or MMC). This mutation results in a stronger binding of the epitope to the MHC class II molecule and leads to more pronounced tolerance and resistance to arthritis induced with rat CII. When the Ncf1 mutation was bred into these mice, tolerance was broken, resulting in enhanced T cell autoreactivity, high titers of anti-CII Abs, and development of severe arthritis. These findings highlight the importance of a sufficient ROS production in maintenance of tolerance to self-Ags, a central mechanism in autoimmune diseases such as rheumatoid arthritis. This is important as we, for the first time, can follow the effect of ROS on molecular mechanisms where T cells are responsible for either protection or promotion of arthritis depending on the level of oxygen species produced.

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, DeltaGT, 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.

NCF1 (p47phox) and NCF1 Pseudogenes Are Not Associated with Inflammatory Bowel Disease

Crohn's disease (CD) and ulcerative colitis (UC) have a strong genetic component, contributing to a patient's susceptibility for inflammatory bowl disease (IBD). Linkage analysis has detected an IBD susceptibility locus in a region on chromosome 7q that encompasses the p47 (NCF1) gene and p47 (PsiNCF1) pseudogenes. Involvement of the NCF1 locus in IBD was supported by the observation that chronic inflammation of the bowel is a feature of chronic granulomatous disease caused by NCF1 mutation in 25% of cases. The pseudogenes have a dinucleotide deletion (PsiGT) at the beginning of exon 2, resulting in a frameshift and premature stop codon. APsiNCF1 (DeltaGT) to NCF1 (GTGT) ratio of 2:1 has been proposed as the predominant ratio in humans; but variability may occur after DNA exchange by recombination between PsiNCF1 and NCF1 to produce a potentially functional gene hybrid (type IIPsiNCF1). A preliminary study suggested an association between individuals with a 1:1 ratio and susceptibility to IBD. The possible presence of type IIPsiNCF1 was proposed as a susceptibility factor. We have now established the PsiNCF1 to NCF1 ratio for a significant number of IBD patients (n = 488) and control subjects (n = 181) and show that there is no statistically significant difference between the frequency of the 1:1 ratio in CD (11.2%) or UC (12.2%) patients and controls (13.4%). The 2:1 ratio was identified as the most common ratio (83.3%). Our data show there is no association of the 1:1 ratio with IBD and that susceptibility is unlikely to be a consequence of an inherited 1:1, rather than a 2:1 (PsiNCF1:NCF1) ratio.

The use of animal models to find and understand arthritis genes

Inbred animals are useful for studies of the identification of genes associated with rheumatoid arthritis (RA) since they are more efficient tools for identification of genes controlling complex diseases. There are several arthritis models, which each may reflect various variants of the heterogeneity of RA in humans. Examples are collagen-induced arthritis (CIA) and pristane-induced arthritis, which both fulfill the clinical diagnostic criteria for RA. Type II collagen (CII) is immunogenic and contains peptides that can be bound to major histocompatibility complex (MHC) class II and can be presented to T cells, whereas pristane is not immunogenic by itself. Both diseases are genetically complex and the susceptibility is, as RA, dependent on many polymorphic genes operating in concert. So far two of these genes have been identified; the MHC class II Ab gene in the mouse [1] and the Ncf1 gene in the rat [2]. The Ncf1 protein is a part of the NADPH oxidase complex involved in generation of the inducible oxidative burst. The discovery of the Ncf1 polymorphism led to a new proposed pathway in which oxygen radicals modify antigen presentation and the resulting activation of autoreactive T cells. This hypothesis has now been further documented by the identification of an Ncf1 mutation in the mouse that reproduces the effects earlier observed in the rat. Mice with the deficient Ncf1 allele, and expressing the MHC class II allele Aq, binding CII peptides, could be shown to be dramatically more susceptible to CIA, and also developed a chronic form of arthritis. Interestingly, the immune response to CII was enhanced by the Ncf1 deficiency linking the Ncf1 pathway to the adaptive immune response.

Autosomal recessive chronic granulomatous disease caused by novel mutations in NCF-2, the gene encoding the p67-phox component of phagocyte NADPH oxidase

Autosomal recessive chronic granulomatous disease caused by novel mutations in NCF-2, the gene encoding the p67-phox component of phagocyte NADPH oxidase