Alternative titles; symbols
HGNC Approved Gene Symbol: NCF4
Cytogenetic location: 22q12.3 Genomic coordinates (GRCh38): 22:36,861,006-36,878,015 (from NCBI)
| Location | Phenotype |
Phenotype MIM number |
Inheritance |
Phenotype mapping key |
|---|---|---|---|---|
| 22q12.3 | Chronic granulomatous disease 3, autosomal recessive | 613960 | Autosomal recessive | 3 |
The NCF4 gene encodes the p40-phox subunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, a multicomponent enzyme system responsible for the oxidative burst in which electrons are transported from NADPH to molecular oxygen, generating reactive oxidant intermediates (summary by Zhan et al., 1996).
Wientjes et al. (1993) cloned the NCF4 gene, which encodes a predicted 339-amino acid protein with a calculated molecular mass of 37 kD. The NCF4 protein has a C-terminal SH3 domain and a large region of sequence similarity with the N-terminus of p47-phox. Immunohistochemical studies showed that the NCF4 protein forms an activation complex with p47-phox (NCF1; 608512) and p67-phox (NCF2; 608515) with which it translocates to the membrane to associate with the flavocytochrome b. The primary association of p40-phox appeared to be with p67-phox.
By Northern blot analysis, Zhan et al. (1996) found that expression of p40-phox mRNA was restricted to hematopoietic cells.
Zhan et al. (1996) determined that the NCF4 gene contains 10 exons spanning approximately 18 kb.
By FISH studies and PCR analysis of a somatic cell hybrid mapping panel, Zhan et al. (1996) localized the NCF4 gene to chromosome 22q13.1.
Zhan et al. (1996) noted that the NADPH oxidase is dormant in resting cells, but upon activation is assembled at the membrane. At the time of activation, the cytosolic complex associates with the membrane subunits gp91-phox (CYBB; 300481) and p22-phox (CYBA; 608508). They cited studies of patients with chronic granulomatous disease (see, e.g., CGD; 306400), an inherited disorder of phagocytic cells, that have unequivocally established the importance of at least 2 cytosolic factors, p47-phox (NCF1; 608512) and p67-phox (NCF2; 608515). The absence of either prevents generation of superoxide by NADPH oxidase and clinically results in the CGD phenotype. The p40-phox protein is a cytosolic component of NADPH oxidase that copurifies with p67-phox. Although its function was not well defined, Zhan et al. (1996) stated that it may be a component of the cytosolic complex.
The NCF4 gene product p40-phox stimulates phagocytosis-induced NADPH oxidase activity via the PX domain at the N terminus that binds to phosphatidylinositol 3-phosphate (PtdIns(3)P), which accumulates on phagosomes (Ellson et al., 2001; Suh et al., 2006; Ellson et al., 2006).
Bravo et al. (2001) determined the 1.7-angstrom x-ray crystal structure of the phox homology (PX) domain from the p40-phox subunit of NADPH oxidase bound to PtdIns(3)P. The crystal structure showed that the PX domain embraces the 3-phosphate on 1 side of a water-filled, positively charged pocket and revealed how 3-phosphoinositide specificity is achieved. A CGD-associated mutation in the p47-phox PX domain (arg42 to gln) that abrogates PtdIns(3)P binding maps to a conserved arg in p40-phox (arg57) that does not directly interact with the phosphoinositide but instead appears to stabilize a critical lipid-binding loop. The SH3 domain present in the full-length protein does not affect soluble PtdIns(3)P binding to the p40-phox PX domain.
Using immunoprecipitation analysis, Kanai et al. (2001) showed that mutation of arg57 in the PX domain of p40-phox or of the analogous residue, arg42, in p47-phox eliminated phosphoinositide binding. Mutation of arg90 in p47-phox markedly reduced, but did not eliminate, phosphoinositide binding. Kanai et al. (2001) concluded that PX domains are specific phosphoinositide-binding modules and that different PX domains have different phosphoinositide specificities.
In a boy with autosomal recessive chronic granulomatous disease-3 (CGD3; 613960), Matute et al. (2009) identified compound heterozygous mutations in the NCF4 gene (10-bp dup, 601488.0001 and R105Q, 601488.0002). In vitro studies of patient neutrophils showed impaired intracellular oxidant production during phagocytosis of serum opsonized S. aureus, although PMA-stimulated superoxide release was normal. The main clinical manifestation in this patient was chronic granulomatous inflammation of the gastrointestinal tract, particularly the colon and rectum, which was difficult to manage. However, he had not had infections with opportunistic pathogens characteristic of other forms of CGD. Matute et al. (2009) noted that genomewide association studies (Rioux et al., 2007) had identified a SNP in the NCF4 gene (rs4821544) that was associated with Crohn disease, suggesting that there may be a relationship between NCF4, phagocytosis-induced oxidant production, and predisposition to inflammatory bowel disease (see IBD; 266600).
In 24 patients from 12 families of various ethnic origins with CGD3, van de Geer et al. (2018) identified homozygous or compound heterozygous mutations in the NCF4 gene (see, e.g., 601488.0002-601488.0006). The mutations, which were found by exome sequencing or targeted panel sequencing and confirmed by Sanger sequencing, segregated with the disorder in the families. There was evidence of age-dependent or incomplete penetrance in 2 families. Mutation types included missense, nonsense, splice site, and a small in-frame deletion. They occurred throughout the gene, and the authors stated that all but 1 (R58C) were absent from the gnomAD database. In vitro functional expression studies in patient-derived cells and EBV-transformed B cells expressing the mutations showed variably impaired production of reactive oxygen species (ROS), indicating dysfunction of the NADPH oxidase complex and a loss-of-function effect. The functional impact depended on the phagocytic cell type and stimulus. Neutrophils and EBV-transformed B cells carrying the mutations showed impaired NADPH oxidase activity. Neutrophils were ineffective against Staphylococcus, had variable activity against E. coli, and retained almost normal defense against fungi. In contrast, NADPH oxidase activity in mononuclear phagocytes was similar to controls. PMA-induced DHR oxidation was normal or only mildly impaired in certain cell types and conditions, reflecting impaired, but not abolished, ROS production.
Ellson et al. (2006) generated p40-phox -/- mice, which were healthy and fertile when kept under pathogen-free barrier conditions. Neutrophils of p40-phox -/- mice showed reduced expression of p67-phox and diminished oxidase responses to a number of stimuli. Defective reactive oxygen species production by p40-phox -/- neutrophils in response to Staphylococcus aureus translated into a severe, CGD-like defect in the killing of this organism in vitro and in vivo. Ellson et al. (2006) concluded that p40-phox is an essential component in bacterial killing.
In a boy with autosomal recessive chronic granulomatous disease-3 (CGD3; 613960), Matute et al. (2009) identified compound heterozygous mutations in the NCF4 gene: a 10-bp duplication (3957_3966dupAAGGAGGATC) in exon 3, resulting in a frameshift and premature termination, and a c.6447G-A transition in exon 4, resulting in an arg105-to-gln substitution (R105Q; 601488.0002) in the PX domain. The frameshift predicts a truncated protein with a molecular mass of 14 kD, a truncated PX domain, and absent SH3 and PB1 domains, which may explain the reduced amount of protein detected in the patient and his carrier father. In vitro studies of patient cells showed impaired intracellular neutrophil oxidant production during phagocytosis of serum opsonized S. aureus, although PMA-stimulated superoxide release was normal. In vitro functional expression studies showed that the mutant R105Q protein remained cytosolic, did not localize to phagosomes or endosomes, and was unable to bind PtdIns(3)P in a lipid-binding assay. Moreover, the R105Q-mutant protein was unable to rescue the NADPH oxidase defect of NCF4 functionally null cells. The main clinical manifestation in this patient was chronic granulomatous inflammation of the gastrointestinal tract, particularly the colon and rectum, which was difficult to manage. However, he had not had infections with opportunistic pathogens characteristic of other forms of CGD.
For discussion of the c.6447G-A transition in the NCF4 gene, resulting in an arg105-to-gln (R105Q) substitution, that was found in compound heterozygous state in a boy with autosomal recessive chronic granulomatous disease-3 (CGD3; 613960) by Matute et al. (2009), see 601488.0001.
In 6 sibs, born of consanguineous Pakistani parents (kindred B), with CGD3, van de Geer et al. (2018) identified a homozygous R105Q mutation in the NCF4 gene. The mutation, which was found by exome sequencing and confirmed by Sanger sequencing, segregated with the disorder in the family. The affected father was also homozygous for the mutation, whereas the unaffected mother was a heterozygous carrier. In vitro studies in transfected cells showed that the mutant protein was expressed at normal levels. EBV-B cells expressing the mutation showed lack of oxygen radical production after PMA stimulation, consistent with severely impaired NADPH oxidase activity and a loss-of-function effect. Additional functional studies showed variably impaired production of reactive oxygen species depending on the cell type and stimulus.
Hamosh (2020) noted that the R105Q variant was present at a low frequency (3.18 x 10(-5)) in only heterozygous state in the gnomAD database (July 6, 2020).
In 3 unrelated patients (P1, P12, and P23) with autosomal recessive chronic granulomatous disease-3 (CGD3; 613960), van de Geer et al. (2018) identified a homozygous G-to-A transition (c.118-1G-A) in intron 2 of the NCF2 gene, resulting in a splicing defect. Analysis of patient cells showed 3 aberrantly spliced variants, all of which were predicted to result in truncated proteins. A fourth minor transcript resulted in a large in-frame deletion (c.118_198del). An asymptomatic 1-year-old sib (P13) of an affected patient (P12) was also homozygous for the mutation, suggesting incomplete or age-dependent penetrance. Two of the families were Pakistani and 1 was Russian; haplotype analysis was consistent with a founder effect. An additional patient (P11) with the disorder was found to be compound heterozygous for this splice site mutation and another splice site mutation (c.759-1G-C; 601488.0004). The latter mutation was predicted to result in splicing abnormalities, frameshifts, and premature termination. The mutations, which were found by whole-exome or targeted sequencing and confirmed by Sanger sequencing, segregated with the disorder in the families; neither mutation was present in the gnomAD database. In vitro functional expression studies of the c.118_198del mutation showed that it caused a loss-of-function effect with impaired NADPH oxidase activity and decreased extracellular H2O2 production, particularly in EBV-B cells. However, there was functional variability depending on the type of phagocytic cell expressing the mutation and the stimulus presented.
For discussion of the G-to-C transversion (c.759-1G-C) in intron 8 of the NCF4 gene that was found in compound heterozygous state in a patient with chronic granulomatous disease-3 (CGD3; 613960) by van de Geer et al. (2018), see 601488.0003.
In 2 sisters (P20 and P21), born of consanguineous Kuwaiti parents (kindred I), with autosomal recessive chronic granulomatous disease-3 (CGD3; 613960), van de Geer et al. (2018) identified a homozygous c.716G-A transition in exon 8a of the NCF4 gene, resulting in a trp239-to-ter (W239X) substitution. The mutation, which was found by exome or targeted sequencing and confirmed by Sanger sequencing, segregated with the disorder in the family. It was not present in the gnomAD database. Western blot analysis of transfected cells showed either undetectable protein levels or very low levels of a truncated protein. Expression of the mutation into EBV-transformed B cells resulted in no detectable oxygen production after PMA stimulation, consistent with a loss-of-function effect. Additional detailed functional studies showed that the mutation caused variably impaired NADPH oxidase activity, depending on the cell type and the stimulus.
In a 5-year-old girl (P14), born of consanguineous Pakistani parents, with autosomal recessive chronic granulomatous disease-3 (CGD3; 613960), van de Geer et al. (2018) identified a homozygous T-to-G transversion in intron 1 of the NCF4 gene (c.32+2T-G). Cellular studies showed absence of the NCF4 transcript, suggesting nonsense-mediated mRNA decay and a complete loss of function. Cells carrying the mutation produced no detectable reactive oxygen species after PMA stimulation, indicating dysfunction of NADPH oxidase and a loss-of-function effect.
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Ellson, C. D., Davidson, K., Ferguson, G. J., O'Connor, R., Stephens, L. R., Hawkins, P. T. Neutrophils from p40-phox-/- mice exhibit severe defects in NADPH oxidase regulation and oxidant-dependent bacterial killing. J. Exp. Med. 203: 1927-1937, 2006. [PubMed: 16880254] [Full Text: https://doi.org/10.1084/jem.20052069]
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Hamosh, A. Personal Communication. Baltimore, Md. 7/6/2020.
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