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Neutrophils are the first line of defense against invading pathogens. Neutrophils execute and modulate immune responses by generating reactive oxygen species (ROS). Chronic granulomatous disease (CGD) is a primary immune deficiency disorder of phagocytes, caused by inherited mutations in the genes of the nicotinamide adenine dinucleotide phosphate reduced oxidase enzyme. These mutations lead to failure of ROS generation followed by recurrent bacterial and fungal infections, frequently associated with hyperinflammatory manifestations. We report a multicenter cumulative experience in diagnosing and treating patients with CGD. From 1986 to 2021, 2918 patients experiencing frequent infections were referred for neutrophil evaluation. Among them, 110 patients were diagnosed with CGD: 56 of Jewish ancestry, 48 of Arabic ancestry, and 6 of non-Jewish/non-Arabic ancestry. As opposed to other Western countries, the autosomal recessive (AR) CGD subtypes were predominant in Israel (71/110 patients). Thirty-nine patients had X-linked CGD, in most patients associated with severe infections (clinical severity score ≥3) and poor outcomes, presenting at a significantly earlier age than AR-CGD subtypes. The full spectrum of infections and hyperinflammatory manifestations is described. Six patients had hypomorphic mutations with significantly milder phenotype, clinical severity score ≤2, and better outcomes. Hematopoietic stem cell transplantation was implemented in 39 of 110 patients (35.5%). Successful engraftment was achieved in 92%, with 82% long-term survival and 71% full clinical recovery. CGD is a complex disorder requiring a multiprofessional team. Early identification of the genetic mutation is essential for prompt diagnosis, suitable management, and prevention.
Leukocyte adhesion deficiency type I is a rare primary immunodeficiency disorder characterized by mutations in the ITGB2 gene encoding CD18. We present clinical and immunological features of 15 patients with leukocyte adhesion deficiency type 1 (LAD-1). Targeted next-generation sequencing was performed with either a primary immunodeficiency gene panel comprising 266 genes or a small LAD-panel consisting of five genes for genetic analysis. To measure the expression level of integrins on the leukocyte surface, flow cytometry analysis was performed. The median age of the patients at diagnosis was 3 (1-48) months. Eleven (73%) of the 15 patients had a LAD-1 diagnosis in their first 6 months and 14 (93%) patients had consanguineous parents. Delayed separation of the umbilical cord was present in 80% (n = 12) of the patients in our cohort, whereas omphalitis was observed in 53% (n = 8) of the patients. Leukocytosis with neutrophil predominance was observed in 73% (n = 11) patients. Nine distinct variants in the ITGB2 gene in 13 of the 15 patients with LAD-1 were characterized, two of which (c.305_306delAA and c.779_786dup) are novel homozygous mutations of ITGB2. Four unrelated patients from Syria had a novel c.305_306delAA mutation that might be a founder effect for patients of Syrian origin. Four (27%) patients underwent hematopoietic stem cell transplantation. Two patients died because of HSCT complications and the other two are alive and well. Early differential diagnosis of the patients is critical in the management of the disease and genetic evaluation provides a basis for family studies and genetic counseling.
, also called Nox2. This protein is encoded by the CYBB gene on the X chromosome. Mutations in this gene are found in about 70% of all CGD patients in Europe and in about 20% in countries with a high ratio of parental consanguinity. This article lists all mutations identified in CYBB and should therefore help in genetic counseling of X-CGD patients' families. Moreover, apparently benign polymorphisms in CYBB are also given, which should facilitate the recognition of disease-causing mutations. In addition, we also include some mutations in G6PD, the gene on the X chromosome that encodes glucose-6-phosphate dehydrogenase, because inactivity of this enzyme may lead to shortage of NADPH and thus to insufficient activity of NADPH oxidase. Severe G6PD deficiency can induce CGD-like symptoms.
Genetic mutations that cause hereditary diseases usually affect the composition of the transcribed mRNA and its encoded protein, leading to instability of the mRNA and/or the protein. Sometimes, however, such mutations affect the synthesis, the processing or the translation of the mRNA, with similar disastrous effects. We here present an overview of mRNA synthesis, its posttranscriptional modification and its translation into protein. We then indicate which elements in these processes are known to be affected by pathogenic mutations, but we restrict our review to mutations in cis, in the DNA of the gene that encodes the affected protein. These mutations can be in enhancer or promoter regions of the gene, which act as binding sites for transcription factors involved in pre-mRNA synthesis. We also describe mutations in polyadenylation sequences and in splice site regions, exonic and intronic, involved in intron removal. Finally, we include mutations in the Kozak sequence in mRNA, which is involved in protein synthesis. We provide examples of genetic diseases caused by mutations in these DNA regions and refer to databases to help identify these regions. The over-all knowledge of mRNA synthesis, processing and translation is essential for improvement of the diagnosis of patients with genetic diseases.
Neutrophils are the first cells recruited at the site of infections, where they phagocytose the pathogens. Inside the phagosome, pathogens are killed by proteolytic enzymes that are delivered to the phagosome following granule fusion, and by reactive oxygen species (ROS) produced by the NADPH oxidase. The NADPH oxidase complex comprises membrane proteins (NOX2 and p22phox), cytoplasmic subunits (p67phox, p47phox, and p40phox) and the small GTPase Rac. These subunits assemble at the phagosomal membrane upon phagocytosis. In resting neutrophils the catalytic subunit NOX2 is mainly present at the plasma membrane and in the specific granules. We show here that NOX2 is also present in early and recycling endosomes in human neutrophils and in the neutrophil-like cell line PLB-985 expressing GFP-NOX2. In the latter cells, an increase in NOX2 at the phagosomal membrane was detected by live-imaging after phagosome closure, probably due to fusion of endosomes with the phagosome. Using super-resolution microscopy in PLB-985 WT cells, we observed that NOX2 forms discrete clusters in the plasma membrane. The number of clusters increased during frustrated phagocytosis. In PLB-985NCF1ΔGT cells that lack p47phox and do not assemble a functional NADPH oxidase, the number of clusters remained stable during phagocytosis. Our data suggest a role for p47phox and possibly ROS production in NOX2 recruitment at the phagosome.
BACKGROUND: Chronic granulomatous disease (CGD) is a rare genetic disorder characterized by failure of phagocytic leukocytes to destroy certain microbes. We present a study on CGD patients enrolled at a single medical center concerning the infectious and noninfectious complications and genetic properties of the disease. METHODS: Icotinamide adenine dinucleotide phosphate oxidase activity and the expression of flavocytochrome b558 were measured by flow cytometry, and clinical outcomes of the patients were listed in relation to the genetic results. RESULTS: The clinical and genetic findings of 32 pediatric cases with CGD from 23 families were enrolled. Pneumonia and anemia were the most common infectious and noninfectious symptoms. Genetic analysis showed that 10 families (43.5%) carried CYBB variants and 13 families (56.5%) have autosomal recessive (AR) CGD, in which 6 families (26%) carried NCF1 variants, 4 (17.4%) carried CYBA variants, and 3 (13%) carried NCF2 variants. The median age of clinical onset was 3.3 and 48 months for patients with X-linked CGD (X-CGD) and AR-CGD, respectively. The onset of symptoms before age 1 year was 94% in X-CGD, 28.5% in AR-CGD, and 12.5% in patients with oxidase residual activity. Moreover, a de novo germline mutation at c.1415delG in CYBB (OMIM#300481) and a novel c.251_263del13bp in CYBA (OMIM#608508) were also investigated. CONCLUSIONS: Ihydrorhodamine-1,2,3 assay could not detect carrier mother in de novo case with CYBB variant. Most X-CGD patients have the onset of symptoms before age 1 year. Additionally, residual oxidase activity in AR-CGD causes a delay in onset, diagnosis, and prophylaxis. The protective role of residual activity is limited while the infection is ongoing and becoming serious.
BACKGROUND: Chronic granulomatous disease (CGD) is a primary immune deficiency caused by mutations in the genes encoding the structural components of the phagocyte NADPH oxidase. As a result, the patients cannot generate sufficient amounts of reactive oxygen species required for killing pathogenic microorganisms. METHODS: We analyzed NADPH oxidase activity and component expression in neutrophils, performed genomic DNA and cDNA analysis, and used mRNA splicing prediction tools to evaluate the impact of mutations. RESULTS: In two patients with CGD, we had previously found mutations that cause aberrant pre-mRNA splicing. In one patient an exonic mutation in a cryptic donor splice site caused the deletion of the 3' part of exon 6 from the mRNA of CYBB. This patient suffers from X-linked CGD. The second patient, with autosomal CGD, has a mutation in the donor splice site of intron 1 of CYBA that activates a cryptic donor splice site downstream in intron 1, causing the insertion of intronic sequences in the mRNA. The third patient, recently analyzed, also with autosomal CGD, has a mutation in intron 4 of CYBA, 15 bp from the acceptor splice site. This mutation weakens a branch site and activates a cryptic acceptor splice site, causing the insertion of 14 intronic nucleotides into the mRNA. CONCLUSION: We found three different mutations, one exonic, one in a donor splice site and one intronic, that all caused missplicing of pre-mRNA. We analyzed these mutations with four different splice prediction programs and found that predictions of splice site strength, splice enhancer and splice silencer protein binding and branch site strength are all essential for correct prediction of pre-mRNA splicing.
Mendelian Susceptibility to Mycobacterial Disease (MSMD) is a rare syndrome with infections-among other complications-after Bacillus Calmette-Guerin (BCG) vaccination in children. We focused on the IL-12/IFN-γ pathway to identify new mutations in our patients. This study included 20 patients by vulnerability to mycobacteria and clinical manifestations of severe, recurrent infections. Blood samples were activated with BCG, BCG + IL-12 and BCG + IFN-γ. Cytokine levels were analyzed by ELISA. Measurements of IL-12Rβ1 and IL-12Rβ2 on the surface of peripheral blood mononuclear cells were performed by flow cytometry. To detect genetic defects, next-generation sequencing was performed by Thermo Fisher immunodeficiency panel. Flow cytometry analysis of 20 patients indicated reduction in IL-12R (β1/β2) expression in seven patients who showed incomplete production of IFN-γ by ELISA. In the patient with reduced IL-12 production, IFN-γR and IL-12R (β1/β2) expression levels were normal. Mutation analysis showed three previously reported mutations, two novel mutations in IL-12 R (β1/β2), and one previously reported mutation in IL-12.
Recurrent severe bacterial and fungal infections are characteristic features of the rare genetic immunodeficiency disorder chronic granulomatous disease (CGD). The disease usually manifests within the first years of life with an incidence of 1 in approximately 200,000 live births. The incidence is higher in Iran and Morocco where it reaches 1.5 per 100,000 live births. Mutations have been described in the 5 subunits of NADPH oxidase, mostly in gp91phox and p47phox, with fewer mutations reported in p67phox, p22phox, and p40phox. These mutations cause loss of superoxide production in phagocytic cells. CYBB, the gene encoding the large gp91phox subunit of the transmembrane component cytochrome b558 of the NADPH oxidase complex, is localized on the X-chromosome. Genetic defects in CYBB are responsible for the disease in the majority of male CGD patients. CGD is associated with the development of granulomatous reactions in the skin, lungs, bones, and lymph nodes, and chronic infections may be seen in the liver, gastrointestinal tract, brain, and eyes. There is usually a history of repeated infections, including inflammation of the lymph glands, skin infections, and pneumonia. There may also be a persistent runny nose, inflammation of the skin, and inflammation of the mucous membranes of the mouth. Gastrointestinal problems can also occur, including diarrhea, abdominal pain, and perianal abscesses. Infection of the bones, brain abscesses, obstruction of the genitourinary tract and/or gastrointestinal tract due to the formation of granulomatous tissue, and delayed growth are also symptomatic of CGD. The prevention of infectious complications in patients with CGD involves targeted prophylaxis against opportunistic microorganisms such as Staphylococcus aureus, Klebsiella spp., Salmonella spp. and Aspergillus spp. In this review, we provide an update on organ involvement and the association with specific isolated microorganisms in CGD patients.
BACKGROUND: Genetic mutations that reduce intracellular superoxide production by granulocytes causes chronic granulomatous disease (CGD). These patients suffer from frequent and severe bacterial and fungal infections throughout their early life. Diagnosis is usually made in the first 2 years of life but is sometimes only diagnosed when the patient is an adult although they may have suffered from symptoms since childhood. CASE PRESENTATION: A 26-year-old man was referred with weight loss, fever, hepatosplenomegaly and coughing. He had previously been diagnosed with lymphadenopathy in the neck at age 8 and prescribed anti-tuberculosis treatment. A chest radiograph revealed extensive right-sided consolidation along with smaller foci of consolidation in the left lung. On admission to hospital he had respiratory problems with fever. Laboratory investigations including dihydrorhodamine-123 (DHR) tests and mutational analysis indicated CGD. Stimulation of his isolated peripheral blood neutrophils (PMN) with phorbol 12-myristate 13-acetate (PMA) produced low, subnormal levels of reactive oxygen species (ROS). Aspergillus terreus was isolated from bronchoalveolar lavage (BAL) fluid and sequenced. CONCLUSIONS: We describe, for the first time, the presence of pulmonary A. terreus infection in an adult autosomal CGD patient on long-term corticosteroid treatment. The combination of the molecular characterization of the inherited CGD and the sequencing of fungal DNA has allowed the identification of the disease-causing agent and the optimal treatment to be given as a consequence.
Neutrophils are short-lived blood cells that play a critical role in host defense against infections. To better comprehend neutrophil functions and their regulation, we provide a complete epigenetic overview, assessing important functional features of their differentiation stages from bone marrow-residing progenitors to mature circulating cells. Integration of chromatin modifications, methylation, and transcriptome dynamics reveals an enforced regulation of differentiation, for cellular functions such as release of proteases, respiratory burst, cell cycle regulation, and apoptosis. We observe an early establishment of the cytotoxic capability, while the signaling components that activate these antimicrobial mechanisms are transcribed at later stages, outside the bone marrow, thus preventing toxic effects in the bone marrow niche. Altogether, these data reveal how the developmental dynamics of the chromatin landscape orchestrate the daily production of a large number of neutrophils required for innate host defense and provide a comprehensive overview of differentiating human neutrophils.
Biallelic loss-of-function (LOF) mutations of the NCF4 gene, encoding the p40phox subunit of the phagocyte NADPH oxidase, have been described in only 1 patient. We report on 24 p40phox-deficient patients from 12 additional families in 8 countries. These patients display 8 different in-frame or out-of-frame mutations of NCF4 that are homozygous in 11 of the families and compound heterozygous in another. When overexpressed in NB4 neutrophil-like cells and EBV-transformed B cells in vitro, the mutant alleles were found to be LOF, with the exception of the p.R58C and c.120_134del alleles, which were hypomorphic. Particle-induced NADPH oxidase activity was severely impaired in the patients' neutrophils, whereas PMA-induced dihydrorhodamine-1,2,3 (DHR) oxidation, which is widely used as a diagnostic test for chronic granulomatous disease (CGD), was normal or mildly impaired in the patients. Moreover, the NADPH oxidase activity of EBV-transformed B cells was also severely impaired, whereas that of mononuclear phagocytes was normal. Finally, the killing of Candida albicans and Aspergillus fumigatus hyphae by neutrophils was conserved in these patients, unlike in patients with CGD. The patients suffer from hyperinflammation and peripheral infections, but they do not have any of the invasive bacterial or fungal infections seen in CGD. Inherited p40phox deficiency underlies a distinctive condition, resembling a mild, atypical form of CGD.
Destruction of cancer cells by therapeutic antibodies occurs, at least in part, through antibody-dependent cellular cytotoxicity (ADCC), and this can be mediated by various Fc-receptor-expressing immune cells, including neutrophils. However, the mechanism(s) by which neutrophils kill antibody-opsonized cancer cells has not been established. Here, we demonstrate that neutrophils can exert a mode of destruction of cancer cells, which involves antibody-mediated trogocytosis by neutrophils. Intimately associated with this is an active mechanical disruption of the cancer cell plasma membrane, leading to a lytic (i.e., necrotic) type of cancer cell death. Furthermore, this mode of destruction of antibody-opsonized cancer cells by neutrophils is potentiated by CD47-SIRPα checkpoint blockade. Collectively, these findings show that neutrophil ADCC toward cancer cells occurs by a mechanism of cytotoxicity called trogoptosis, which can be further improved by targeting CD47-SIRPα interactions.
Background Retrotransposable elements are stretches of DNA that encode proteins with the inherent ability to insert their own RNA or another RNA by reverse transcriptase as DNA into a new genomic location. In humans, the only autonomous retrotransposable elements are members of the Long INterspersed Element‐1 (LINE‐1) family. LINE‐1s may cause gene inactivation and human disease. Design We present a brief summary of the published knowledge about LINE‐1s in humans and the RNAs that these elements can transpose, and we focus on the effect of LINE‐1‐mediated retrotransposition on human neutrophil function. Results Retrotransposons can cause genetic disease by two primary mechanisms: (1) insertional mutagenesis and (2) nonallelic homologous recombination. The only known neutrophil function affected by retrotransposition is that of NADPH oxidase activity. Four patients with chronic granulomatous disease (CGD) are known with LINE‐1‐mediated insertional inactivation of CYBB, the gene that encodes the gp91phox component of the phagocyte NADPH oxidase. In addition, 5 CGD patients had a large deletion in the NCF2 gene, encoding the p67phox component, and 2 CGD patients had a similar deletion in NCF1, encoding p47phox. These deletions were caused by nonallelic homologous recombination between 2 Alu elements at the borders of each deletion. Alu elements have spread throughout the human genome by LINE‐1 retrotransposition. Conclusions Probably, the occurrence of LINE‐1‐mediated insertions causing autosomal CGD has been underestimated. It might be worthwhile to reinvestigate the DNA from autosomal CGD patients with missplice mutations and large deletions for indications of LINE‐1‐mediated insertions.
BACKGROUND AND AIM: Leukocyte adhesion deficiency type 1 is a rare, autosomal recessive disorder that results from mutations in the ITGB2 gene. This gene encodes the CD18 subunit of β2 integrin leukocyte adhesion cell molecules. Leukocyte adhesion deficiency type 1 is characterized by recurrent bacterial infections, impaired wound healing, inadequate pus formation, and delayed separation of the umbilical cord. MATERIALS AND METHODS: Blood samples were taken from 13 patients after written consent had been obtained. Genomic DNA was extracted, and ITGB2 exons and exon-intron boundaries were amplified by polymerase chain reaction. The products were examined by Sanger sequencing. RESULTS: In this study, 8 different previously reported mutations (intron7+1G>A, c.715G>A, c.1777 C>T, c.843del C, c.1768T>C, c.1821C>A, Intron7+1G>A, c.1885G>A) and 2 novel mutations (c.1821C>A; p.Tyr607Ter and c.1822C>T; p.Gln608Ter) were found. CONCLUSIONS: c.1821C>A (p.Tyr607Ter) and c.1822C>T (p.Gln608Ter) mutations should be included in the panel of carrier detection and prenatal diagnosis.
Chronic granulomatous disease (CGD) is an inherited disease of the innate immune system that results from defects in 1 of the 5 subunits of nicotinamide adenine dinucleotide phosphate oxidase complex and leads to life-threatening infections with granuloma formation. During 3 years of study, we recognized 10 male patients with X-linked CGD from a tertiary referral center for immune deficiencies in Iran. The CGD patients were diagnosed according to clinical features and biochemical tests, including nitroblue tetrazolium and dihydrorhodamine-1, 2, 3 tests, performed on patients and their mothers. In all patients, Western blot analysis showed a gp91 phenotype. Mutation screening by single strand conformation polymorphism and multiplex ligation-dependent probe amplification analysis of the CYBB gene encoding gp91, followed by sequencing, showed 9 different mutations, 4 of them novel as far as we know.
Summary Neutrophils are short-lived blood cells that play a critical role in host defense against infections. To better comprehend neutrophil functions and their regulation, we provide a complete epigenetic and functional overview of their differentiation stages from bone marrow-residing progenitors to mature circulating cells. Integration of epigenetic and transcriptome dynamics reveals an enforced regulation of differentiation, through cellular functions such as: release of proteases, respiratory burst, cell cycle regulation and apoptosis. We observe an early establishment of the cytotoxic capability, whilst the signaling components that activate antimicrobial mechanisms are transcribed at later stages, outside the bone marrow, thus preventing toxic effects in the bone marrow niche. Altogether, these data reveal how the developmental dynamics of the epigenetic landscape orchestrate the daily production of large number of neutrophils required for innate host defense and provide a comprehensive overview of the epigenomes of differentiating human neutrophils. Key points Dynamic acetylation enforces human neutrophil progenitor differentiation. Neutrophils cytotoxic capability is established early at the (pro)myelocyte stage. Coordinated signaling component expression prevents unwanted toxic effects to the bone marrow niche.
In a male patient suffering from X-linked chronic granulomatous disease (CGD) we found a c.389G>T mutation in exon 5 of the CYBB gene. We have analyzed why 95% of the transcripts of this gene lacked exon 5, leading to a frameshift and premature termination codon. The mutation was located in a region comprising three putative exonic splicing enhancer binding sites, for SRSF1, SRFS2 and SRFS6, according to the ESEfinder Tool (http://rulai.cshl.edu/cgi-bin/tools/ESE3/esefinder.cgi). With the Analyser Splice Tool we calculated the probability of skipping of exon 5 in CYBB mRNA, and by means of Sroogle the number of putative binding motifs for splicing enhancer and splicing silencer proteins (http://astlab.tau.ac.il/index.php). These analyses clarify why this exon was skipped in the majority of the mRNA. The normally spliced transcript contains an amino acid change p.Arg130Leu. This poorly expressed transcript gives rise to a protein with low expression but presumably normal activity, leading to a respiratory burst activity in the patient's neutrophils of about 15% of normal.
Hermansky-Pudlak syndrome type 2 (HPS2) is a syndrome caused by mutations in the beta-3A subunit of the adaptor protein (AP)-3 complex (AP3B1 gene). We describe five unreported cases with four novel mutations, one of which caused aberrant pre-mRNA splicing. A point mutation c.2702C>G in exon 23 of the AP3B1 gene caused deletion of 112 bp in the mRNA in two siblings. This mutation activates a cryptic donor splice site that overrules the wild-type donor splice site of this exon. Three other novel mutations in AP3B1 were identified, that is, a nonsense mutation c.716G>A (p.Trp239Ter), a 1-bp and a 4-bp deletion c.177delA and c.1839_1842delTAGA, respectively, both causing frameshift and premature termination of translation. Mass spectrometry in four of these HPS2 patients demonstrated the (near) absence of all AP-3 complex subunits. Immunoelectron microscopy on the neutrophils of two of these patients showed abnormal granule formation. We found clear mislocalization of myeloperoxidase in the neutrophils even though the content of this protein but not the activity seemed to be present at normal levels. In sum, HPS2 is the result of the absence of the entire AP-3 complex, which results in severe neutropenia with a defect in granule formation as the major hematological finding.
BACKGROUND: Primary immunodeficiency (PID) is a heterogeneous group of inheritable genetic disorders with increased susceptibility to infections, autoimmunity, uncontrolled inflammation and malignancy. Timely precise diagnosis of these patients is very essential since they may not be able to live with their congenital immunity defects; otherwise, they could survive with appropriate treatment. DNA biobanks of such patients could be used for molecular and genetic testing, facilitating the detection of underlying mutations in known genes as well as the discovery of novel genes and pathways. METHODS: According to the last update of the International Union of Immunological Societies (IUIS) classification, patients are registered in our biobank during a period of 15 years. All patients' data were collected via questionnaire and their blood samples were taken in order to extract and protect their DNA content. RESULTS: Our study comprised 197 patients diagnosed with PID. Antibody deficiency in 50 patients (25.4%), phagocytic defect in 47 patients (23.8%) and combined immunodeficiency with associated/syndromic feature in 19 patients (9.6%) were the most common PID diagnoses, respectively. The most common variant of PID in our study is common variable immunodeficiency, which accounted for 20 cases (10.1%), followed by chronic mucocutaneous candidiasis in 15 patients (7.9%) and congenital neutropenia in 13 patients (7%). Mean age at onset of disease was 4 years and mean age of diagnosis was 9.6 years. The average diagnostic delay was 5.5 years, with a range of 6 months to 46 years. Parental consanguinity and history of PID in family were observed in 70.2 and 48.9% of the patients, respectively. The majority of PID patients (93.3%) were from families with low socioeconomic status. CONCLUSION: This prospective study was designed to establish a PID Biobank in order to have a high quality DNA reservoir of these patients, shareable for international diagnostic and therapeutic collaborations. This article emphasizes the need to raise the awareness of society and general practitioners to achieve timely diagnosis of these patients and prevent current mismanagements.