Genotype-Phenotype Correlations in Chronic Granulomatous Disease: Insights From a Large National Cohort.

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 NADPH oxidase enzyme. These mutations lead to failure of ROS generation followed by recurrent bacterial and fungal infections, frequently associated with hyper-inflammatory manifestations. We report a multi-center cumulative experience in diagnosing and treating patients with CGD. From 1986 to 2021, 2,918 patients suffering from 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 non-Jewish/non-Arabic. 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 hyper-inflammatory manifestations are described. Six patients had hypomorphic mutations with significantly milder phenotype, clinical severity score ≤2, and better outcomes. Hematopoietic stem cell transplantation was implemented in 39/110 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 multi-professional team. Early identification of the genetic mutation is essential for prompt diagnosis, suitable management and prevention.

A Novel Deletion in FERMT3 Causes LAD-III in a Turkish Family.

Leukocyte adhesion deficiency-III (LAD-III) is an extremely rare autosomal recessive syndrome caused by mutations in FERMT3, the gene encoding kindlin-3. The genetic alterations in this gene lead to abnormal expression or activity of kindlin-3 in leukocytes and platelets. Kindlin-3 acts as an important regulator of integrin activation. LAD-III has features of the bleeding syndrome of Glanzmann and also of leukocyte adhesion deficiency. In this study, we report on two families, one of Turkish and one of Syrian origin, with clinical features of LAD-III, loss of kindlin-3 protein expression, and a functional leukocyte defect. A novel, homozygous deletion in FERMT3 (c.921delC, p.Ser307Argfs*21) was found in the Turkish patient. The parents were carriers of the mutation, consistent with an autosomal recessive inheritance. A common c.1525C > T (p.Arg509*) mutation was found in the Syrian patient. In conclusion, beside the variant c.1525C > T in the FERMT3 gene, which was previously found in more than 15 patients in Anatolia, our study is the first to identify the novel homozygous variant c.921delC in the FERMT3 gene.

Hematologically important mutations: Leukocyte adhesion deficiency (second update).

Leukocyte adhesion deficiency (LAD) is an immunodeficiency caused by defects in the adhesion of leukocytes (especially neutrophils) to the blood vessel wall. As a result, patients with LAD suffer from severe bacterial infections and impaired wound healing, accompanied by neutrophilia. In LAD-I, characterized directly after birth by delayed separation of the umbilical cord, mutations are found in ITGB2, the gene that encodes the ß subunit (CD18) of the ß2 integrins. In the rare LAD-II disease, the fucosylation of selectin ligands is disturbed, caused by mutations in SLC35C1, the gene that encodes a GDP-fucose transporter of the Golgi system. LAD-II patients lack the H and Lewis Lea and Leb blood group antigens. Finally, in LAD-III, the conformational activation of the hematopoietically expressed ß integrins is disturbed, leading to leukocyte and platelet dysfunction. This last syndrome is caused by mutations in FERMT3, encoding the kindlin-3 protein in all blood cells, involved in the regulation of ß integrin conformation. This article contains an update of the mutations that we consider to be relevant for the various forms of LAD.

Hematologically important mutations: The autosomal forms of chronic granulomatous disease (third update).

Chronic granulomatous disease (CGD) is an immunodeficiency disorder affecting about 1 in 250,000 individuals. CGD patients suffer from severe, recurrent bacterial and fungal infections. The disease is caused by mutations in the genes encoding the components of the leukocyte NADPH oxidase. This enzyme produces superoxide, which is subsequently metabolized to hydrogen peroxide and other reactive oxygen species (ROS). These products are essential for intracellular killing of pathogens by phagocytic leukocytes (neutrophils, eosinophils, monocytes and macrophages). The leukocyte NADPH oxidase is composed of five subunits, four of which are encoded by autosomal genes. These are CYBA, encoding p22phox, NCF1, encoding p47phox, NCF2, encoding p67phox and NCF4, encoding p40phox. This article lists all mutations identified in these genes in CGD patients. In addition, cytochrome b558 chaperone-1 (CYBC1), recently recognized as an essential chaperone protein for the expression of the X-linked NADPH oxidase component gp91phox (also called Nox2), is encoded by the autosomal gene CYBC1. Mutations in this gene also lead to CGD. Finally, RAC2, a small GTPase of the Rho family, is needed for activation of the NADPH oxidase, and mutations in the RAC2 gene therefore also induce CGD-like symptoms. Mutations in these last two genes are also listed in this article.

Clinical and laboratory findings in patients with leukocyte adhesion deficiency type I: A multicenter study in Turkey.

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.

Hematologically important mutations: X-linked chronic granulomatous disease (fourth update).

Chronic granulomatous disease (CGD) is an immunodeficiency disorder affecting about 1 in 250,000 individuals. CGD patients suffer from severe bacterial and fungal infections. The disease is caused by a lack of superoxide production by the leukocyte enzyme NADPH oxidase. Superoxide and subsequently formed other reactive oxygen species (ROS) are instrumental in killing phagocytosed micro-organisms in neutrophils, eosinophils, monocytes and macrophages. The leukocyte NADPH oxidase is composed of five subunits, of which the enzymatic component is gp91phox, 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.

Mutations in cis that affect mRNA synthesis, processing and translation.

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.

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

BACKGROUND: Chronic granulomatous disease (CGD), one of the phagocytic system defects, is the primary immunodeficiency caused by dysfunction of the NADPH oxidase complex which generates reactive oxygen species (ROS), which are essential for killing pathogenic microorganisms, especially catalase-positive bacteria and fungi. OBJECTIVE: The objective of our study was to assess the clinical and laboratory characteristics, treatment modalities, and prognosis of patients with CGD. METHODS: We retrospectively reviewed 63 patients with CGD who have been diagnosed, treated, and/or followed-up between 1984 and 2018 in Hacettepe University, Ankara, in Turkey, as a developing country. RESULTS: The number of female and male patients was 26/37. The median age at diagnosis was 3.8 (IQR: 1.0-9.6) years. The rate of consanguinity was 63.5%. The most common physical examination finding was lymphadenopathy (44/63), growth retardation (33/63), and hepatomegaly (27/63). One adult patient had squamous cell carcinoma of the lung. The most common infections were lung infection (53/63), skin abscess (43/63), and lymphadenitis (19/63). Of the 63 patients with CGD, 6 patients had inflammatory bowel disease (IBD). Twelve of the 63 patients died during follow-up. CYBA, NCF1, CYBB, and NCF2 mutations were detected in 35%, 27.5%, 25%, and 12.5% of the patients, respectively. CONCLUSION: We identified 63 patients with CGD from a single center in Turkey. Unlike other cohort studies in Turkey, due to the high consanguineous marriage rate in our study group, AR form of CGD was more frequent, and gastrointestinal involvement were found at relatively lower rates. The rate of patients who treated with HSCT was lower in our research than in the literature. A majority of the patients in this study received conventional prophylactic therapies, which highlight on the outcome of individuals who have not undergone HSCT.

Membrane Dynamics and Organization of the Phagocyte NADPH Oxidase in PLB-985 Cells.

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.

Genetic Characteristics, Infectious, and Noninfectious Manifestations of 32 Patients with Chronic Granulomatous Disease.

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.

Activation of cryptic splice sites in three patients with chronic granulomatous disease.

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.

Chronic Granulomatous Disease.

Chronic granulomatous disease is a clinical condition that stems from inactivating mutations in NOX2 and its auxiliary proteins. Together, these proteins form the phagocyte NADPH oxidase enzyme that generates superoxide. Superoxide (O2c-) and its reduced product hydrogen peroxide (H2O2) give rise to several additional reactive oxygen species (ROS), which together are necessary for adequate killing of pathogens. Thus, CGD patients, with a phagocyte NADPH oxidase that is not properly functioning, suffer from recurrent, life-threatening infections with certain bacteria, fungi, and yeasts. Here, I give a short survey of the genetic mutations that underlie CGD, the effect of these mutations on the activity of the leukocyte NADPH oxidase, the clinical symptoms of CGD patients, and the treatment options for these patients.

Characterization of two novel mutations in IL-12R signaling in MSMD patients.

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-12Rß1 and IL-12Rß2 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 (ß1/ß2) 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 (ß1/ß2) expression levels were normal. Mutation analysis showed three previously reported mutations, two novel mutations in IL-12 R (ß1/ß2), and one previously reported mutation in IL-12.

Common Infections and Target Organs Associated with Chronic Granulomatous Disease in Iran.

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.

Genetic Analysis of 13 Iranian Families With Leukocyte Adhesion Deficiency Type 1.

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 ß2 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.

An adult autosomal recessive chronic granulomatous disease patient with pulmonary Aspergillus terreus infection.

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.

Dynamics of Transcription Regulation in Human Bone Marrow Myeloid Differentiation to Mature Blood Neutrophils.

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.

Neutrophils Kill Antibody-Opsonized Cancer Cells by Trogoptosis.

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.

Retrotransposable genetic elements causing neutrophil defects.

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.