Mutations In CYBB Research Articles

Profile of juvenile systemic lupus erythematosus patients with a special reference to monogenic lupus and lupus nephritis: a cross-sectional study.

To study the clinical, laboratory profile and outcome of juvenile Systemic Lupus Erythematosus (jSLE) patients at a tertiary care centre in South India. A retrospective review of the medical records of all jSLE patients visiting the Pediatric Immunology and Rheumatology Unit, Aster CMI Hospital, India from February 2017 to December 2023 was performed. The clinical characteristics, treatment and outcomes were recorded and tabulated. Seventy patients diagnosed with jSLE were included in the study. The female-to-male ratio was 4.4:1. Mean age at onset and delay in diagnosis were 120.1 (+/- 56.8) and 11.7 (+/- 22.7) months respectively. The median follow-up period was 13 months (range 4, 29 months). Nine patients presented with early onset SLE (< 5 years). Most common manifestations were constitutional symptoms (n = 56), followed by haematologic (n = 55), and mucocutaneous(n = 50) involvement. Immunological workup showed SLE-specific antibody positivity in 38 patients, hypocomplementemia in 40 patients, and anti-phospholipid antibody positivity in 13 patients. Mortality was observed in five patients with LN while there was no mortality in the non-nephritis group (p 0.004). C1q deficiency was the most common cause of monogenic lupus seen in 5/9 patients; protein kinase C delta (PRKCD) defect and chronic granulomatous disease (CYBB mutation) were seen in one patient each. We describe a large cohort of jSLE from Southern India. Lupus nephritis was noted in 35.7% of our cohort and had a direct correlation with mortality. 10% of patients had monogenic lupus. Serious infections were more frequent in patients with monogenic lupus.

Spontaneous NETosis and type I IFN signaling activation in resting neutrophils of chronic granulomatous disease patients with CYBB mutations

Spontaneous NETosis and type I IFN signaling activation in resting neutrophils of chronic granulomatous disease patients with CYBB mutations

Hematopoietic stem cell transplantation for CYBB heterozygous mutation resulting in very early onset inflammatory bowel disease in children: a case report

BackgroundInflammatory bowel disease (IBD) is a heterogeneous group of disorders associated with environmental triggers and dysregulated immune responses resulting in chronic, recurrent intestinal inflammation. Very early-onset IBD (VEO-IBD) refers to patients with symptoms or diagnosis before the age of 6 years and is widely thought to be associated with monogenic mutations. Traditional drug therapy is often ineffective in this patient population, while hematopoietic stem cell transplantation (HSCT) represents the definitive cure for patients with gene mutations.Case presentationWe report a case of VEO-IBD associated with a monogenic mutation in a 2-year-old girl presenting mainly with gastrointestinal symptoms, including recurrent hematochezia and abdominal pain for more than 3 months. A gastroscopy revealed erosive gastritis and bulbar duodenitis, while a colonoscopy indicated erosive colitis. Abnormal results were obtained from the dihydrohodamine (DHR) assay and immunoglobulin testing. Whole-exome sequencing identified a heterozygous and de novo nonsense mutation (c.388 C > T; p.R130X) in the CYBB gene leading to deficiency of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) (encoded by CYBB), a critical component of phagocytes. HSCT was performed successfully, and the DHR assay showed that normal neutrophil function was restored. Six months after HSCT, clinical remission was observed, and a repeat colonoscopy revealed intestinal mucosal healing was attained.ConclusionsPatients with CYBB mutations often develop recurrent or severe bacterial or fungal infections, mostly in the lungs, skin, lymph nodes, and liver. Here, we report on a young female child with CYBB mutations presenting predominantly with gastrointestinal symptoms. This study explores the mechanisms of inflammatory bowel disease caused by a monogenic mutation in CYBB to improve early diagnosis and effective treatment rates of this patient population.

CRISPR-gene-engineered CYBB knock-out PLB-985 cells, a useful model to study functional impact of X-linked chronic granulomatous disease mutations: application to the G412E X91+-CGD mutation.

Chronic granulomatous disease (CGD) is a rare primary immune disorder caused by mutations in one of the five subunits of the NADPH oxidase complex expressed in phagocytes. Two-thirds of CGD cases are caused by mutations in CYBB that encodes NOX2 or gp91phox. Some rare X91+-CGD point mutations lead to a loss of function but with a normal expression of the mutated NOX2 protein. It is therefore necessary to ensure that this mutation is indeed responsible for the loss of activity in order to make a safe diagnosis for genetic counselling. We previously used the X-CGD PLB-985 cell model of M.C. Dinauer obtained by homologous recombination in the original PLB-985 human myeloid cell line, in order to study the functional impact of such mutations. Although the PLB-985 cell line was originally described by K.A. Tucker et al. in1987 as a distinct cell line isolated from a patient with acute nonlymphocytic leukemia, it is actually identified as a subclone of the HL-60 cells. In order to use a cellular model that meets the quality standard for the functional study of X91+-CGD mutations in CGD diagnosis, we developed our own model using the CRISPR-Cas9 technology in a certified PLB-985 cell line from DSMZ-German Collection of Microorganisms and Cell Cultures. Thanks to this new X-CGD model, we demonstrated that the G412E mutation in NOX2 found in a X91+-CGD patient prohibits access of the electron donor NADPH to its binding site explaining the absence of superoxide production in his neutrophils.

Variant Type X91+ Chronic Granulomatous Disease: Clinical and Molecular Characterization in a Chinese Cohort

PurposeWe aimed to report the clinical and immunological characteristics of variant type X91+ chronic granulomatous disease (CGD) in a Chinese cohort.MethodsThe clinical manifestations and immunological phenotypes of patients with X91+ CGD were collected. A dihydrorhodamine (DHR) analysis was performed to evaluate neutrophil function. Gp91phox protein expression was determined using extracellular staining with the monoclonal antibody (mAb) 7D5 and flow cytometry.ResultsPatients with X91+ CGD accounted for 8% (7/85) of all patients with CGD. The median age of onset in the seven patients with X91+ CGD was 4 months. Six patients received the BCG vaccine, and 50% (3/6) had probable BCG infections. Mycobacterium tuberculosis infection was prominent. The most common sites of infection were the lung (6/7), lymph nodes (5/7), and soft tissue (3/7). Two patients experienced recurrent oral ulcers. The stimulation index (SI) of the patients with X91+ CGD ranged widely from 1.9 to 67.3. The difference in the SI among the three groups of patients (X91+ CGD, X91− CGD, and X910 CGD) was statistically significant (P = 0.0071). The three groups showed no significant differences in onset age, diagnosis age, or severe infection frequency. CYBB mutations associated with X91+ CGD were commonly located in the second transmembrane or intracellular regions. Three novel X91+ CGD–related mutations (c.1462–2 A > T, c.1243C > T, and c.925G > A) were identified.ConclusionsVariant type X91+ CGD may result in varied clinical manifestations. Moreover, the laboratory findings might indicate a moderate neutrophil SI. We should deepen our understanding of variant X91+ CGD to prevent missed diagnoses.

False-positive HIV serology, Candida lusitaniae pneumonia, and a novel mutation in the CYBB gene

BackgroundChronic granulomatous disease (CGD) presents with a myriad of clinical manifestations pertaining to both immunodeficiency and hyperinflammation. Although Candida infection is a signature organism for patients with CGD, C. lusitaniae pneumonia in CGD has rarely been reported. C. lusitaniae is a ubiquitous ascomycete predominantly infecting immunocompromised hosts and has the potential to rapidly develop multi-drug resistance during therapy. Additionally, C. lusitaniae is recognized for its variable resistance against amphotericin B. To date, C. lusitaniae infections in patients with CGD have not been reviewed in detail. False-positive HIV serology, resulting from polyclonal hypergammaglobulinemia, has been reported in association with several infections, auto-immune diseases, and malignancies. Although CGD is often associated with hypergammaglobulinemia, a false-positive HIV serology in CGD has not been reported previously. ProcedureWe report a combination of unique findings in a child with CGD – a false-positive HIV serology, Candida lusitaniae pneumonia, and a novel CYBB mutation. We also provide a detailed review of C. lusitaniae infections in patients with CGD. ResultsIn patients with CGD, C. lusitaniae has been reported to cause lymphadenitis (cervical, abdominal), fungemia, meningoencephalitis, or abscesses in the liver and spleen. Many CGD patients with C. lusitaniae infection have associated inflammatory complications of the gut (inflammatory bowel disease, colitis). Additionally, almost all C. lusitaniae infections in CGD have been reported in young infants or in patients receiving long-term immunosuppressive therapy. This reflects that further immunocompromise (in addition to the underlying immune deficiency in CGD) may specifically predispose to C. lusitaniae infection (unlike other candidal infections). Most of the CGD patients with documented C. lusitaniae infection have X-linked form of the disease which generally has been postulated to have a more severe clinical phenotype than the autosomal recessive forms of the disease. ConclusionsHIV serology may be positive in patients with CGD and other inborn errors of immunity as a result of hypergammaglobulinemia. C. lusitaniae, which may have peculiar and evolving antimicrobial sensitivity patterns, needs to be considered in patients with CGD and pneumonia. Lastly, to reiterate, CGD should to be considered in patients with proven C. lusitaniae infection.

Second Report of Chronic Granulomatous Disease in Jordan: Clinical and Genetic Description of 31 Patients From 21 Different Families, Including Families From Lybia and Iraq.

Chronic granulomatous Disease (CGD) is a rare innate immunodeficiency disorder caused by mutations in one of the six genes (CYBA, CYBB, NCF1, NCF2, NCF4, and CYBC1/EROS) encoding the superoxide-producing nicotinamide adenine dinucleotide phosphate (NADPH)—oxidase complex in phagocytes. In the Western population, the most prevalent form of CGD (about two-thirds of all cases) is the X-linked form (X-CGD) caused by mutations in CYBB. The autosomal recessive forms (AR-CGD), due to mutations in the other genes, collectively account for the remaining one-third of CGD cases. We investigated the clinical and molecular features of 22 Jordanian, 7 Libyan, and 2 Iraqi CGD patients from 21 different families. In addition, 11 sibling patients from these families were suspected to have been died from CGD as suggested by their familial and clinical history. All patients except 9 were children of consanguineous parents. Most of the patients suffered from AR-CGD, with mutations in CYBA, NCF1, and NCF2, encoding p22phox, p47phox, and p67phox proteins, respectively. AR-CGD was the most frequent form, in Jordan probably because consanguineous marriages are common in this country. Only one patient from non-consanguineous parents suffered from an X910 CGD subtype (0 indicates no protein expression). AR670 CGD and AR220 CGD appeared to be the most frequently found sub-types but also the most severe clinical forms compared to AR470 CGD. As a geographical clustering of 11 patients from eight Jordanian families exhibited the c.1171_1175delAAGCT mutation in NCF2, segregation analysis with nine polymorphic markers overlapping NCF2 indicates that a common ancestor has arisen ~1,075 years ago.

Four novel mutations in the gene encoding gp91-phox of human NADPH oxidase: consequences for oxidase assembly

The superoxide-forming nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase of human phagocytes comprises membrane-bound and cytosolic proteins, which, upon cell activation, assemble on the plasma membrane to form the active enzyme. Patients with chronic granulomatous disease (CGD) are defective in one of the phagocyte oxidase (phox) components, p47-phox or p67-phox, which reside in the cytosol of resting phagocytes, or gp91-phox or p22-phox, which constitute the membrane-bound cytochrome b(558). In four X-linked CGD patients we have identified novel missense mutations in CYBB, the gene encoding gp91-phox. These mutations were associated with normal amounts of nonfunctional cytochrome b(558) in the patients' neutrophils. In phorbol-myristate-stimulated neutrophils and in a cell-free translocation assay with neutrophil membranes and cytosol, the association of p47-phox and p67-phox with the membrane fraction of the cells with Cys369-->Arg, Gly408-->Glu, and Glu568--> Lys substitutions was strongly disturbed. Only a Thr341-->Lys substitution, residing in a region of gp91-phox involved in flavin adenine dinucleotide (FAD) binding, supported a normal translocation. Thus, the introduction or reversal of charge at residues 369, 408, and 568 in gp91-phox destroys the correct binding of p47-phox and p67-phox to cytochrome b(558). Based on mutagenesis studies of structurally related flavin-dependent oxidoreductases, we propose that the Thr341-->Lys substitution results in impaired hydride transfer from NADPH to FAD. Because we found no electron transfer in solubilized neutrophil plasma membranes from any of the four patients, we conclude that all four amino acid replacements are critical for electron transfer. Apparently, an intimate relation exists between domains of gp91-phox involved in electron transfer and in p47/p67-phox binding. (Blood. 2000;95:666-673)

Primary Immunodeficiencies in India: Molecular Diagnosis and the Role of Next-Generation Sequencing.

Primary immunodeficiency diseases (PIDs) are a group of clinically and genetically heterogeneous disorders showing ethnic and geographic diversities. Next-generation sequencing (NGS) is a comprehensive tool to diagnose PID. Although PID is common in India, data on the genetic spectrum of PIDs are limited due to financial restrictions. The study aims to characterize the clinical and genetic spectrum of PID patients in India and highlight the importance of a cost-effective targeted gene panel sequencing approach for PID in a resource-limited setting. The study includes 229 patients with clinical and laboratory features suggestive of PIDs. Mutation analysis was done by Sanger sequencing and NGS targeting a customized panel of genes. Pathogenic variants were identified in 97 patients involving 42 different genes with BTK and IL12RB1 being the most common mutated genes. Autosomal recessive and X-linked recessive inheritance were seen in 51.6% and 23.7% of patients. Mendelian susceptibility to mycobacterial diseases (MSMD) and IL12RB1 mutations was more common in our population compared to the Western world and the Middle East. Two patients with hypomorphic RAG1 mutations and one female with skewed CYBB mutation were also identified. Another 40 patients had variants classified as variants of uncertain significance (VUS). The study shows that targeted NGS is an effective diagnostic strategy for PIDs in countries with limited diagnostic resources. Molecular diagnosis of PID helps in genetic counseling and to make therapeutic decisions including the need for a stem cell transplantation.

Clinical, functional and genetic characterization of 16 patients suffering from chronic granulomatous disease variants-identification of 11 novel mutations in CYBB.

Chronic granulomatous disease (CGD) is a rare inherited disorder in which phagocytes lack nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. The most common form is the X-linked CGD (X91-CGD), caused by mutations in the CYBB gene. Clinical, functional and genetic characterizations of 16 CGD cases of male patients and their relatives were performed. We classified them as suffering from different variants of CGD (X910 , X91- or X91+ ), according to NADPH oxidase 2 (NOX2) expression and NADPH oxidase activity in neutrophils. Eleven mutations were novel (nine X910 -CGD and two X91- -CGD). One X910 -CGD was due to a new and extremely rare double missense mutation Thr208Arg-Thr503Ile. We investigated the pathological impact of each single mutation using stable transfection of each mutated cDNA in the NOX2 knock-out PLB-985 cell line. Both mutations leading to X91- -CGD were also novel; one deletion, c.-67delT, was localized in the promoter region of CYBB; the second c.253-1879A>G mutation activates a splicing donor site, which unveils a cryptic acceptor site leading to the inclusion of a 124-nucleotide pseudo-exon between exons 3 and 4 and responsible for the partial loss of NOX2 expression. Both X91- -CGD mutations were characterized by a low cytochrome b558 expression and a faint NADPH oxidase activity. The functional impact of new missense mutations is discussed in the context of a new three-dimensional model of the dehydrogenase domain of NOX2. Our study demonstrates that low NADPH oxidase activity found in both X91- -CGD patients correlates with mild clinical forms of CGD, whereas X910 -CGD and X91+ -CGD cases remain the most clinically severe forms.

Genetic, Immunological, and Clinical Features of the First Mexican Cohort of Patients with Chronic Granulomatous Disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by an inability of phagocytes to produce reactive oxygen species, impairing their killing of various bacteria and fungi. We summarize here the 93 cases of CGD diagnosed in Mexico from 2011 to 2019. Thirteen Mexican hospitals participated in this study. We describe the genetic, immunological, and clinical features of the 93 CGD patients from 78 unrelated kindreds. Eighty-two of the patients (88%) were male. All patients developed bacterial infections and 30% suffered from some kind of fungal infection. Fifty-four BCG-vaccinated patients (58%) presented infectious complications of BCG vaccine. Tuberculosis occurred in 29%. Granulomas were found in 56% of the patients. Autoimmune and inflammatory diseases were present in 15% of patients. A biological diagnosis of CGD was made in 89/93 patients, on the basis of NBT assay (n = 6), DHR (n = 27), and NBT plus DHR (n = 56). The deficiency was complete in all patients. The median age of biological diagnosis was 17months (range, 0-186months). A genetic diagnosis was made in 83/93 patients (when material was available), corresponding to CYBB (n = 64), NCF1 (n = 7), NCF2 (n = 7), and CYBA (n = 5) mutations. The clinical manifestations in these Mexican CGD patients were similar to those in patients elsewhere. This cohort is the largest in Latin America. Mycobacterial infections are an important cause of morbidity in Mexico, as in other countries in which tuberculosis is endemic and infants are vaccinated with BCG. X-linked CGD accounted for most of the cases in Mexico, as in other Latin American countries. However, a significant number of CYBA and NCF2 mutations were identified, expanding the spectrum of known causal mutations.

CRISPR gene-engineered CYBBko THP-1 cell lines highlight the crucial role of NADPH-induced reactive oxygen species for regulating inflammasome activation

Summary CRISPR-Cas9 engineered CYBBko THP-1 cell lines display an inflammatory profile with increased IL-1β, IL-6 and TNF-α secretion as consequence of NADPH-induced ROS deficiency. This phenotype is reminiscent of the one observed in phagocytes from CGD patients.

Role of Allogeneic Hematopoietic Stem Cell Transplant for Chronic Granulomatous Disease (CGD): a Report of the United States Immunodeficiency Network.

Chronic granulomatous disease (CGD) is a primary immunodeficiency for which allogeneic hematopoietic stem cell transplant (HSCT) offers potential cure. Direct comparison of HSCT to non-HSCT management in the North American population was performed to identify clinical factors associated with overall survival (OS) and transplant-related survival (TRS). Retrospective review of CGD subjects enrolled in the United States Immunodeficiency Network. Survival was estimated by the Kaplan-Meier method and modeled by proportional hazards regression. We identified 507 patients (66% CYBB mutants) diagnosed in 1953-2016. Fifty underwent allogeneic HSCT. Median follow-up was 9.1years after diagnosis (0-45.8years). OS was negatively associated with CYBB mutation (HR=6.25; p=0.034) and not associated with HSCT (88% v. 85% ± HCT) (HR=1.26; p=0.65). Transplant at ≤ 14years old was associated with improved TRS (93% v. 82% at T + 60months) (HR=- 4.51; p=0.035). Patients transplanted before 15years old had fewer severe infections pre-HSCT (mean 0.95 v. 2.13; p=0.047). No mortality was reported in patients receiving stem cells from matched siblings. Infection incidence declined post-HSCT in subjects with greater than or equal to four infections pre-HSCT (p=0.0010). Compared to non-HSCT patients ≥ 15years old, post-transplant survivors had higher mean performance score (93.2 v. 85.9; p=0.0039) and lower frequency of disability (11% v. 52%; p=0.014). Allogeneic HSCT was associated with reduced infection incidence and improved functional performance, but not with a change in overall survival. Transplant-related survival was elevated in patients undergoing HSCT before 15years old. Consider HSCT prior to late adolescence in patients with severely diminished reactive oxygen intermediate synthesis, particularly if a matched sibling is available.

Genetic and Clinical Profiles of Disseminated Bacillus Calmette-Guérin Disease and Chronic Granulomatous Disease in China.

Background: Disseminated Bacillus Calmette-Guérin disease (D-BCG) in children with chronic granulomatous disease (CGD) can be fatal, while its clinical characteristics remain unclear because both diseases are extremely rare. The patients with CGD receive BCG vaccination, because BCG vaccination is usually performed within 24 h after delivery in China.Methods: We prospectively followed-up Chinese patients with CGD who developed D-BCG to characterize their clinical and genetic characteristics. The diagnoses were based on the patients' clinical, genetic, and microbiological characteristics.Results: Between September 2009 and September 2016, we identified 23 patients with CGD who developed D-BCG. Their overall 10-year survival rate was 34%. We created a simple dissemination score to evaluate the number of infected organ systems and the survival probabilities after 8 years were 62 and 17% among patients with simple dissemination scores of ≤3 and >3, respectively (p = 0.0424). Survival was not significantly associated with the CGD stimulation index or interferon-γ treatment. Eight patients underwent umbilical cord blood transplantation and 5 of them were successfully treated. The genetic analyses found mutations in CYBB (19 patients), CYBA (1 patient), NCF1 (1 patient), and NCF2 (1 patient). We identified 6 novel highly likely pathogenic mutations, including 4 mutations in CYBB and 2 mutations in NCF1.Conclusions: D-BCG is a deadly complication of CGD. The extent of BCG spreading is strongly associated with clinical outcomes, and hematopoietic stem cell transplantation may be a therapeutic option for this condition.

Next Generation Proteomics of Human Neutrophil Granulocytes in Monogenic Disease

Background and objectiveNeutrophil granulocytes (NG) are critical mediators of innate immunity and tissue regeneration. Few monogenic diseases affecting their differentiation and/or function have been described, yet a systems-biology perspective has not yet been opened. We set out to define the proteome and transcriptome of NG from healthy individuals and exome-sequenced patients aiming to develop a multi-dimensional perspective for diagnostic purposes.Patients and methodsWe analyzed highly purified peripheral blood neutrophil granulocytes from 68 healthy individuals, 6 children with severe congenital neutropenia (SCN) associated with ELANE mutations, 5 children with chronic granulomatous disease (CGD) with CYBA (2) or CYBB (3) mutations and 3 children with leukocyte adhesion deficiency (LAD, 2x ITGB2 mutation, 1x SLC35C1 mutation). In addition we analyzed proteomes of 2 children with genetically undetermined neutrophil defects. Cells were isolated from fresh venous blood using magnetic bead-based negative selection (purity >99%). Whole cell proteome analysis was done by data-independent acquisition using a Thermo Fisher QExactive HF mass spectrometer.ResultsWe quantified a total of ~4300 proteins in each sample. This is the first study to analyze highly purified neutrophil granulocytes from a large cohort of healthy individuals as well as from patients with rare diseases affecting differentiation and/or function of NG. Intraindividual reproducibility was shown for one donor sampled at 3 different time points with a correlation coefficient of ~0.89. Principal component analysis demonstrated separation of the proteome of healthy and diseased cells. Interestingly, only 6 proteins constituted for 50% of the neutrophils proteome with the antimicrobial proteins DEF3A and S100A8 being the most abundant.Stringent differential expression analysis (p<0.01) showed specific differences between the neutrophil proteome of healthy individuals and patients with monogenic diseases. In patients with LAD, only few proteins were found to be present in decreased (7 proteins) or increased (5 proteins) abundance. Of note, proteins with lowest expression levels in LAD1 (ITGB2 mutation) were the integrins ITGB2, ITGAM and ITGAX while in LAD2 (SLC35C1 mutation) integrin expression was not affected. In contrast we noticed lack of PRCP, HDHD2 and XRCC4 protein. In patients with CGD, CYBA and CYBB were decreased in expression, independent of the underlying genotype. In contrast, the other members of the NADPH oxidase complex (NCF1/2/4, Rac1/2) were not found to be differentially expressed. This suggests that both membrane-bound proteins CYBB and CYBA stabilize each other. In total, CGD neutrophils showed limited aberrations with 23 overexpressed and 11 underexpressed proteins. In striking contrast to LAD and CGD, SCN patients with ELANE mutations had a much higher number of proteins with increased (93 proteins) or decreased (47 proteins) expression levels. It is currently unknown whether these imbalances are due to the immaturity of peripheral blood neutrophils in SCN or rather secondary to unfolded protein response and cellular stress mechanisms.In ongoing studies, we analyze the transcriptome of healthy neutrophil granulocytes in correlation to the proteome. Preliminary data indicate that there is poor correlation (PCC 0.11) between protein and mRNA levels, indicating that granule proteins are synthesized during earlier steps of differentiation.In two patients where exome sequencing did not reveal any underlying mutation, proteome analysis showed decreased expression of NCF1 and RAB27A, respectively. In both patients we were able to use targeted sequencing of amplified DNA to show the causative mutations in NCF1 and RAB27A. Multi-omic analysis may therefore be superior to unidimensional genomic sequencing to establish a molecular diagnosis.ConclusionIn-depth mass spectrometry-based proteomics provides new insights into disease-specific alterations of neutrophil granulocytes and may help to refine molecular diagnostics. DisclosuresNo relevant conflicts of interest to declare.

Preliminary study on chronic granulomatous disease in Sri Lanka

BackgroundChronic granulomatous disease (CGD) is a rare primary immunodeficiency of the phagocytic cells, which results in absent or diminished levels of microbicidal reactive oxygen species. The disease occurs due to germline mutations in the genes encoding the five subunits of NADPH oxidase complex. The present study is a pilot study to understand the clinical and genetic aspects of CGD in Sri Lanka.MethodsClinical records of thirteen CGD patients were analysed and compared with similar studies performed in different countries and regions to identify patterns in demographics, clinical manifestations and infectious agents. Genomic DNA and cDNA were analysed in eight patients to identify mutations in CYBB and NCF1 genes, thereby to ascertain the potential X-linked and autosomal recessive (AR) CGD patients.ResultsThe onset of symptoms in the patient cohort was very early (mean 4.6 months) compared to 20 months in India and 23.9 months in Latin America. Similarly, the age at diagnosis was lower (mean 1.6 years after birth) compared to other studies; 4.5 years in India and 6.1 years in Europe. Pulmonary manifestations were the most common (85%), followed by skin/subcutaneous infections (77%) and lymphadenopathy (62%). The death rate of local patients (38%) was higher than other countries (India 35%, Europe 20%). Majority (77%) were treated for tuberculosis at some point in life. Genetic analysis confirmed six out of eight patients as X-linked CGD cases with mutations in CYBB gene. A novel splice site mutation was identified in P-07 at position c.141+6 which resulted in the deletion of entire exon 2. Two siblings (P-05 and P-06) from consanguineous parents, were identified with AR-CGD based on the homozygous GT deletion mutation in NCF1 gene.ConclusionsThe clinical presentation, manifestations and genetic subtypes in the local cohort, appear to be comparable with global trends. Mycobacterial infections should be investigated and treated with more prominence. Effective treatment options are required to control the high mortality rate.

A Cohort of 169 Chronic Granulomatous Disease Patients Exposed to BCG Vaccination: a Retrospective Study from a Single Center in Shanghai, China (2004-2017).

Clinical diagnosis and treatment for chronic granulomatous disease (CGD) have advanced greatly in recent years. However, CGD patients in China have unique clinical features and infection spectrums, which are challenging to their caretakers. Here, we summarized the clinical characteristics, genetic features, treatment, and prognosis of CGD in a single center in Shanghai. One hundred sixty-nine CGD patients were recruited between January 2004 and May 2017 based on clinical diagnosis. Electronic medical charts were reviewed to collect clinical data. Among the 169 patients recruited, CYBB mutations were identified in 150 cases, whereas CYBA mutations were identified in 7 cases, NCF1 in 5, and NCF2 in 7. The medium age at onset was 1month (interquartile range 1-3). The medium age at diagnosis was 8months (interquartile range 3-19). The most common infection sites were the lung (95.9%), lymph node (58.5%), skin (45.4%), intestinal (43.1%), and perianal (38.5%). Bacillus Calmette-Guérin (BCG) infections were common (59.2%). In addition, other non-infectious complications were also common, including anemia (55.4%) and impaired liver functions (34.6%). Thirty-one patients received stem cell transplantation. By the end of this study, 83/131 patients survived. Similar to other non-consanguineous populations, X-linked CGD accounted for the majority of the cases in China. However, BCG infections were a clinical challenge unique to China. In addition, severe infections were the major cause of death and the overall mortality was still high in China.

Clinical and Genomic Correlates of Neutrophil Reactive Oxygen Species Production in Pediatric Patients With Crohn’s Disease

Individuals with monogenic disorders of phagocyte function develop chronic colitis that resembles Crohn's disease (CD). We tested for associations between mutations in genes encoding reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, neutrophil function, and phenotypes of CD in pediatric patients. We performed whole-exome sequence analysis to identify mutations in genes encoding NADPH oxidases (such as CYBA, CYBB, NCF1, NCF2, NCF4, RAC1, and RAC2) using DNA from 543 pediatric patients with inflammatory bowel diseases. Blood samples were collected from an additional 129 pediatric patients with CD and 26 children without IBD (controls); we performed assays for neutrophil activation, reactive oxygen species (ROS) production, and bacteria uptake and killing. Whole-exome sequence analysis was performed using DNA from 46 of the children with CD to examine associations with NADPH gene mutations; RNA sequence analyses were performed using blood cells from 46 children with CD to test for variations in neutrophil gene expression associated with ROS production. We identified 26 missense mutations in CYBA, CYBB, NCF1, NCF2, and NCF4. Patients with CD who carried mutations in these genes were 3-fold more likely to have perianal disease (P= .0008) and stricturing complications (P= .002) than children with CD without these mutations. Among patients withCD with none of these mutations, 9% had undergone abdominal surgery; among patients with mutations in these NADPH oxidase genes, 31% had undergone abdominal surgery (P=.0004). A higher proportion of neutrophils from children with CD had low ROS production (47%) than from controls (15%) among the 129 patients tested for ROS (P= .002). Minor alleles of the NADPH genes were detected in 7% of children with CD whose neutrophils produced normal levels of ROS vs 38% of children whose neutrophils produced low levels of ROS (P= .009). Neutrophils that produced low levels of ROS had specific alterations in genes that regulate glucose metabolism and antimicrobial responses. We identified missense mutations in genes that encode NADPH oxidases in children with CD; these were associated with a more aggressive disease course and reduced ROS production by neutrophils from the patients.

Identification of IL2RG and CYBB mutations in two Chinese primary immunodeficiency patients by whole-exome sequencing

ABSTRACT Background Primary immunodeficiency diseases are a group of genetic disorders that lead to increased propensity to a variety of infections, sometimes with fatal outcomes. Method In this study, whole-exome sequencing (WES) was used to identify mutations in two patients suspected of having primary immunodeficiency. Sanger sequencing was used to confirm the results in the patients and their family. Result One patient was diagnosed as X-linked severe combined immunodeficiency (X-SCID) and another patient as X-linked chronic granulomatous disease (X-CGD) by WES. Sequencing analysis of IL2RG gene revealed a novel mutation (c.794T>A, p.I265N) and CYBB gene revealed a missense mutation (c.935T>A, p.M312K). Discussion and conclusion This study identifies one novel mutation in the IL2RG gene and another, previously described mutation in the CYBB genes. It is the first report establishing a diagnosis of X-SCID and X-CGD using WES in Chinese patients.

The three CYBA variants (rs4673, rs1049254 and rs1049255) are benign: new evidence from a patient with CGD

BackgroundChronic granulomatous disease (CGD) is an inherited immunodeficiency disease caused by the defect of NADPH oxidase. Mutations in CYBB or CYBA gene may result in membrane subunits, gp91phox or p22phox, expression failure respectively and NADPH oxidase deficiency. Previous study showed that three variants, c.214 T > C (rs4673), c.521 T > C (rs1049254) and c.*24G > A (rs1049255), in CYBA gene form a haplotype, which are associated with decreased reactive oxygen species generation. The study aims to confirm the three above mentioned variants are benign and report a novel mutation in CYBB gene.MethodsA patient with CGD and his family members were enrolled in the study. NADPH oxidase activity and gp91phox protein expression of neutrophils were analyzed by flow cytometry. Direct sequencing was used to detect CYBB and CYBA gene mutations.ResultsThe patient was diagnosed with CGD according to clinical and immune phenotype. The case has a novel homozygous mutation in CYBB gene and the above mentioned three variants in CYBA gene. The mutation in CYBB gene was confirmed to be pathogenic, and the three variants in CYBA gene to be benign.ConclusionsThe study not only reported a novel mutation in CYBB, which results in CGD, but also confirmed the above mentioned three variants in CYBA are benign.