CYBB Research Articles

The Role of rs2238296 of the Mitochondrial DNA Polymerase Gamma Gene in Combination with Polymorphic Variants of Antioxidant Defense Genes in the Development of Postinfarction Left Ventricular Aneurysm

Studied the relationship of the polymorphic variant of the mitochondrial DNA polymerase gamma gene (POLG rs2238296) in combination with single-nucleotide polymorphic variants of the genes of the antioxidant system of the body (mitochondrial transcription factor A (TFAM rs1937), superoxide dismutase (SOD2 rs4880), glutathione peroxidase (GPX1 rs1050450), catalase (CAT rs1001179), paraoxonase 1 (PON1 rs854560) and NADP-H oxidase (CYBA rs4673)) with features of postinfarction remodeling of the left ventricle (LV). One hundred and fifty-three patients with coronary heart disease (137 men and 16 women) aged 56 (50; 60.5) years were examined. Genotyping was carried out using a polymerase chain reaction followed by analysis of the polymorphism of the lengths of restriction fragments. No significant difference was found in the SOD2, GPX1, CAT, PON1, TFAM genes in the studied groups. Significant differences were found with respect to the POLG and CYBA genes: the CC rs2238296 genotype of the POLG gene was found in every third patient with LV aneurysm (30.3%), where as in the group without aneurysm – only in every eighth case (12.3%, p = 0.006). The CC rs4673 genotype of the CYBA gene was found in every second patient with an aneurysm (51.8%) and in 32% without LV aneurysm (p = 0.01). Patients with a combination of CC (POLG) and CC (CYBA) genotypes were represented exclusively by younger men, who were characterized by a less burdened comorbid background, in comparison with patients with a different genotype of these genes. At the same time, the LV ejection fraction in such patients was significantly lower (40 (27; 52) and 50 (40; 61), p = = 0.006), and the development of LV aneurysm was recorded in 73% of cases.

ПОЛИМОРФИЗМЫ ГЕНОВ NOS3 И CYBA В РАЗВИТИИ ЦИРРОЗА ПЕЧЕНИ У БОЛЬНЫХ ХРОНИЧЕСКИМ ВИРУСНЫМ ГЕПАТИТОМ В В ЯКУТСКОЙ ПОПУЛЯЦИИ

Chronic viral hepatitis B is a significant public health problem. Hepatitis virus carrier factors affect the course of chronic hepatitis B and odds of progression to liver cirrhosis. So, the research for the early development genetic predictors of complications of chronic hepatitis B is relevant. NOS3 and CYBA genes can act as predictor genes for early fibrotic processes in the liver. Researches confirm the role of oxidative stress and the mechanisms of cellular aging in the development of liver fibrosis. The purpose of this study is to identify the relationship between the carriage of polymorphisms of the NOS3 and CYBA genes and the development of liver fibrosis in patients with chronic viral hepatitis B in the Yakut population. Molecular genetic study of SNPs rs1799983 and rs1801131 of the NOS3 gene, rs4673 of the CYBA gene was carried out on 417 people. The collection of representative material was carried out on the basis of the Infectious Diseases Department of the Yakut Republican Clinical Hospital and the NEFU Clinic. As a result of a genetic study, it was found that in carriers of the mutant allele for the rs1799983 polymorphism of the NOS3 gene, the probability of developing liver cirrhosis is 1.81 times higher (0.176–0.098, CI 95%,), in carriers of the rs4673 mutant allele of the CYBA gene, the probability of developing liver cirrhosis is higher by 1.9 times (0.211–0.111, CI 95%), in carriers of the mutant allele for the rs2070744 polymorphism of the NOS3 gene, the probability of developing liver cirrhosis is 2.03 times higher (0.181–0.089, CI 95%). Thus, the obtained results indicate the need for additional studies of these genes, since they can be predictors of the development of liver cirrhosis in patients with chronic viral hepatitis B in the Yakut population.

Polymorphic Variants of AGT, ABCA1, and CYBA Genes Influence the Survival of Patients with Coronary Artery Disease: A Prospective Cohort Study.

Genetic factors can influence the risk of coronary artery disease (CAD) and the survival of patients. Our previous research led to the identification of genetic variants predisposing to CAD in the Polish population. Since many of them affect the clinical phenotype of the disease, the aim of this study was searching for genetic factors potentially influencing survival in patients with CAD. The study included 276 patients hospitalized due to coronary artery disease. The database of medical history and genotypic results of 29 polymorphisms were used. The endpoint was defined as death from cardiovascular causes. Survival was defined as the period from angiographic confirmation of CAD to death from cardiovascular causes. Three of all the analyzed genes were associated with survival. In the case of the AGT (rs699) and ABCA1 (rs2230806) genes polymorphisms, the risk of death was higher in GG homozygotes compared to the A allele carriers in the 10-year period. In the case of the CYBA (rs72811418) gene polymorphism, the effect on mortality was shown in both 5- and 10-year periods. The TA heterozygotes were predisposed to a higher risk of death than the TT homozygotes. Concluding, the AGT, ABCA1, and CYBA genes polymorphisms influence the risk of death in patients with CAD.

Lentiviral gene therapy for X-linked chronic granulomatous disease recapitulates endogenous CYBB regulation and expression

AbstractX-linked chronic granulomatous disease (X-CGD) is a primary immunodeficiency caused by mutations in the CYBB gene, resulting in the inability of phagocytic cells to eliminate infections. To design a lentiviral vector (LV) capable of recapitulating the endogenous regulation and expression of CYBB, a bioinformatics-guided approach was used to elucidate the cognate enhancer elements regulating the native CYBB gene. Using this approach, we analyzed a 600-kilobase topologically associated domain of the CYBB gene and identified endogenous enhancer elements to supplement the CYBB promoter to develop MyeloVec, a physiologically regulated LV for the treatment of X-CGD. When compared with an LV currently in clinical trials for X-CGD, MyeloVec showed improved expression, superior gene transfer to hematopoietic stem and progenitor cells (HSPCs), corrected an X-CGD mouse model leading to complete protection against Burkholderia cepacia infection, and restored healthy donor levels of antimicrobial oxidase activity in neutrophils derived from HSPCs from patients with X-CGD. Our findings validate the bioinformatics-guided design approach and have yielded a novel LV with clinical promise for the treatment of X-CGD.

CHRONIC GRANULOMATOUS DISEASE PRESENTING AS COW'S MILK ALLERGY AND EOSINOPHILIC ESOPHAGITIS

<h3>Introduction</h3> An infant with failure to thrive was diagnosed with cow's milk allergy (CMA) and eosinophilic esophagitis (EOE). Further work up by multiple specialists considered inborn error of metabolism, cystic fibrosis, infection, or malignancy. He was diagnosed with chronic granulomatous disease (CGD) after 3 weeks in the hospital. <h3>Case</h3> A 2 month-old male with history of formula intolerance was admitted for poor weight gain, vomiting, and bloody stools in the setting of social challenges. The infant was pale-appearing with baseline tachypnea however chest x-ray was unremarkable. Initial immune workup was limited to immunoglobulin levels which were normal. Given his elevated fecal calprotectin, he was diagnosed with CMA. EGD revealed EOE and gastritis. He had persistent leukocytosis to 59,000/mm^3 with 32% eosinophils, anemia, thrombocytosis, and elevated inflammatory markers. This prompted suggestion of bone marrow biopsy to evaluate for leukemia. Prior to this, dihydrorhodamine (DHR) assay confirmed absence of superoxide production in neutrophils. Genetic testing revealed hemizygous pathogenic variant c.469C>T in the CYBB gene associated with X-linked CGD. Mother's DHR was normal suggesting de novo mutation. Chest CT after diagnosis showed right lower lobe inflammatory chronic granulomatous process which was confirmed with lung biopsy. Bacterial and fungal prophylaxis were initiated and curative treatment with hematopoietic is currently being pursued. This patient's genetic variant has been described in a case series to cause very early onset GI manifestations, hypereosinophilia, and hyperleukocytosis with no infections. Persistent failure to thrive and enteropathy with leukocytosis warrants an extensive workup as CGD and other primary immunodeficiencies may present with inflammatory findings.

Entamoeba histolytica HM-1: IMSS gene expression profiling identifies key hub genes, potential biomarkers, and pathways in Amoebiasis infection: a systematic network meta-analysis.

Entamoeba histolytica (E. histolytica) is an anaerobic parasite that causes Amoebiasis in the intestine or extraintestinal, with immunology, genetics, and environmental variables all playing a part in the disease's development, but its molecular mechanism is unknown. One of the primary obstacles in understanding the etiology of Amoebiasis will be identifying the genetics profiling that controls the Amoebiasis network. By examining the gene expression profile of Amoebiasis and comparing it with healthy controls, we could identify differentially expressed genes (DEGs). DEGs were used to build the Amoebiasis protein interaction network and calculated its network topological properties. We discovered nine key hub genes (KHGs): JUN, PTGS2, FCGR3A, MNDA, CYBB, EGR1, CCL2, TLR8, and LRRK2 genes. The genes JUN and EGR1 were transcriptional factors (TFs) and up-regulated, others down-regulated. hsa-miR-155-5p, hsa-miR-101-3p, hsa-miR-124-3p, hsa-miR-26b-5p, and hsa-miR-16-5p are also among the essential miRNAs that have been demonstrated to be targeted by KHGs. These KHGs were primarily enriched in the IL-17 signaling pathway, TNF signaling pathway, NOD-like receptor signaling pathway, and Toll-like receptor signaling pathway. miRNAs were grouped in various pathways, focusing on the TGF-β signaling pathway, human immunodeficiency virus 1 infection, insulin signaling pathway, signaling pathways regulating pluripotency of stem cells, etc. Amoebiasis KHGs (JUN, PTGS2, CCL2, and MNDA) and their associated miRNAs are the primary targets for therapeutic methods and possible biomarkers. Furthermore, we identified drugs for genes JUN, PTGS2, FCGR3A, CCL2, and LRRK2. KHGs, on the other hand, required experimental validation to prove their efficacy.

GENETIC AND CLINICAL PROFILING OF MENDELIAN SUSCEPTIBILITY TO MYCOBACTERIAL DISEASE PATIENTS; SINGLE CENTER EXPERIENCE

Objective: Mendelian susceptibility to mycobacterial disease (MSMD) is a subgroup of primary immunodeficiencies which develops with the Bacille Calmette–Guérin (BCG) vaccine or non-tuberculous mycobacterial infections. The clinical symptoms have a broad spectrum, from localized to disseminated infections. Materials and Methods: Herein, we performed whole-exome sequencing (WES) on 13 patients with MSMD phenotype. All variants were confirmed by Sanger sequencing. The mean age was 8.41 years (min 3 – max 14 years), and the mean age of symptom onset was 4.6 years in our cohort. Results: We found previously identified IFNGR1 (n=1), IFNGR2 (n=1), TYK2 (n=1), IL12RB1 (n=1), and CYBB (n=1) gene variants in nine patients. Our patients mostly suffered from lymphadenitis (61.5%), osteomyelitis (38%), and miliary tuberculosis (31%). All patients except one had had the BCG vaccination. Two patients developed BCGitis after vaccination. Three patients suffered from disseminated BCG infection (BCGosis). Conclusion: Our findings show the importance of molecular diagnosis in patients with severe infections as an approach for understanding the genetic basis of infectious diseases and deciding on treatment options. The deficiency of IFN-mediated immunity genes plays a crucial role in the pathogenesis of MSMD and must be considered in pediatric patients with BCGitis.

A Novel Assay in Whole Blood Demonstrates Restoration of Mitochondrial Activity in Phagocytes After Successful HSCT in Hyperinflamed X-Linked Chronic Granulomatous Disease

X-linked chronic granulomatous disease is a rare disease caused by mutations in the CYBB gene. While more extensive knowledge is available on genetics, pathogenesis, and possible therapeutic options, mitochondrial activity and its implications on patient monitoring are still not well-characterized. We have developed a novel protocol to study mitochondrial activity on whole blood of XCGD patients before and after transplantation, as well as on XCGD carriers. Here we present results of these analyses and of the restoration of mitochondrial activity in hyperinflamed X-linked Chronic Granulomatous Disease after hematopoietic stem cell transplantation. Moreover, we show a strong direct correlation between mitochondrial activity, chimerism, and DHR monitored before and after transplantation and in XCGD carriers. In conclusion, based on these findings, we suggest testing this new ready-to-use marker to better characterize patients before and after treatment and to investigate disease expression in carriers.

РОЛЬ ПОЛИМОРФИЗМОВ ГЕНОВ NOS3 И CYBA В ФОРМИРОВАНИИ ОСЛОЖНЕНИЙ ХРОНИЧЕСКОГО ВИРУСНОГО ГЕПАТИТА В Название объекта Название объекта в переводе ФИО автора / список авторов Место работы автора ORCID Ключевые слова Аннотация

In the Republic of Sakha (Yakutia), persistently high incidence rates of CVH remain, as well as mortality rates from complications of liver cirrhosis (LC), as the most common complication. It seems relevant to search for genetic predictors of early development of severe complications in CVH patients in the Yakut population. They can be polymorphic variants of the genes for endothelial dysfunction and oxidative stress NOS3 and CYBA. The collection of representative material was carried out on the basis of the Infectious Diseases Department of the State Budgetary Institution of the Republic of Sakha (Yakutia) of the Yakut Republican Clinical Hospital. In total, 47 DNA samples of patients with chronic viral hepatitis В and liver cirrhosis in its outcome, hospitalized in the Infectious Diseases Department from September 2020 to June 2021, were studied. The control group consisted of patients not infected with viral hepatitis, as well as without pathology of the hemostasis system. The experimental part of the study was carried out by real-time PCR with the melting of hybridization products with a fluorescent label, followed by analysis of the RealBest-Genetics melting curves. As a result of the PCR-RFLP analysis of the rs1799983 polymorphism of the NOS3 gene among patients with chronic viral hepatitis, the T allele frequency was 0.09, and the G allele frequency was 0.91. The frequency of the C allele of the rs2070744 polymorphism of the NOS3 gene was 0.05, the frequency of the T allele was 0.95. The frequency of occurrence of the major allele C of the rs4673 polymorphism of the CYBA gene was 0.78, the minor allele T was 0.22. The relationship between the carriage of polymorphisms of these genes and the disease was not found. However, it was found that carriers of the mutant allele for the rs1799983 polymorphism of the NOS3 gene were 1.81 times more likely to develop liver cirrhosis (95 % CI, odds in the groups 0.176–0.098), carriers of the mutant allele for the rs4673 polymorphism of the CYBA gene were more likely to develop cirrhosis liver cirrhosis is 1.9 times higher (95 % CI, odds in groups 0.211–0.111), in carriers of the mutant allele for the rs2070744 polymorphism of the NOS3 gene, the probability of developing liver cirrhosis is 2.03 times higher (95 % CI, odds in groups 0.181–0.089 ). In carriers of the mutant allele for the rs4673 polymorphism of the CYBA gene, shortening of PT and APTT (17.79 ± 10.26 and 35.13 ± 7.82 sec) is observed, which may indicate a greater tendency of this group to hypercoagulability. In carriers of the mutant allele for the rs2070744 polymorphism of the NOS3 gene, shortening of PT and APTT (11.56 ± 0.99 and 30.12 ± 4.45 sec) is observed, which may also indicate a greater tendency of this group to hypercoagulability.

A granular view of X-linked chronic granulomatous disease exploiting single-cell transcriptomics

Abstract X-linked chronic granulomatous disease (X-CGD) is a rare monogenetic immunodeficiency primarily affecting phagocytes. Precipitated by mutations in the CYBB gene, patients exhibit a compromised oxidative burst, leading to recurrent infections which can be life-threatening. Curiously, autoimmune manifestations are also common in patients and carriers. Here, exploiting the cell type-specific nature of this disorder, we characterize X-CGD on a transcriptional level using single-cell sequencing. Peripheral blood from 14 X-CGD probands and 10 carriers signed onto IRB approved protocol NCT00404560, as well as from 15 controls was sampled, and PBMCs and isolated monocytes were subjected to single-cell sequencing. Probands exhibited a strong differential expression signal relative to controls. This was composed of not only genes previously described to be up-regulated in X-CGD such as IFI27, and indeed an autoimmunity-associated broader type I interferon response, but also previously undescribed genes involved in monocyte function (ARG1), antimicrobial proteins (CAMP, SLPI), and inflammasome components (AIM2). Surprisingly, expression variability was not greater in carriers relative to probands or controls, indicating a lack of cell autonomous effects from the deletion of CYBB. Interestingly, aggregate expression of differentially expressed genes in the probands was able to classify carriers from sex-matched controls with high accuracy (AUROC = 0.92), indicating the presence of an X-CGD-specific gene signature. This gene signature was also strongly co-expressed across 17 chordate species, pointing towards the disruption of ancestral pathways important in antimicrobial immunity in X-CGD probands and carriers. This work was partially supported by a Swiss National Science Foundation fellowship to S.M.

De novo mutation of CYBB gene in a boy presenting as intra-abdominal infection of Burkholderia contaminans: a case report

BackgroundChronic granulomatous disease (CGD) is an inborn error of immunity. It is characterized by recurrent bacterial or fungal infections, including infections by Burkholderia species. This is due to respiratory burst dysfunction of phagocytes. Currently, there is no report on Burkholderia contaminans (B. Contaminans) infection in children with CGD.Case presentationWe present a previously healthy, 17-month-old Chinese boy infected with B. Contaminans in the intra-abdominal regions. Immunological screening, including assessment of cellular immunity and humoral immunity did not yield conclusive results. The level of nicotinamide adenine dinucleotide phosphatase (NADPH) activity was decreased and whole-exome sequencing identified a de novo mutation in the CYBB gene.ConclusionsFor specific pathogens such as B. Contaminans, immune assessment should be carried out even if there is no positive medical history or specificity in basic immunity screening.

In silico identification of single nucleotide variations at CpG sites regulating CpG island existence and size

Genetic and epigenetic modifications of genes involved in the key regulatory pathways play a significant role in the pathophysiology and progression of multifactorial diseases. The present study is an attempt to identify single nucleotide variations (SNVs) at CpG sites of promoters of ACAT1, APOB, APOE, CYBA, FAS, FLT1, KSR2, LDLR, MMP9, PCSK9, PHOX2A, REST, SH2B3, SORT1 and TIMP1 genes influencing CpG island (CGI) existence and size associated with the pathophysiology of Diabetes mellitus, Coronary artery disease and Cancers. Promoter sequences located between −2000 to + 2000 bp were retrieved from the EPDnew database and predicted the CpG island using MethPrimer. Further, SNVs at CpG sites were accessed from NCBI, Ensembl while transcription factor (TF) binding sites were accessed using AliBaba2.1. CGI existence and size were determined for each SNV at CpG site with respect to wild type and variant allele by MethPrimer. A total of 200 SNVs at CpG sites were analyzed from the promoters of ACAT1, APOB, APOE, CYBA, FAS, FLT1, KSR2, LDLR, MMP9, PCSK9, PHOX2A, REST, SH2B3, SORT1 and TIMP1 genes. Of these, only 17 (8.5%) SNVs were found to influence the loss of CGI while 70 (35%) SNVs were found to reduce the size of CGI. It has also been found that 59% (10) of CGI abolishing SNVs are showing differences in binding of TFs. The findings of the study suggest that the candidate SNVs at CpG sites regulating CGI existence and size might influence the DNA methylation status and expression of genes involved in molecular pathways associated with several diseases. The insights of the present study may pave the way for new experimental studies to undertake challenges in DNA methylation, gene expression and protein assays.

Germline Variants of CYBA and TRPM4 Predispose to Familial Colorectal Cancer.

Simple SummaryWhole-genome sequencing and bioinformatics analysis on unique colorectal cancer families revealed two attractive candidate predisposition genes, CYBA and TRPM4, each with a loss-of-function variant. Supported by our functional studies, we suggest that the two gene defects mechanistically involve intestinal barrier integrity through reactive oxygen species and mucus biology, which converges in chronic bowel inflammation, a known risk factor for colorectal cancer.Familial colorectal cancer (CRC) is only partially explained by known germline predisposing genes. We performed whole-genome sequencing in 15 Polish families of many affected individuals, without mutations in known CRC predisposing genes. We focused on loss-of-function variants and functionally characterized them. We identified a frameshift variant in the CYBA gene (c.246delC) in one family and a splice site variant in the TRPM4 gene (c.25–1 G > T) in another family. While both variants were absent or extremely rare in gene variant databases, we identified four additional Polish familial CRC cases and two healthy elderly individuals with the CYBA variant (odds ratio 2.46, 95% confidence interval 0.48–12.69). Both variants led to a premature stop codon and to a truncated protein. Functional characterization of the variants showed that knockdown of CYBA or TRPM4 depressed generation of reactive oxygen species (ROS) in LS174T and HT-29 cell lines. Knockdown of TRPM4 resulted in decreased MUC2 protein production. CYBA encodes a component in the NADPH oxidase system which generates ROS and controls, e.g., bacterial colonization in the gut. Germline CYBA variants are associated with early onset inflammatory bowel disease, supported with experimental evidence on loss of intestinal mucus barrier function due to ROS deficiency. TRPM4 encodes a calcium-activated ion channel, which, in a human colonic cancer cell line, controls calcium-mediated secretion of MUC2, a major component of intestinal mucus barrier. We suggest that the gene defects in CYBA and TRPM4 mechanistically involve intestinal barrier integrity through ROS and mucus biology, which converges in chronic bowel inflammation.

Overlapping infection of Nocardia farcinica and Aspergillus fumigatus in a child with X-linked chronic granulomatous disease: a case report

BackgroundChronic granulomatous disease (CGD) is a rare inherited primary immunodeficiency syndrome, manifested as recurrent infections and inflammatory complications. Although prophylactic treatment with antibiotics and antifungals improved the outcome of CGD patients, infections remain the major cause of mortality.Case presentationA boy aged 3 years and 8 months was admitted to hospital complaining of lip swelling with fever for half a month and neck abscess for 11 days. After a thorough examination, severe pneumonia, respiratory failure, oral and maxillofacial space infection, and perianal abscess were confirmed. However, his condition didn’t improve after initial comprehensive therapy. Subsequently, overlapping infections of Nocardia farcinica and Aspergillus fumigatus were identified by metagenomic next-generation sequencing. He was treated with imipenem, linezolid, and voriconazole intravenously, plus taking oral compound sulfamethoxazole. Later, his condition improved. Through whole-exome sequencing, the child was ultimately diagnosed as X-linked chronic granulomatous disease (X-CGD) caused by CYBB gene mutation. Allogeneic hematopoietic stem cell transplantation was the potential sanative approach but there were no available human leukocyte antigen compatible donors for the child. The family requested to transfer to a superior hospital for further treatment. Two months later, we followed up the child’s family. Unfortunately, the child had expired due to severe infection.ConclusionTo our knowledge, this is the first case of overlapping infection of Nocardia farcinica and Aspergillus fumigatus identified by metagenomic next-generation sequencing in a child with X-CGD from China. For infectious pathogens that are hard to diagnosis by traditional detection methods, metagenomic next-generation sequencing is recommended as an adminicle or indispensable approach for microbial identification. Patients with X-CGD have poor prognosis, early diagnosis and intervention of X-CGD may reduce the mortality.

Identification and Validation of CYBB, CD86, and C3AR1 as the Key Genes Related to Macrophage Infiltration of Gastric Cancer.

Gastric cancer (GC) is rampant around the world. Most of the GC cases are detected in advanced stages with poor prognosis. The identification of marker genes for early diagnosis is of great significance. Studying the tumor environment is helpful to acknowledge the process of tumorigenesis, development, and metastasis. Twenty-two kinds of immune cells were calculated by CIBERSORT from Gene Expression Omnibus (GEO) database. Subsequently, higher infiltration of macrophages M0 was discovered in GC compared with normal tissues. WGCNA was utilized to construct the network and then identify key modules and genes related to macrophages in TCGA. Finally, 18 hub genes were verified. In the PPI bar chart, the top 3 genes were chosen as hub genes involved in most pathways. On the TIMER and THPA websites, it is verified that the expression levels of CYBB, CD86, and C3AR1 genes in tumor tissues were higher than those in normal tissues. These genes may work as biomarkers or targets for accurate diagnosis and treatment of GC in the future. Our findings may be a new strategy for the treatment of GC.

Di-Tyrosine Crosslinking and NOX4 Expression as Oxidative Pathological Markers in the Lungs of Patients with Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a noninflammatory progressive lung disease. Oxidative damage is a hallmark of IPF, but the sources and consequences of oxidant generation in the lungs are unclear. In this study, we addressed the link between the H2O2-generating enzyme NADPH oxidase 4 (NOX4) and di-tyrosine (DT), an oxidative post-translational modification in IPF lungs. We performed immunohistochemical staining for DT and NOX4 in pulmonary tissue from patients with IPF and controls using validated antibodies. In the healthy lung, DT showed little or no staining and NOX4 was mostly present in normal vascular endothelium. On the other hand, both markers were detected in several cell types in the IPF patients, including vascular smooth muscle cells and epithelium (bronchial cells and epithelial cells type II). The link between NOX4 and DT was addressed in human fibroblasts deficient for NOX4 activity (mutation in the CYBA gene). Induction of NOX4 by Transforming growth factor beta 1 (TGFβ1) in fibroblasts led to moderate DT staining after the addition of a heme-containing peroxidase in control cells but not in the fibroblasts deficient for NOX4 activity. Our data indicate that DT is a histological marker of IPF and that NOX4 can generate a sufficient amount of H2O2 for DT formation in vitro.

Utility of Immunohistochemistry and Immunofluorescence in Determining the Pathogenic Variants of Chronic Granulomatous Disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency disorder of phagocytes due to defects in any of the five subunits of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. An initial diagnosis of CGD is made by flow cytometry-based dihydrorhodamine assay or nitro blue tetrazolium test, which is further confirmed by molecular assays. Expression of five subunits of NADPH oxidase components by either flow cytometric or western blot analysis provides clues toward the potential gene targets which are subsequently confirmed by various genetic assays. Immunohistochemistry (IHC) and immunofluorescence (IF) have never been earlier used to determine the expression of different subunits of NADPH oxidase system. We evaluated the utility of IHC and IF in determining the underlying pathogenic variants of CGD. Twelve genetically confirmed cases of CGD, comprising of biopsy specimens (n = 6), tissue blocks from autopsy cases (n = 3), and cellblocks of cell pellet prepared from peripheral blood (n = 4) were included. IHC for p67phox and p47phox subunits and IF for cytochrome b558 were performed. All 4 cases with pathogenic variation of NCF2 gene showed loss of expression for p67phox subunit. Two cases with pathogenic variation of NCF1 gene showed loss of expression for p47phox subunit. Five cases, except a single case with CYBB gene pathogenic variation, showed loss of expression for cytochrome b558 on IF. Thus, loss of expression consistently matched with the underlying genetic defects assessed by sequencing. Our results confirm our hypothesis that IHC and IF are two rapid, economical, pathologist-friendly techniques providing pertinent information regarding the underlying pathogenic variants and such immuno-analysis can be easily performed on the tissue.

Late Onset of Chronic Granulomatous Disease Revealed by Paecilomyces lilacinus Cutaneous Infection

Chronic granulomatous disease (CGD) is an inherited immunodeficiency due to defective leukocyte NADPH responsible for recurrent infections and aberrant inflammation. Mutations in the CYBB gene are responsible for the X-linked CGD and account for approximately 70% of the cases. CGD is diagnosed during childhood in males. Female carriers may have biased X-inactivation and may present with clinical manifestations depending on the level of residual NADPH oxidase activity. We report the case of a previously asymptomatic female carrier who was diagnosed at age 67 with a skin infection with the rare fungus Paecilomyces lilacinus as the first manifestation of CGD. Dihydrorhodamine 123 (DHR) activity was below 10%. Next-generation sequencing (NGS) revealed mutations in DNMT3A, ASXL1, and STAG2 suggesting that clonal hematopoiesis could be responsible for a progressive loss of NADPH oxidase activity and the late onset of X-linked CGD in this patient. Long-term follow-up of asymptomatic carrier women seems to be essential after 50 years old.

Granuloma Formation in a Cyba-Deficient Model of Chronic Granulomatous Disease Is Associated with Myeloid Hyperplasia and the Exhaustion of B-Cell Lineage.

Haematopoiesis is a paradigm of cell differentiation because of the wide variety and overwhelming number of mature blood cells produced daily. Under stress conditions, the organism must adapt to a boosted demand for blood cells. Chronic granulomatous disease (CGD) is a genetic disease caused by inactivating mutations that affect the phagocyte oxidase. Besides a defective innate immune system, CGD patients suffer from recurrent hyper-inflammation episodes, circumstances upon which they must face emergency haematopoiesis. The targeting of Cybb and Ncf1 genes have produced CGD animal models that are a useful surrogate when studying the pathophysiology and treatment of this disease. Here, we show that Cyba−/− mice spontaneously develop granuloma and, therefore, constitute a CGD animal model to complement the existing Cybb−/− and Ncf1−/− models. More importantly, we have analysed haematopoiesis in granuloma-bearing Cyba−/− mice. These animals showed a significant loss of weight, developed remarkable splenomegaly, bone marrow myeloid hyperplasia, and signs of anaemia. Haematological analyses showed a sharped decrease of B-cells and a striking development of myeloid cells in all compartments. Collectively, our results show that granuloma inflammatory lesions dramatically change haematopoiesis homeostasis. Consequently, we suggest that besides their defective innate immunity, the alteration of haematopoiesis homeostasis upon granuloma may contribute to the dismal outcome of CGD.

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.