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Chronic granulomatous disease is caused by mutations in any of the 6 components of the phagocytic NADPH oxidase complex including gp91 , p47 , p22 , p40 , p67 , or EROS. Functional assays include reactive oxygen species (ROS) production, flow cytometry, and immunoblotting for NADPH proteins. The advent of high-throughput sequencing allows genetic diagnosis for all components except (p47 ) due to two, nearly identical, pseudogenes ( , ). The majority of NCF1-CGD patients carry a 2-base deletion caused by crossover between and or . Currently, NCF1 deficiency is diagnosed functionally: a characteristic DHR with low levels of residual ROS, loss of p47 on immunoblot, or digital droplet PCR or Gene-scan to enumerate intact (GTGT) or deleted (ΔGT). While this provides patients a clinical CGD diagnosis, for the 20% of NCF1-CGD patients with a non-ΔGT mutation a definitive genetic diagnosis is still lacking. We developed a bioinformatic method using existing short or long-read sequencing data from 48 NCF1-CGD patients or carriers. We identified both ΔGT and non-ΔGT gene mutations. Additionally, we confirm that the presence of ΔGT in is due to pseudogene copy into the locus. We compare sequence from NCF1-CGD patients to cohorts of non-NCF1-CGD and healthy controls (1000Genomes), demonstrating pseudogene replacement of in NCF1-CGD as well as the reciprocal replacement of or by in some healthy controls. With this method, reanalysis of existing sequence data may provide genetic diagnosis to NCF1-CGD patients. This technique may be modified for other diagnostically relevant pseudogenes.

Chronic granulomatous disease (CGD) is a rare genetic disorder causing recurrent infections. More than one-quarter of patients develop hepatic abscesses and liver dysfunction. Recent reports suggest that disease-modifying treatment with corticosteroids is effective for these abscesses. Comparison of corticosteroid therapy to traditional invasive treatments has not been performed. Records of 268 patients with CGD treated at the National Institutes of Health from 1980 to 2014 were reviewed. Patients with liver involvement and complete records were included. We recorded residual reactive oxygen intermediate (ROI) production by neutrophils, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase germline mutation status, laboratory values, imaging characteristics, time to repeat hepatic interventions, and overall survival among 3 treatment cohorts: open liver surgery (OS), percutaneous liver-directed interventional radiology therapy (IR), and high-dose corticosteroid management (CM). Eighty-eight of 268 patients with CGD suffered liver involvement. Twenty-six patients with a median follow-up of 15.5 years (8.5-32.9 years of follow-up) had complete records and underwent 100 standard interventions (42 IR and 58 OS). Eight patients received a treatment with high-dose corticosteroids only. There were no differences in NADPH genotype, size, or number of abscesses between patients treated with OS, IR, or CM. Time to repeat intervention was extended in OS compared with IR (18.8 vs 9.5 months, P = .04) and further increased in CM alone (median time to recurrence not met). Impaired macrophage and neutrophil function measured by ROI production correlated with shorter time to repeat intervention (r = 0.6, P = .0019). Treatment of CGD-associated liver abscesses with corticosteroids was associated with fewer subsequent hepatic interventions and improved outcome compared to invasive treatments.

Autosomal dominant hyper IgE syndrome (AD-HIES), or Job's syndrome, is a primary immune deficiency caused by dominant-negative mutations in STAT3. Recurrent Staphylococcus aureus skin abscesses are a defining feature of this syndrome. A widely held hypothesis that defects in peripheral Th17 differentiation confer this susceptibility has never been directly evaluated. To assess the cutaneous immune response in AD-HIES, we induced suction blisters in healthy volunteers (HVs) and patients with AD-HIES and then challenged the wound with lethally irradiated bacteria. We show that cutaneous production of IL-17A and IL-17F was normal in patients with AD-HIES. Overproduction of TNF-α differentiated the responses in AD-HIES from HVs. This was associated with reduced IL-10 family signaling in blister-infiltrating cells and defective epithelial cell function. Mouse models of AD-HIES recapitulated these aberrant epithelial responses to S. aureus and involved defective epithelial-to-mesenchymal transition (EMT) rather than a failure of bacterial killing. Defective responses in mouse models of AD-HIES and primary keratinocyte cultures from patients with AD-HIES could be reversed by TNF-α blockade and by drugs with reported modulatory effects on EMT. Our results identify these as potential therapeutic approaches in patients with AD-HIES suffering S. aureus infections.

Aspergillus fumigatus is the most common etiologic agent of invasive aspergillosis in immunocompromised patients. Several studies have addressed the mechanism involved in host defense but only few have investigated the pathogen's response to attack by the host cells. To our knowledge, this is the first study that investigates the genes differentially expressed in conidia vs hyphae of A. fumigatus in response to neutrophils from healthy donors as well as from those with chronic granulomatous disease (CGD) which are defective in the production of reactive oxygen species. Transcriptional profiles of conidia and hyphae exposed to neutrophils, either from normal donors or from CGD patients, were obtained by using the genome-wide microarray. Upon exposure to either normal or CGD neutrophils, 244 genes were up-regulated in conidia but not in hyphae. Several of these genes are involved in the degradation of fatty acids, peroxisome function and the glyoxylate cycle which suggests that conidia exposed to neutrophils reprogram their metabolism to adjust to the host environment. In addition, the mRNA levels of four genes encoding proteins putatively involved in iron/copper assimilation were found to be higher in conidia and hyphae exposed to normal neutrophils compared to those exposed to CGD neutrophils. Deletants in several of the differentially expressed genes showed phenotypes related to the proposed functions, i.e. deletants of genes involved in fatty acid catabolism showed defective growth on fatty acids and the deletants of iron/copper assimilation showed higher sensitivity to the oxidative agent menadione. None of these deletants, however, showed reduced resistance to neutrophil attack. This work reveals the complex response of the fungus to leukocytes, one of the major host factors involved in antifungal defense, and identifies fungal genes that may be involved in establishing or prolonging infections in humans.

In chronic granulomatous disease of childhood, killing of microorganisms is impaired because of defects in the production of hydrogen peroxide. In this controlled study, 39 patients were treated in alternate years with either itraconazole or placebo, once per day. There were seven serious infections during treatment with placebo, and only one during treatment with itraconazole. Chronic granulomatous disease of childhood is a rare group of inherited disorders of phagocytic cells characterized clinically by recurrent life-threatening infections and excessive granuloma formation. 1 Phagocyte migration and phagocytosis are normal, but killing of microorganisms is impaired because of defective production of hydrogen peroxide and related products of oxygen metabolism. A variety of biochemical defects lead to the disorder. The current mortality rate is 2 to 5 percent per year. 2 In the absence of prophylaxis with antibiotics or interferon gamma, patients with chronic granulomatous disease have severe infections due to catalase-positive bacteria and fungi about once a year. Prophylaxis with . . .

Principles and Practice of Clinical Research, Fourth Edition has been thoroughly revised to provide a comprehensive look at both the fundamental principles and expanding practice of clinical research.

Background. Chronic granulomatous disease (CGD) is due to defective nicotinamide adenine dinucleotide phosphate oxidase activity and characterized by recurrent infections with a limited spectrum of bacteria and fungi as well as inflammatory complications. To understand the impact of common severe infections in CGD, we examined the records of 268 patients followed at a single center over 4 decades. Methods. All patients had confirmed diagnoses of CGD, and genotype was determined where possible. Medical records were excerpted into a standard format. Microbiologic analyses were restricted to Staphylococcus, Burkholderia, Serratia, Nocardia, and Aspergillus. Results. Aspergillus incidence was estimated at 2.6 cases per 100 patient-years; Burkholderia, 1.06 per 100 patient-years; Nocardia, 0.81 per 100 patient-years; Serratia, 0.98 per 100 patient-years, and severe Staphylococcus infection, 1.44 per 100 patient-years. Lung infection occurred in 87% of patients, whereas liver abscess occurred in 32%. Aspergillus incidence was 55% in the lower superoxide-producing quartiles (quartiles 1 and 2) but only 41% in the higher quartiles (rate ratio, <0.0001). Aspergillus and Serratia were somewhat more common in lower superoxide producing gp91phox deficiency. The median age at death has increased from 15.53 years before 1990 to 28.12 years in the last decade. Fungal infection carried a higher risk of mortality than bacterial infection and was the most common cause of death (55%).Gastrointestinal complications were not associated with either infection or mortality. Conclusions. Fungal infections remain a major determinant of survival in CGD. X-linked patients generally had more severe disease, and this was generally in those with lower residual superoxide production. Survival in CGD has increased over the years, but infections are still major causes of morbidity and mortality.

The third edition of this innovative work again provides a unique perspective on the clinical discovery process by providing input from experts within the NIH on the principles and practice of clinical research.

Survival in chronic granulomatous disease is related to the level of NADPH oxidase activity, which correlates with the altered site on the mutated gene. Assessed NADPH oxidase activity is a more powerful prognostic factor than reported clinical features of the disease. Chronic granulomatous disease, first described in the 1950s, 1 is a rare genetic disease (approximately 1 case in 200,000 people) without ethnic preference. The risk of death among persons with chronic granulomatous disease is 1 to 5% per year, and the extent of risk has been thought to depend on whether inheritance is an autosomal recessive trait or an X-linked trait. 2 Chronic granulomatous disease is caused by defects in any one of five subunits of phagocyte-derived NADPH oxidase, including gp91phox ( CYBB [cytochrome b-245, beta polypeptide], in approximately 65% of patients), p22phox ( CYBA [cytochrome b-245, alpha polypeptide], <5%), p47phox ( . . .

IntroductionChediak-Higashi syndrome (CHS) is a rare autosomal recessive disorder characterised by partial oculocutaneous albinism, a bleeding diathesis, immunological dysfunction and neurological impairment. Bi-allelic loss-of-function variants in LYST cause CHS. LYST encodes the lysosomal trafficking regulator, a highly conserved 429 kDa cytoplasmic protein with an unknown function.MethodsTo further our understanding of the pathogenesis of CHS, we conducted clinical evaluations on individuals with CHS enrolled in our natural history study. Using genomic DNA Sanger sequencing, we identified novel pathogenic LYST variants. Additionally, we performed an extensive literature review to curate reported LYST variants and classified these novel and reported variants according to the American College of Medical Genetics/Association for Molecular Pathology variant interpretation guidelines.ResultsOur investigation unveiled 11 novel pathogenic LYST variants in eight patients with a clinical diagnosis of CHS, substantiated by the presence of pathognomonic giant intracellular granules. From these novel variants, together with a comprehensive review of the literature, we compiled a total of 147 variants in LYST, including 61 frameshift variants (41%), 44 nonsense variants (30%), 23 missense variants (16%), 13 splice site variants or small genomic deletions for which the coding effect is unknown (9%), 5 in-frame variants (3%) and 1 start-loss variant (1%). Notably, a genotype–phenotype correlation emerged, whereby individuals harbouring at least one missense or in-frame variant generally resulted in milder disease, while those with two nonsense or frameshift variants generally had more severe disease.ConclusionThe identification of novel pathogenic LYST variants and improvements in variant classification will provide earlier diagnoses and improved care to individuals with CHS.