Identification of patients with RAG mutations previously diagnosed with common variable immunodeficiency disorders.

PURPOSE: Combined immunodeficiency (CID) presents a unique challenge to clinicians. Two patients presented with the prior clinical diagnosis of common variable immunodeficiency (CVID) disorder marked by an early age of presentation, opportunistic infections, and persistent lymphopenia. Due to the presence of atypical clinical features, next generation sequencing was applied documenting RAG deficiency in both patients. METHODS: Two different genetic analysis techniques were applied in these patients including whole exome sequencing in one patient and the use of a gene panel designed to target genes known to cause primary immunodeficiency disorders (PIDD) in a second patient. Sanger dideoxy sequencing was used to confirm RAG1 mutations in both patients. RESULTS: Two young adults with a history of recurrent bacterial sinopulmonary infections, viral infections, and autoimmune disease as well as progressive hypogammaglobulinemia, abnormal antibody responses, lymphopenia and a prior diagnosis of CVID disorder were evaluated. Compound heterozygous mutations in RAG1 (1) c256_257delAA, p86VfsX32 and (2) c1835A>G, pH612R were documented in one patient. Compound heterozygous mutations in RAG1 (1) c.1566G>T, p.W522C and (2) c.2689C>T, p. R897X) were documented in a second patient post-mortem following a fatal opportunistic infection. CONCLUSION: Astute clinical judgment in the evaluation of patients with PIDD is necessary. Atypical clinical findings such as early onset, granulomatous disease, or opportunistic infections should support the consideration of atypical forms of late onset CID secondary to RAG deficiency. Next generation sequencing approaches provide powerful tools in the investigation of these patients and may expedite definitive treatments.

Severely depressed interleukin-17 production by human neonatal mononuclear cells.

BACKGROUND: The role of T-helper 17 cells (Th17) in neonatal host defense remains to be fully elucidated. Interleukin (IL)-17 plays an important role in the immune response to bacterial and fungal pathogens by promoting inflammation. METHODS: We examined neonatal production of IL-17 in mixed mononuclear cells (MMCs) isolated from umbilical cord blood for comparison with adult peripheral blood mononuclear cell controls. RESULTS: IL-17 production was profoundly diminished in MMCs isolated from cord blood when compared with MMCs from adult blood. This was associated with a marked reduction in the population of CCR6+ IL-17(+) T-cells in the neonatal cord blood. We also found diminished intracellular formation of IL-17, and diminished IL-17 responses to both group B streptococci (GBS) and Escherichia coli. Neonatal mononuclear cells were found to adequately phosphorylate signal transducer and activator of transcription 3, pY705, and pS727. We and others have reported markedly reduced interferon-γ production by neonate mononuclear cells exposed to GBS. Here, we correct that profound abnormality with added IL-17. CONCLUSION: Our results suggest that profound deficiency of IL-17 production associated with a marked decrease in Th17 cells likely contributes significantly to the increased susceptibility of human neonates to invasive bacterial and fungal infections.

Germline mutations in NFKB2 implicate the noncanonical NF-κB pathway in the pathogenesis of common variable immunodeficiency.

Common variable immunodeficiency (CVID) is a heterogeneous disorder characterized by antibody deficiency, poor humoral response to antigens, and recurrent infections. To investigate the molecular cause of CVID, we carried out exome sequence analysis of a family diagnosed with CVID and identified a heterozygous frameshift mutation, c.2564delA (p.Lys855Serfs(∗)7), in NFKB2 affecting the C terminus of NF-κB2 (also known as p100/p52 or p100/p49). Subsequent screening of NFKB2 in 33 unrelated CVID-affected individuals uncovered a second heterozygous nonsense mutation, c.2557C>T (p.Arg853(∗)), in one simplex case. Affected individuals in both families presented with an unusual combination of childhood-onset hypogammaglobulinemia with recurrent infections, autoimmune features, and adrenal insufficiency. NF-κB2 is the principal protein involved in the noncanonical NF-κB pathway, is evolutionarily conserved, and functions in peripheral lymphoid organ development, B cell development, and antibody production. In addition, Nfkb2 mouse models demonstrate a CVID-like phenotype with hypogammaglobulinemia and poor humoral response to antigens. Immunoblot analysis and immunofluorescence microscopy of transformed B cells from affected individuals show that the NFKB2 mutations affect phosphorylation and proteasomal processing of p100 and, ultimately, p52 nuclear translocation. These findings describe germline mutations in NFKB2 and establish the noncanonical NF-κB signaling pathway as a genetic etiology for this primary immunodeficiency syndrome.

Rare duplication or deletion of exons 6, 7 and 8 in CYBB leading to X-linked chronic granulomatous disease in two patients from different families.

Chronic granulomatous disease (CGD) is a rare congenital disorder in which phagocytes cannot generate superoxide (O(2)(-)) and other microbicidal oxidants due to mutations in one of the five components of the O(2)(-)-generating NADPH oxidase complex. The most common form is caused by mutations in CYBB on the X chromosome, encoding gp91phox, the enzymatic subunit of the phagocyte NADPH oxidase. Here, we report two rare cases of male X-linked CGD patients, one caused by a 5.7-kb duplication of a region containing CYBB exons 6 to 8 and the other caused by a deletion of this same region. We found both the duplication in patient 1 and the deletion in patient 2 to be bordered by a GT repeat. Indeed, in control DNA, the 3' part of CYBB intron 5 contains a GT repeat and the 5' part of intron 8 also contains such a repeat. Duplication of exons 6, 7 and 8 in patient 1 was probably caused by a non-homologous crossing over between the two GT repeats. The deletion found in patient 2 probably arose from a similar misalignment. The results found in these patients were confirmed by multiplex ligation-dependent probe amplification. The clinical profile of XCGD is severe in both patients.

Diffuse large B cell lymphoma in hyper-IgE syndrome due to STAT3 mutation.

The Job or hyper-immunoglobulinemia E syndrome is a primary immunodeficiency that is usually inherited in an autosomal dominant fashion. With the discovery of mutations in the STAT3 gene in the majority of autosomal dominant cases, it is now possible to make a molecular diagnosis of hyper-IgE syndrome. Both primary and secondary immunodeficiencies, including hyper-IgE syndrome, may predispose for malignancies, especially lymphomas, mainly mature B cell lymphomas, and classical Hodgkin lymphoma. Here, we report of a 48-year-old male with hyper-IgE syndrome who developed a primary parotid gland diffuse large B cell lymphoma. Analysis for STAT3 mutations demonstrated that the causal mutation of hyper-IgE syndrome, R382Q, arose de novo in the patient and it was transmitted to three of his five children, all three of whom are clinically affected. We review the literature regarding lymphoma in hyper-IgE syndrome and the possible etiologic relationship with STAT3 mutations.

Rapid genetic analysis of x-linked chronic granulomatous disease by high-resolution melting.

High-resolution melting analysis was applied to X-linked chronic granulomatous disease, a rare disorder resulting from mutations in CYBB. Melting curves of the 13 PCR products bracketing CYBB exons were predicted by Poland's algorithm and compared with observed curves from 96 normal individuals. Primer plates were prepared robotically in batches and dried, greatly simplifying the 3- to 6-hour workflow that included DNA isolation, PCR, melting, and cycle sequencing of any positive products. Small point mutations or insertions/deletions were detected by mixing the hemizygous male DNA with normal male DNA to produce artificial heterozygotes, whereas detection of gross deletions was performed on unmixed samples. Eighteen validation samples and 22 clinical kindreds were analyzed for CYBB mutations. All blinded validation samples were correctly identified. The clinical probands were identified after screening for neutrophil oxidase activity. Nineteen different mutations were found, including seven near intron-exon boundaries predicting splicing defects, five substitutions within exons, three small deletions predicting premature termination, and four gross deletions of multiple exons. Ten novel mutations were found, including (c.) two missense (730T>A, 134T>G), one nonsense (90C>A), four splice site defects (45 + 1G>T, 674 + 4A>G, 1461 + 2delT, and 1462-2A>C), two small deletions (636delT, 1661_1662delCT), and one gross deletion of exons 6 to 8. High-resolution melting can provide timely diagnosis at low cost for effective clinical management of rare, genetic primary immunodeficiency disorders.

Rapid molecular analysis of the STAT3 gene in Job syndrome of hyper-IgE and recurrent infectious diseases.

With the recent discovery of mutations in the STAT3 gene in the majority of patients with classic Hyper-IgE syndrome, it is now possible to make a molecular diagnosis in most of these cases. We have developed a PCR-based high-resolution DNA-melting assay to scan selected exons of the STAT3 gene for mutations responsible for Hyper-IgE syndrome, which is then followed by targeted sequencing. We scanned for mutations in 10 unrelated pedigrees, which include 16 patients with classic Hyper-IgE syndrome. These pedigrees include both sporadic and familial cases and their relatives, and we have found STAT3 mutations in all affected individuals. High-resolution melting analysis allows a single day turn-around time for mutation scanning and targeted sequencing of the STAT3 gene, which will greatly facilitate the rapid diagnosis of the Hyper-IgE syndrome, allowing prompt and appropriate therapy, prophylaxis, improved clinical outcome, and accurate genetic counseling.

Multiplex analysis of toll-like receptor-stimulated neonatal cytokine response.

BACKGROUND: The human neonate's increased susceptibility to bacterial infections is not completely understood. Toll-like receptors (TLRs) have been recognized as pattern-recognition receptors critical to the innate immune response. TLR function in neonates, however, remains incompletely defined. OBJECTIVE: To examine regulatory and proinflammatory cytokine responses to TLR-1-6 stimulation of cord blood compared to adult blood. METHODS: We stimulated cord blood with ligands for each of TLRs 1-6 and compared these responses to adult controls. The following TLR ligands were utilized: Pam3CSK4 (TLR-1 and 2), zymosan (TLR-2 and 6), poly I:C (TLR-3), LPS (TLR-4), and flagellin (TLR-5). Cytokine production was measured with an assay developed in-house utilizing multi-analyte technology. RESULTS: TLR-1-6 stimulation produced higher concentrations of proinflammatory cytokines (IL-1beta, IL-6, and IL-8) in cord blood compared to adult blood, with the exception of TLR-4-stimulated TNF-alpha production, which was significantly lower in cord blood (319 pg/ml) compared to adult blood (645 pg/ml; p = 0.027). In contrast, TLR-1-6 stimulation resulted in decreased concentrations of Th1 and Th2 cytokines in cord blood compared to adult blood, with significantly diminished production of IL-12 (TLRs 1/2, 2/6, 3 and 4), IL-13 (TLR-1-6), and IL-10 (TLR-4). CONCLUSION: Cord blood production of regulatory Th1 and Th2 cytokines following TLR stimulation is decreased compared to that of adult blood. In contrast, TLR-stimulated proinflammatory cytokine production was markedly higher in neonates than in adults, with the exception of TLR-4-induced TNF-alpha production. The human neonate's increased susceptibility to bacterial infections may be related to abnormal TLR responsiveness, with enhanced proinflammatory and decreased regulatory cytokine production.

Family clusters of variant X-linked chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a rare inherited immunodeficiency disorder. The clinical presentation is varied depending on the degree of involvement of the NADPH oxidase system responsible for the oxidative burst of neutrophils. We present 3 cases of variant X-linked CGD in an effort to introduce the disease and highlight the importance and limitations of CGD screening. The variant X-linked form of CGD results in a less severe phenotype and frequently presents later in life. Variant X-linked CGD is difficult to diagnose, but is becoming more readily recognized based on improved testing methods. A high index of suspicion in the setting of unusual infections such as Burkholderia cepacia pneumonia is essential to make the diagnosis. Family screening can lead to early intervention, prophylaxis, and appropriate genetic counseling.

Comprehensive analysis of antibody responses to streptococcal and tissue antigens in patients with acute rheumatic fever.

Acute rheumatic fever (ARF) is an autoimmune disease occurring in individuals following untreated group A streptococcal infection believed to be triggered by antibodies to bacterial components that cross-react with human tissues. We developed a multiplexed immunoassay for the simultaneous quantitation of antibodies to nine streptococcal-related antigens including streptolysin O (SLO), DNase B, collagen I and IV, fibronectin, myosin, group A carbohydrate, M6 protein and streptococcal C5a peptidase. Utilizing this method, we examined serum from 49 ARF, 58 pharyngitis patients and age- and sex-matched controls in samples collected at initial disease onset, and at 4 weeks, 6 months and 1 year after diagnosis. Antibody responses were significantly higher for SLO, DNase B, M6 protein, group A carbohydrate and the cross-reactive antigens collagen I and myosin in ARF compared with pharyngitis patients (P

Comparison of ATP production in whole blood and lymphocyte proliferation in response to phytohemagglutinin.

Lymphocyte proliferation in response to mitogens, phytohemagglutinin (PHA), concanavalin A, pokeweed, and/or specific antigens has been the method of choice for in vitro diagnosis of cell-mediated immune dysfunction. Recently, an assay to measure intracellular adenosine triphosphate (ATP) production in response to PHA has been developed that requires a shorter, overnight incubation. We compared a standard 5- to 7-day lymphocyte mitogen stimulation assay utilizing tritiated thymidine (3H-thy) incorporation to one in which ATP production in response to PHA by CD4-positive cells is measured in a luminometer that requires only 18-24 hr. A total of 20 patient samples suspected of having decreased cell-mediated immunity submitted for mitogen induced lymphocyte proliferation and 21 normal controls were tested in both assays. A comparison of these two methods has demonstrated that the screening ATP assay has a sensitivity at 24 hr of 100% in detecting decreased PHA induced lymphocyte proliferation at 5 days and a specificity of 85% in the samples obtained from normal controls. The data indicate that the ATP assay may be a useful screening tool for more rapid detection of blood samples with decreased cell-mediated immune responses. However, a positive screen should always be confirmed by 3H-thy uptake using mitogens and recall antigens like candida and tetanus.

Development of a multiplexed fluorescent immunoassay for the quantitation of antibody responses to group A streptococci.

The host immunologic response to group A streptococcal infections gives rise to numerous antibodies directed against cellular and extracellular bacterial antigens. For determining individual immune status, or studying the pathogenesis of group A streptococcal associated diseases, such as acute rheumatic fever (ARF), an assay capable of determining antibodies responses to multiple antigens would be of great advantage. We have developed a microsphere based, multiplexed immunoassay for the simultaneous quantitation of antibodies to nine different extracellular, ARF related tissue and group A streptococci specific antigens using only 5 microl of sample. Through the selection of microspheres and serum diluent, non-specific antibody binding was reduced by 17%. Different formulations of the coupling buffer were found to greatly influence the efficiency of coupling antigens to the carboxylated microspheres. Monoclonal antibodies against the different antigens demonstrated assay specificity as well as sensitivities of less than 1 ng/ml of antibody. This multiplexed assay should be a powerful research and clinical tool in determining antibody responses to group A streptococcal infections and in potentially determining the role of a variety of cross-reactive antigens in rheumatic fever and rheumatic heart disease.

D8/17 and CD19 expression on lymphocytes of patients with acute rheumatic fever and Tourette's disorder.

D8/17, an alloantigen found on B lymphocytes, has been reported to be elevated in patients susceptible to rheumatic fever and may be associated with autoimmune types of neuropsychiatric disorders. The pediatric-autoimmune-neuropsychiatric-disorders-associated-with-streptococci model is a putative model of pathogenesis for a group of children whose symptoms of obsessive-compulsive disorder and Tourette's disorder (TD) are abrupt and may be triggered by an infection with group A streptococci. As a test of this model, we have examined D8/17 levels on the B cells of patients with TD and acute rheumatic fever (ARF) along with those on the B cells of normal controls by flow cytometry. We have utilized several different preparations of D8/17 antibody along with a variety of secondary antibodies but have been unable to show an association with an elevated percentage of D8/17-positive, CD19-positive B cells in either ARF or TD. We did find, however, that the percentages of CD19-positive B cells in ARF and TD patients were significantly elevated compared to those in normal controls. Group A streptococcal pharyngitis patients also had an elevated percentage of CD19 B cells, however. These studies failed to confirm the utility of determining the percentage of B cells expressing the D8/17 alloantigen in ARF patients or our sample of TD patients. In contrast, the percentage of CD19-positive B cells was significantly elevated in ARF and TD patients, as well as group A streptococcal pharyngitis patients, suggesting a role for inflammation and/or autoimmunity in the pathogenesis of these disorders.

Defective production of IL-18 and IL-12 by cord blood mononuclear cells influences the T helper-1 interferon gamma response to group B Streptococci.

Human neonates are uniquely susceptible to group B streptococcal (GBS) infections. We have shown that neonatal mixed mononuclear cells have a deficiency in the production of the T helper-1 (Th-1) cytokine, interferon gamma (IFN-gamma), and that incubation of neonatal neutrophils with recombinant IFN-gamma corrects these neutrophil defects. IL-12 and the more recently described IL-18 are also Th-1 type cytokines that are able to induce the production of IFN-gamma in the presence of bacteria and bacterial products. We examine the ability of GBS to induce the production of IFN-gamma, IL-18, and IL-12 by cord blood mixed mononuclear cells and compared these results with the IFN-gamma, IL-18, and IL-12 response of mixed mononuclear cells from adult blood. We demonstrate that cord blood mixed mononuclear cells produced significantly less IFN-gamma, IL-18, and IL-12 in response to GBS compared with mixed mononuclear cells from adults. Cord blood mixed mononuclear cells' production of IFN-gamma is enhanced by added recombinant IL-18 and IL-12. The maximal cord blood cell production of IFN-gamma, in response to GBS, is achieved by priming the cells with both IL-18 and IL-12. We conclude that neonatal mixed mononuclear cells exhibit deficiencies in three main Th-1 type cytokine responses, IFN-gamma, IL-12, and IL-18. This combined Th-1 type cytokine deficiency may contribute to the enhanced susceptibility of the human neonate to GBS and other microbial infections.