BACKGROUND Common variable immunodeficiency (CVID) is characterized by late-onset hypogammaglobulinemia in the absence of predisposing factors. The genetic cause is unknown in the majority of cases, and less than 10% of patients have a family history of the disease. Most patients have normal numbers of B cells but lack plasma cells. METHODS We used whole-exome sequencing and array-based comparative genomic hybridization to evaluate a subset of patients with CVID and low B-cell numbers. Mutant proteins were analyzed for DNA binding with the use of an electrophoretic mobility-shift assay (EMSA) and confocal microscopy. Flow cytometry was used to analyze peripheral-blood lymphocytes and bone marrow aspirates. RESULTS Six different heterozygous mutations in IKZF1, the gene encoding the transcription factor IKAROS, were identified in 29 persons from six families. In two families, the mutation was a de novo event in the proband. All the mutations, four amino acid substitutions, an intragenic deletion, and a 4.7-Mb multigene deletion involved the DNA-binding domain of IKAROS. The proteins bearing missense mutations failed to bind target DNA sequences on EMSA and confocal microscopy; however, they did not inhibit the binding of wild-type IKAROS. Studies in family members showed progressive loss of B cells and serum immunoglobulins. Bone marrow aspirates in two patients had markedly decreased early B-cell precursors, but plasma cells were present. Acute lymphoblastic leukemia developed in 2 of the 29 patients. CONCLUSIONS Heterozygous mutations in the transcription factor IKAROS caused an autosomal dominant form of CVID that is associated with a striking decrease in B-cell numbers. (Funded by the National Institutes of Health and others.)

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.

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.

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Group B streptococci (GBS) are a major cause of severe infection in newborns, pregnant females, and other immunocompromised hosts. Infection often includes septicemia, shock, pneumonia, and respiratory failure. In previous studies, we have reported that GBS induce marked production of tumor necrosis factor alpha (TNF-␣) by human mononuclear cells. The present study was designed to measure the production of TNF-␣ as well as additional cytokines, including interleukin 1␤ (IL-1␤), IL-6, IL-8, IL-12, and gamma interferon (IFN-␥) but also to determine from what cells and at what time point during incubation with GBS that these cytokines are produced. Mixed mononuclear cells were incubated with heat-killed GBS, media alone, or 1 g of Escherichia coli lipopolysaccharide (LPS). Brefeldin A was added to each sample prior to staining, which prevented the export of cytokines by the Golgi apparatus. The cells were then stained with the appropriate conjugated antibodies and analyzed by using a flow cytometer. Results indicate that intracellular cytokines appear, in almost all cases, simultaneous to or before secreted proteins are detected. In contrast to the response to LPS, where TNF-␣, IL-1␤, IL-6, and IL-8 appear almost simultaneously, the human monocyte response to GBS results in the production of TNF-␣ but delayed appearance of IL-1␤, IL-6, and IL-8. The lymphocyte response to GBS was also strikingly different from that to LPS in that both secreted IFN-␥ and IL-12 was detected, while LPS failed to induce production of these critical cytokines. This suggests an important role for TNF-␣, IFN-␥, and IL-12 in GBS pathogenesis and/or immunity.

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-␥), and that incubation of neonatal neutrophils with recombinant IFN-␥ 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-␥ in the presence of bacteria and bacterial products. We examine the ability of GBS to induce the production of IFN-␥, IL-18, and IL-12 by cord blood mixed mononuclear cells and compared these results with the IFN-␥, IL-18, and IL-12 response of mixed mononuclear cells from adult blood. We demonstrate that cord blood mixed mononuclear cells produced significantly less IL-18 is a recently described member of the IL-1 cytokine family, which was initially defined as IFN-␥-inducing factor (1). IL-18 gene expression and/or protein secretion has been observed in macrophages (2), dendritic cells (3), mononuclear cells (4), keratinocytes (5), chondrocytes (6), pituitary and adrenal cells (7), astrocytes and microglia (8), and intestinal epithelial cells (9). Studies have elucidated a broad array of effector functions implicating IL-18 as an important regulator of both innate and acquired immune responses (10, 11). In animals, IL-18 contributes to protective immunity against a variety of pathogens, including Cryptococcus, Leishmania, Staphylococcus, Salmonella,.IL-12 is also an integral immune regulator, which promotes Th-1 responses while suppressing Th-2 responses (16,17). IL-12 is primarily produced by macrophages and dendritic cells and has been shown to induce the production of IFN-␥ by T cells and natural killer (NK) cells (18). Recent studies have focused on the interaction between IL-18 and IL-12 in certain inflammatory responses. In Th-1 immune responses, IL-18 and IL-12 are important cytokines that may synergistically stimulate IFN-␥ production and enhance NK and T cell-mediated cytotoxicity (19). Recent studies implicate the interaction of these Th-1 cytokines in the development of autoimmune diseases and suggest that regulating their function may be therapeutically beneficial (20,21).Early-onset GBS infections in neonates often lead to sepsis and severe septic shock, with an approximate 5-15% mortality rate (22)(23)(24)

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.