Loss of B Cells in Patients with Heterozygous Mutations in IKAROS.

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.).

Frequent mutations in SH2D1A (XLP) in males presenting with high-grade mature B-cell neoplasms.

X-linked lymphoproliferative syndrome (XLP) is caused by mutations in SH2D1A, and is associated with overwhelming infectious mononucleosis, aplastic anemia, hypogammaglobulinemia, and B-cell lymphomas. However, the frequency of SH2D1A mutations in males who present with B NHL is unknown. Five cases of XLP were diagnosed among 158 males presenting with B NHL (approximately 3.2%). Four of the patients had two episodes of B NHL and one had a single episode of B NHL followed by aggressive infectious mononucleosis. Prospective screening for XLP in males with B-cell lymphoma at the time of initial diagnosis should be considered.

Membranous glomerulopathy in an adult patient with X-linked agammaglobulinemia receiving intravenous gammaglobulin.

BACKGROUND: Immune complex deposition in the subepithelial zone of glomerular capillaries can lead to membranous glomerulopathy. OBJECTIVE: To present the case of a 23-year-old man with X-linked agammaglobulinemia (XLA) who developed idiopathic membranous glomerulopathy while receiving intravenous immunoglobulin (IVIG). METHODS: We performed an immunological workup, genetic testing, and a renal biopsy. RESULTS: XLA was confirmed with less than 0.02% CD19+ cells in the blood after sequence analysis revealed a nonfunctional BTK gene. The patient presented with microhematuria, which persisted for 3 years and spanned treatment with 5 different preparations of intravenous gammaglobulin. Immunohistochemistry revealed membranous glomerulopathy. CONCLUSION: Although endogenous serum immunoglobulin (Ig) production is severely impaired in XLA, rare B lymphocytes that have managed to mature can produce functional IgG antibodies. The pathogenic immune complexes could reflect IVIG reacting with polymorphic autoantigens, an endogenous IgG-producing clone reacting with a common idiotype present in the IVIG, or both.

A minimally hypomorphic mutation in Btk resulting in reduced B cell numbers but no clinical disease.

Reduced B cell numbers and a mutation in Btk are considered sufficient to make the diagnosis of X-linked agammaglobulinaemia. In the process of conducting family studies, we identified a 58-year-old healthy man with an amino acid substitution, Y418H, in the adenosine-5'-triphosphate binding site of Btk. Immunofluorescence studies showed that this man had 0.85% CD19+ B cells (normal 4-18%) in the peripheral circulation and his monocytes were positive for Btk. He had borderline low serum immunoglobulins but normal titres to tetanus toxoid and multiple pneumococcal serotypes. To determine the functional consequences of the amino acid substitution, a Btk- chicken B cell line, DT40, was transfected with expression vectors producing wild-type Btk or Y418H Btk. The transfected cells were stimulated with anti-IgM and calcium flux and inositol triphosphate (IP3) production were measured. Cells bearing the mutant protein demonstrated consistently a 15-20% decrease in both calcium flux and IP3 production. These findings indicate that even a modest decrease in Btk function can impair B cell proliferation or survival. However, a mutation in Btk and reduced numbers of B cells are not always associated with clinical disease.

Diffuse CNS vasculopathy with chronic Epstein-Barr virus infection in X-linked lymphoproliferative disease.

We report a patient with X-linked lymphoproliferative disease (XLP) who developed multiple central nervous system (CNS) manifestations of Epstein-Barr virus infection. XLP, or Duncan syndrome, is a rare inherited disorder characterized by the inability to clear Epstein-Barr virus infection. In addition to Epstein-Barr virus encephalitis, CNS lymphoproliferative disease, and lymphoma, this patient also developed MR angiographic evidence of diffuse fusiform aneurysmal dilation of intracranial vessels.

Quantitative effects of palivizumab and donor-derived T cells on chronic respiratory syncytial virus infection, lung disease, and fusion glycoprotein amino acid sequences in a patient before and after bone marrow transplantation.

A patient with respiratory syncytial virus (RSV) infection and severe combined immunodeficiency was studied during a 3-month period of bone marrow transplantation and palivizumab infusion. No RSV isolates with palivizumab escape mutations were identified. Donor lymphocytes, including CD8 cells, appeared to markedly reduce the RSV load but increased the pulmonary symptoms. Immunosuppressive therapy ameliorated lung disease but allowed the RSV load to rebound.

Negative selection at the pre-BCR checkpoint elicited by human mu heavy chains with unusual CDR3 regions.

Approximately 9% of in-frame mu heavy chain transcripts found in normal human pro-B cells encode proteins that can be expressed on the cell surface in the absence of surrogate or conventional light chains. These unusual mu heavy chains demonstrate preferential use of certain VH genes (VH3-23), frequent expression of DH regions in underrepresented reading frames, and an increased number of positively charged amino acids within the CDR3 region. Transcripts for these proteins are not found in pre-B cells or in mature B cells. When expressed in Jurkat T cells with the Ig(alpha)/Ig(beta) signal transduction module, these aberrant mu heavy chains induce cell activation and apoptosis. These results suggest that some mu heavy chains elicit negative selection at the pro-B cell to pre-B cell transition.

X-linked anhidrotic ectodermal dysplasia with immunodeficiency is caused by impaired NF-kappaB signaling.

The molecular basis of X-linked recessive anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID) has remained elusive. Here we report hypomorphic mutations in the gene IKBKG in 12 males with EDA-ID from 8 kindreds, and 2 patients with a related and hitherto unrecognized syndrome of EDA-ID with osteopetrosis and lymphoedema (OL-EDA-ID). Mutations in the coding region of IKBKG are associated with EDA-ID, and stop codon mutations, with OL-EDA-ID. IKBKG encodes NEMO, the regulatory subunit of the IKK (IkappaB kinase) complex, which is essential for NF-kappaB signaling. Germline loss-of-function mutations in IKBKG are lethal in male fetuses. We show that IKBKG mutations causing OL-EDA-ID and EDA-ID impair but do not abolish NF-kappaB signaling. We also show that the ectodysplasin receptor, DL, triggers NF-kappaB through the NEMO protein, indicating that EDA results from impaired NF-kappaB signaling. Finally, we show that abnormal immunity in OL-EDA-ID patients results from impaired cell responses to lipopolysaccharide, interleukin (IL)-1beta, IL-18, TNFalpha and CD154. We thus report for the first time that impaired but not abolished NF-kappaB signaling in humans results in two related syndromes that associate specific developmental and immunological defects.

Hypogammaglobulinemia and reduced numbers of B-cells in children with myelodysplastic syndrome.

BACKGROUND: Immunodeficiency in pediatric patients with myelodysplastic syndrome (MDS) has not been described. We report the clinical course of three children with MDS, hypogammaglobulinemia, and reduced numbers of B-cells and B-cell precursors. OBSERVATIONS: Three patients with recurrent infection who were younger than 1-year-old had MDS of the refractory anemia (RA) subtype diagnosed. All had reduced numbers of circulating B-cells and hypogammaglobulinemia. In two patients, cytogenetic studies revealed a monosomy 7 karyotype and bone marrow studies showed decreased numbers of CD34+ progenitor cells and CD 19+ B-cells. Both patients had prolonged courses (7 yrs 10 mos and 6 yrs 9 mos) characterized by recurrent infection and slowly progressive pancytopenia. Both received allogeneic bone marrow transplantation (BMT). The third patient had normal cytogenetic studies and a normal number of CD34+ progenitors but decreased CD19+ B-cells in the bone marrow. She had a stable course with refractory anemia over the course of 7 years. CONCLUSIONS: Pediatric patients with MDS may have hypogammaglobulinemia and reduced numbers of B-cells. These findings do not preclude a relatively stable and prolonged clinical course. Children with newly diagnosed MDS should have an immunologic evaluation in addition to their hematologic assessment.

The transcription factor Bright associates with Bruton's tyrosine kinase, the defective protein in immunodeficiency disease.

Binding of the transcription factor Bright to Ig heavy chain loci after B cell activation is associated with increased heavy chain transcription. We now report that Bright coprecipitates with Bruton's tyrosine kinase (Btk), the defective enzyme in X-linked immunodeficiency disease (xid). Furthermore, we observed Btk in the nucleus of activated murine B cells, and mobility shift assays suggest that it is a component of the Bright DNA-binding complex. While BRIGHT protein was synthesized in activated spleen cells from xid mice, it did not bind DNA or associate stably with Btk. These data suggest that deficiencies in BRIGHT DNA-binding activity may contribute to the defects in Ig production seen in xid mice.

Molecular evidence of ocular Epstein-Barr virus infection.

Ocular manifestations have been attributed to the Epstein-Barr virus (EBV), largely on the basis of seroepidemiologic data. Two patients who developed conjunctival disease as the presenting feature of EBV infection are reported, each confirmed by in situ hybridization of EBV genome in affected tissue biopsy specimens. Recognition of EBV-induced ocular disease as an initial presentation of clinical EBV infection is important to the practitioner because of the ubiquitous nature of this herpesvirus.

Genetics of primary immunodeficiency diseases.

The primary immunodeficiencies are a heterogeneous group of disorders that affect either the development or the function of the immune system. In the last ten years, the genes responsible for many of the most common and best studied immunodeficiencies have been identified. As might be expected, the expression of most of these genes is limited to the hematopoietic system. Although most are members of gene families, their association with disease indicates that they do not perform redundant functions. Some immunodeficiencies involve the effector functions of the immune system, for example the NADPH oxidase system or perforin; however, a striking number of the disorders involve signal transduction pathways. These include defects in ligands, transmembrane receptors, kinases, adaptor proteins and transcription factors. Mutations for each disorder tend to be highly variable and the specific mutation in a gene is only one of the factors that influence the clinical phenotype. Polymorphic variations in susceptibility genes may also contribute to the disease phenotype. Not all genes responsible for immunodeficiency have been identified. As many as 20 to 30% of patients with clinical and laboratory evidence of single gene defects of the immune system do not fit any well described clinical disorder.

An essential role for BLNK in human B cell development.

The signal transduction events that control the progenitor B cell (pro-B cell) to precursor B cell (pre-B cell) transition have not been well delineated. In evaluating patients with absent B cells, a male with a homozygous splice defect in the cytoplasmic adapter protein BLNK (B cell linker protein) was identified. Although this patient had normal numbers of pro-B cells, he had no pre-B cells or mature B cells, indicating that BLNK plays a critical role in orchestrating the pro-B cell to pre-B cell transition. The immune system and overall growth and development were otherwise normal in this patient, suggesting that BLNK function is highly specific.

Correction of X-linked immunodeficient mice by competitive reconstitution with limiting numbers of normal bone marrow cells.

Gene therapy for inherited disorders is more likely to succeed if gene-corrected cells have a proliferative or survival advantage compared with mutant cells. We used a competitive reconstitution model to evaluate the strength of the selective advantage that Btk normal cells have in Btk-deficient xid mice. Whereas 2,500 normal bone marrow cells when mixed with 497,500 xid cells restored serum IgM and IgG3 levels to near normal concentrations in 3 of 5 lethally irradiated mice, 25,000 normal cells mixed with 475,000 xid cells reliably restored serum IgM and IgG3 concentrations and the thymus-independent antibody response in all transplanted mice. Reconstitution was not dependent on lethal irradiation, because sublethally irradiated mice all had elevated serum IgM and IgG3 by 30 weeks postreconstitution when receiving 25,000 normal cells. Furthermore, the xid defect was corrected with as few as 10% of the splenic B cells expressing a normal Btk. When normal donor cells were sorted into B220(+)/CD19(+) committed B cells and B220(-)/CD19(-) cell populations, only the B220(-)/CD19(-) cells provided long-term B-cell reconstitution in sublethally irradiated mice. These findings suggest that even inefficient gene therapy may provide clinical benefit for patients with XLA.

Mutations in Igalpha (CD79a) result in a complete block in B-cell development.

Mutations in Btk, mu heavy chain, or the surrogate light chain account for 85-90% of patients with early onset hypogammaglobulinemia and absent B cells. The nature of the defect in the remaining patients is unknown. We screened 25 such patients for mutations in genes encoding components of the pre-B-cell receptor (pre-BCR) complex. A 2-year-old girl was found to have a homozygous splice defect in Igalpha, a transmembrane protein that forms part of the Igalpha/Igbeta signal-transduction module of the pre-BCR. Studies in mice suggest that the Igbeta component of the pre-BCR influences V-DJ rearrangement before cell-surface expression of mu heavy chain. To determine whether Igalpha plays a similar role, we compared B-cell development in an Igalpha-deficient patient with that seen in a mu heavy chain-deficient patient. By immunofluorescence, both patients had a complete block in B-cell development at the pro-B to pre-B transition; both patients also had an equivalent number and diversity of rearranged V-DJ sequences. These results indicate that mutations in Igalpha can be a cause of agammaglobulinemia. Furthermore, they suggest that Igalpha does not play a critical role in B-cell development until it is expressed, along with mu heavy chain, as part of the pre-BCR.

Transcriptional regulatory elements within the first intron of Bruton's tyrosine kinase.

Defects in the gene for Bruton's tyrosine kinase (Btk) result in the disorder X-linked agammaglobulinemia (XLA). Whereas XLA is characterized by a profound defect in B-cell development, Btk is expressed in both the B lymphocyte and myeloid cell lineages. We evaluated a patient with XLA who had reduced amounts of Btk transcript but no abnormalities in his coding sequence. A single base-pair substitution in the first intron of Btk was identified in this patient, suggesting that this region may contain regulatory elements. Using reporter constructs we identified two transcriptional control elements in the first 500 bp of intron 1. A strong positive regulator, active in both pre-B cells and B cells, was identified within the first 43 bp of the intron. Gel-shift assays identified two Sp1 binding sites within this element. The patient's mutation results in an altered binding specificity of the proximal Sp1 binding site. A negative regulator, active in pre-B cells only, was located between base pairs 281 and 491 of the intron. These findings indicate that regulation of Btk transcription is complex and may involve several transcriptional regulatory factors at the different stages of B-cell differentiation.

Expression and activation of the nonreceptor tyrosine kinase Tec in human B cells.

The tyrosine kinase Tec belongs to a new group of structurally related nonreceptor tyrosine kinases that also includes Btk and Itk. Previous studies have suggested that these kinases have lineage-specific roles, with Tec being involved mainly in the regulation of cytokine-mediated myeloid cell growth and differentiation. In this study, we investigated expression and activation of Tec in human B-lymphoid cell lines representing different stages of B-cell maturation, including pro-B (RS4;11, 380, REH), pre-B (NALM6), and mature B (Ramos, and one Epstein-Barr virus [EBV]-transformed lymphoblastoid line) cells. Like Btk, Tec protein was expressed in all B-cell lines tested. Tec was also highly expressed in two EBV-transformed lymphoblastoid lines derived from patients with X-linked agammaglobulinemia (XLA) lacking Btk expression, as well as in tonsillar lymphoid cells. In surface immunoglobulin-positive B cells (Ramos), ligation of the B-cell antigen receptor (BCR) with anti-IgM antibodies caused marked tyrosine phosphorylation of Tec and increased Tec tyrosine kinase activity. Likewise, cross-linking of CD19 with a monoclonal antibody in BCR-negative pro-B (RS4;11, 380) and pre-B (NALM6) cells induced Tec tyrosine phosphorylation and increased Tec autophosphorylation, as well as Btk activation. Tyrosine phosphorylation of Tec, but not of Btk, was detectable in RS4;11 cells after CD38 ligation, suggesting that these kinases are regulated differently. We conclude that Tec is expressed and can be stimulated throughout human B-cell differentiation, implying that this tyrosine kinase plays a role in B-cell development and activation.

Mutations in the human lambda5/14.1 gene result in B cell deficiency and agammaglobulinemia.

B cell precursors transiently express a pre-B cell receptor complex consisting of a rearranged mu heavy chain, a surrogate light chain composed of lambda5/14.1 and VpreB, and the immunoglobulin (Ig)-associated signal transducing chains, Igalpha and Igbeta. Mutations in the mu heavy chain are associated with a complete failure of B cell development in both humans and mice, whereas mutations in murine lambda5 result in a leaky phenotype with detectable humoral responses. In evaluating patients with agammaglobulinemia and markedly reduced numbers of B cells, we identified a boy with mutations on both alleles of the gene for lambda5/14.1. The maternal allele carried a premature stop codon in the first exon of lambda5/14.1 and the paternal allele demonstrated three basepair substitutions in a 33-basepair sequence in exon 3. The three substitutions correspond to the sequence in the lambda5/14. 1 pseudogene 16.1 and result in an amino acid substitution at an invariant proline. When expressed in COS cells, the allele carrying the pseudogene sequence resulted in defective folding and secretion of mutant lambda5/14.1. These findings indicate that expression of the functional lambda5/14.1 is critical for B cell development in the human.

Seven novel mutations in the adenosine deaminase (ADA) gene in patients with severe and delayed onset combined immunodeficiency: G74C, V129M, G140E, R149W, Q199P, 462delG, and E337del. Mutations in brief no. 142. Online.

The degree of immunodeficiency associated with deficiency of adenosine deaminase (ADA) is variable. Most patients are infants with severe combined immunodeficiency (SCID), but in about 20 percent immune dysfunction becomes manifest later in childhood ("delayed-onset"); several patients with "late" or "adult" onset of immune dysfunction have been diagnosed at 15-39 years. Over 40 ADA gene mutations have thus far been identified. To better define the genotype-phenotype relationship, we report 7 novel ADA mutations, including 5 missense mutations (G74C, V129M, G140E, R149W, Q199P) and two short deletions (462delG, E337del). These were identified among 7 patients (3 with SCID and 4 with delayed-onset). A homozygote for 462delG had SCID, whereas patients homozygous or heterozyous for V129M had delayed-onset. Two other delayed-onset patients, one heterozygous for G74C and the other for Q199P, each had a second allele carrying the previously reported "severe" mutation G216R. These findings are consistent with previous observations suggesting that, in general, SCID occurs when both alleles eliminate ADA function, and a milder phenotype when at least one allele can supply a low level of function.