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.

A possible bichromatid mutation in a male gamete giving rise to a female mosaic for two different mutations in the X-linked gene WAS.

In genetic disorders caused by point mutations or small frameshift mutations, affected members of the same family are expected to have the same mutation in the causative gene. We have recently evaluated a family in which this was not the case. Maternal cousins with Wiskott-Aldrich syndrome (WAS; MIM 301000) had two different but contiguous single base pair deletions in WAS. The proband had an A deletion in codon 242 in exon 7 of WAS; his two cousins had a C deletion in codon 241. The mother of the proband was heterozygous for the A deletion allele, but her three sisters, including the mother of the affected cousins, were heterozygous for the C deletion. Both deletions occurred on the haplotype from the unaffected maternal great-grandfather. The maternal grandmother, who was a carrier of WAS, based on a non-random pattern of X chromosome inactivation in T cells, was mosaic for both deletions. These findings are most consistent with the mutations originating in a male gamete with different mutations on the two strands of DNA, a bichromatid mutation.

A newly recognized, likely autosomal recessive syndrome comprising agammaglobulinemia, microcephaly, craniosynostosis, severe dermatitis, and other features.

We present a novel, likely autosomal recessive, multi-system disorder seen in three siblings, two males and one female, born to nonconsanguineous parents. The disease manifests as agammaglobulinemia with marked microcephaly, significant developmental delay, craniosynostosis, a severe dermatitis, cleft palate, narrowing of the choanae, and blepharophimosis. The constellation of clinical signs seen in this family likely represents a new and recognizable form of agammaglobulinemia due to a defect in early B-cell maturation.

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.

Analysis of SWAP-70 as a candidate gene for non-X-linked hyper IgM syndrome and common variable immunodeficiency.

SWAP-70 is a recently identified protein that functions as the only B cell-specific component of an isotype switch recombination complex called SWAP. The SWAP complex has specificity for the switch regions upstream of the constant region immunoglobulin genes and it facilitates the transfer of DNA between switch regions. These features suggested that mutations in the gene encoding SWAP-70 might result in humoral immunodeficiency. To test this hypothesis we determined the genomic structure of this gene and used single-stranded conformational polymorphism (SSCP) analysis to screen DNA from 38 patients with either non-X-linked hyper IgM syndrome or common variable immunodeficiency. The results demonstrated that SWAP-70 consists of 12 exons spread over 89 kb at chromosome 11p15.2. SSCP analysis of the patient population revealed five polymorphic variants in the gene, one of which (Q505E) is an amino acid substitution in the putative nuclear export signal of SWAP-70. However, none of the alterations appeared to be associated with disease in the patients screened.

Variations in the human phospholipase Cgamma2 gene in patients with B-cell defects of unknown etiology.

Our recent studies using targeted gene disruption have shown that defects in phospholipase Cgamma2 (PLCgamma2) result in a B-cell abnormality that is very similar to that seen in Btk-deficient mice. Null mutations in either PLCG2 or BTK are associated with decreased numbers of mature B cells, failure to make antibodies to some T cell-independent antigens and the absence of CD5+ peritoneal B cells. Mutations in BTK in humans cause a more severe defect in B-cell development characterized by almost complete absence of B cells in the peripheral circulation, profound hypogammaglobulinemia and an inability to produce antibodies to any antigens. However, not all patients with severe defects in B-cell development have mutations in BTK or the components of the B-cell signal transduction complex. To explore the possibility that some patients with defects in B-cell development of unknown etiology might have mutations in PLCG2, we determined the genomic structure of this gene and established conditions to analyze the 32 exons of the gene and the flanking sequences by single-strand conformation polymorphism. Although 24 polymorphic variants of this gene were found in 35 patients, we did not identify any alterations that were likely to be the cause of disease.

A contiguous deletion syndrome of X-linked agammaglobulinemia and sensorineural deafness.

Hearing loss in patients with X-linked agammaglobulinemia is often attributed to recurrent infections. However, recent genetic studies suggest a different etiology in some patients. We present three unrelated patients, 6, 9, and 14 years of age, with large deletions of the terminal portion of the Bruton tyrosine kinase (Btk) gene extending 4.2-19 kb beyond the 3' end of the gene. The DNA immediately downstream of the 3' end of Btk contains the deafness-dystonia protein gene (DDP). Mutations in this gene have recently been shown to underlie the Mohr-Tranebjaerg syndrome, which is characterized by sensorineural deafness, dystonia, and mental deficiency. Besides the immunodeficiency, our patients exhibited progressive sensorineural deafness. The clue to an associated hearing problem was delayed development of speech in one patient and post-lingual deafness noticed between the age of 3-4 years in the other two. These patients have not yet exhibited significant associated neurologic deficits.

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.

V(D)J recombination defects in lymphocytes due to RAG mutations: severe immunodeficiency with a spectrum of clinical presentations.

Severe combined immunodeficiency (SCID) comprises a heterogeneous group of primary immunodeficiencies, a proportion of which are due to mutations in either of the 2 recombination activating genes (RAG)-1 and -2, which mediate the process of V(D)J recombination leading to the assembly of antigen receptor genes. It is reported here that the clinical and immunologic phenotypes of patients bearing mutations in RAGs are more diverse than previously thought and that this variability is related, in part, to the specific type of RAG mutation. By analyzing 44 such patients from 41 families, the following conclusions were reached: (1) null mutations on both alleles lead to the T-B-SCID phenotype; (2) patients manifesting classic Omenn syndrome (OS) have missense mutations on at least one allele and maintain partial V(D)J recombination activity, which accounts for the generation of residual, oligoclonal T-lymphocytes; (3) in a third group of patients, findings were only partially compatible with OS, and these patients, who also carried at least one missense mutation, may be considered to have atypical SCID/OS; (4) patients with engraftment of maternal T cells as a complication of a transplacental transfusion represented a fourth group, and these patients, who often presented with a clinical phenotype mimicking OS, may be observed regardless of the type of RAG gene mutation. Analysis of the RAG genes by direct sequencing is an effective way to provide accurate diagnosis of RAG-deficient as opposed to RAG-independent V(D)J recombination defects, a distinction that cannot be made based on clinical and immunologic phenotype alone.

Mutations in activation-induced cytidine deaminase in patients with hyper IgM syndrome.

Recent studies have shown that mutations in a newly described RNA editing enzyme, activation-induced cytidine deaminase (AID), can cause an autosomal recessive form of hyper IgM syndrome. To determine the relative frequency of mutations in AID, we evaluated a group of 27 patients with hyper IgM syndrome who did not have defects in CD40 ligand and 23 patients with common variable immunodeficiency. Three different mutations in AID were identified in 18 patients with hyper IgM syndrome, including 14 French Canadians, 2 Lumbee Indians, and a brother and sister from Okinawa. No mutations were found in the remaining 32 patients. In the group of patients with hyper IgM syndrome, the patients with mutations in AID were older at the age of diagnosis, were more likely to have positive isohemagglutinins, and were less likely to have anemia, neutropenia, or thrombocytopenia. Lymphoid hyperplasia was seen in patients with hyper IgM syndrome and normal AID as well as the patients with hyper IgM syndrome and defects in AID.

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.