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The United States Immunodeficiency Network (USIDNET) patient registry was used to characterize the presentation, genetics, phenotypes, and treatment of patients with Hyper IgM Syndrome (HIGM). The USIDNET Registry was queried for HIGM patient data collected from October 1992 to July 2015. Data fields included demographics, criteria for diagnosis, pedigree analysis, mutations, clinical features, treatment and transplant records, laboratory findings, and mortality. Fifty-two physicians entered data from 145 patients of ages 2 months to 62 years (median 12 years); 131 were males. Using patients’ age at last entry, data from 2072 patient years are included. Mutations were recorded for 85 subjects; 82 were in CD40LG. Eighteen subjects had non-X-linked HIGM. 40 % had a normal serum IgM and 15 %, normal IgA. Infections were reported for 91 %, with pulmonary, ear, and sinus infections being the most common. 42 % had Pneumocystis jirovecii pneumonia; 6 % had Cryptosporidium. 41 % had neutropenia. 78 % experienced non-infectious complications: chronic diarrhea (n = 22), aphthous ulcers (n = 28), and neoplasms (n = 8) including colon cancer, adrenal adenoma, liver adenocarcinoma, pancreatic carcinoid, acute myeloid leukemia, hepatoma, and, in a female with an autosomal dominant gain of function mutation in PIK3CD, an ovarian dysgerminoma. Thirteen patients had a hematopoietic marrow or stem cell transplant; three had solid organ transplants. Thirteen were known to have died (median age = 14 years). Analysis of the USIDNET Registry provides data on the common clinical features of this rare syndrome, and in contrast with previously published data, demonstrates longer survival times and reduced gastrointestinal manifestations.

Despite the known importance of zinc for human immunity, molecular insights into its roles have remained limited. Here we report a novel autosomal recessive disease characterized by absent B cells, agammaglobulinemia and early onset infections in five unrelated families. The immunodeficiency results from hypomorphic mutations of SLC39A7, which encodes the endoplasmic reticulum-to-cytoplasm zinc transporter ZIP7. Using CRISPR-Cas9 mutagenesis we have precisely modeled ZIP7 deficiency in mice. Homozygosity for a null allele caused embryonic death, but hypomorphic alleles reproduced the block in B cell development seen in patients. B cells from mutant mice exhibited a diminished concentration of cytoplasmic free zinc, increased phosphatase activity and decreased phosphorylation of signaling molecules downstream of the pre-B cell and B cell receptors. Our findings highlight a specific role for cytosolic Zn2+ in modulating B cell receptor signal strength and positive selection.

There are now nearly 300 single-gene inborn errors of immunity underlying phenotypes as diverse as infection, malignancy, allergy, auto-immunity, and auto-inflammation. For each of these five categories, a growing variety of phenotypes are ascribed to Primary Immunodeficiency Diseases (PID), making PIDs a rapidly expanding field of medicine. The International Union of Immunological Societies (IUIS) PID expert committee (EC) has published every other year a classification of these disorders into tables, defined by shared pathogenesis and/or clinical consequences. In 2013, the IUIS committee also proposed a more user-friendly, phenotypic classification, based on the selection of key phenotypes at the bedside. We herein propose the revised figures, based on the accompanying 2015 IUIS PID EC classification.

Despite the known importance of zinc for human immunity, molecular insights into its roles have remained limited. Here we report a novel autosomal recessive disease characterized by absent B cells, agammaglobulinemia and early onset infections in five unrelated families. The immunodeficiency results from hypomorphic mutations of SLC39A7, which encodes the endoplasmic reticulum-to-cytoplasm zinc transporter ZIP7. Using CRISPR-Cas9 mutagenesis we have precisely modeled ZIP7 deficiency in mice. Homozygosity for a null allele caused embryonic death, but hypomorphic alleles reproduced the block in B cell development seen in patients. B cells from mutant mice exhibited a diminished concentration of cytoplasmic free zinc, increased phosphatase activity and decreased phosphorylation of signaling molecules downstream of the pre-B cell and B cell receptors. Our findings highlight a specific role for cytosolic Zn

Casanova and colleagues discuss the importance of single-patient genetic studies in the discovery of novel primary immunodeficiencies and offer insight into the standards and criteria that should accompany these studies., Can genetic and clinical findings made in a single patient be considered sufficient to establish a causal relationship between genotype and phenotype? We report that up to 49 of the 232 monogenic etiologies (21%) of human primary immunodeficiencies (PIDs) were initially reported in single patients. The ability to incriminate single-gene inborn errors in immunodeficient patients results from the relative ease in validating the disease-causing role of the genotype by in-depth mechanistic studies demonstrating the structural and functional consequences of the mutations using blood samples. The candidate genotype can be causally connected to a clinical phenotype using cellular (leukocytes) or molecular (plasma) substrates. The recent advent of next generation sequencing (NGS), with whole exome and whole genome sequencing, induced pluripotent stem cell (iPSC) technology, and gene editing technologies—including in particular the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology—offer new and exciting possibilities for the genetic exploration of single patients not only in hematology and immunology but also in other fields. We propose three criteria for deciding if the clinical and experimental data suffice to establish a causal relationship based on only one case. The patient’s candidate genotype must not occur in individuals without the clinical phenotype. Experimental studies must indicate that the genetic variant impairs, destroys, or alters the expression or function of the gene product (or two genetic variants for compound heterozygosity). The causal relationship between the candidate genotype and the clinical phenotype must be confirmed via a relevant cellular phenotype, or by default via a relevant animal phenotype. When supported by satisfaction of rigorous criteria, the report of single patient–based discovery of Mendelian disorders should be encouraged, as it can provide the first step in the understanding of a group of human diseases, thereby revealing crucial pathways underlying physiological and pathological processes.

Approximately 90% of patients with isolated agammaglobulinemia and failure of B cell development have mutations in genes required for signaling through the pre–B cell and B cell receptors. The nature of the gene defect in the majority of remaining patients is unknown. We recently identified 4 patients with agammaglobulinemia and markedly decreased numbers of peripheral B cells. The B cells that could be detected had an unusual phenotype characterized by the increased expression of CD19 but the absence of a B cell receptor. Genetic studies demonstrated that all 4 patients had the exact same de novo mutation in the broadly expressed transcription factor E47. The mutant protein (E555K) was stable in patient-derived EBV-transformed cell lines and cell lines transfected with expression vectors. E555K in the transfected cells localized normally to the nucleus and resulted in a dominant negative effect when bound to DNA as a homodimer with wild-type E47. Mutant E47 did permit DNA binding by a tissue-specific heterodimeric DNA-binding partner, myogenic differentiation 1 (MYOD). These findings document a mutational hot-spot in E47 and represent an autosomal dominant form of agammaglobulinemia. Further, they indicate that E47 plays a critical role in enforcing the block in development of B cell precursors that lack functional antigen receptors.

There is no clear consensus about the definition of the term primary immunodeficiency in 2011. Although there is general agreement that defects in both adaptive and innate immunity should be included, issues related to the frequency of primary immunodeficiencies, the modes of inheritance, the other types of cells involved, and the required clinical phenotype are more contentious. Three friends with an interest in both the clinical and scientific aspects of primary immunodeficiency carried out a discussion or trialogue to address some of these issues.

Primary immunodeficiency disorders are a recognized public health problem worldwide. The prototype of these conditions is X-linked agammaglobulinemia (XLA) or Bruton's disease. XLA is caused by mutations in Bruton's tyrosine kinase gene (BTK), preventing B cell development and resulting in the almost total absence of serum immunoglobulins. The genetic profile and prevalence of XLA have not previously been studied in Eastern and Central European (ECE) countries. We studied the genetic and demographic features of XLA in Belarus, Croatia Hungary, Poland, Republic of Macedonia, Romania, Russia, Serbia, Slovenia, and Ukraine. We collected clinical, immunological, and genetic information for 122 patients from 109 families. The BTK gene was sequenced from the genomic DNA of patients with a high susceptibility to infection, almost no CD19(+) peripheral blood B cells. and low or undetectable levels of serum immunoglobulins M, G, and A, compatible with a clinical and immunological diagnosis of XLA. BTK sequence analysis revealed 98 different mutations, 46 of which are reported for the first time here. The mutations included single nucleotide changes in the coding exons (35 missense and 17 nonsense), 23 splicing defects, 13 small deletions, 7 large deletions, and 3 insertions. The mutations were scattered throughout the BTK gene and most frequently concerned the SH1 domain: no missense mutation was detected in the SH3 domain. The prevalence of XLA in ECE countries (total population 145,530,870) was found to be 1 per 1,399,000 individuals. This report provides the first comprehensive overview of the molecular genetic and demographic features of XLA in Eastern and Central Europe. (C) 2009 Elsevier Ltd. All rights reserved.

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.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.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.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.).

WOS: 000371897500033, PubMed: 26478008, Background: CD19 is a B cell-specific molecule that serves as a major costimulatory molecule for amplifying B-cell receptor (BCR) responses. Biallelic CD19 gene mutations cause common variable immunodeficiency in human subjects. BCR- and Toll-like receptor (TLR) 9-induced B-cell responses are impaired in most patients with common variable immunodeficiency. Objective: We sought to analyze whether CD19 is required for TLR9 function in human B cells. Methods: Expression of surface activation markers was assessed after anti-IgM or CpG stimulation by using flow cytometry on B cells from patients with 1 or 2 defective CD19 alleles, which decrease or abrogate CD19 expression, respectively. The phosphorylation or interaction of signaling molecules was analyzed by using phospho flow cytometry, immunoblotting, or co-immunoprecipitation in CD19-deficient or control B cells and in a B-cell line in which CD19 has been knocked down with lentivirus-transduced short hairpin RNA. Results: B cells from subjects with 1 or 2 defective CD19 alleles showed defective upregulation in vitro of CD86, transmembrane activator and CAML interactor (TACI), and CD23 activation markers after TLR9 stimulation. TLR9 ligands normally induce phosphorylation of CD19 through myeloid differentiation primary response gene-88 (MYD88)/proline-rich tyrosine kinase 2 (PYK2)/LYN complexes, which allows recruitment of phosphoinositide 3-kinase (PI3K) and phosphorylation of Bruton tyrosine kinase (BTK) and AKT in human B cells with a different kinetic than that of BCRs. In addition, inhibition of PI3K, AKT, or BTK, as well as BTK deficiency, also resulted in TLR9 activation defects in B cells similar to those in patients with CD19 deficiency. Conclusion: CD19 is required for TLR9-induced B-cell activation. Hence CD19/PI3K/AKT/BTK is an essential axis integrating BCRs and TLR9 signaling in human B cells., National Institute of Health (NIH)United States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [AI061093, AI071087, AI082713, AI095848]; German Research FoundationGerman Research Foundation (DFG) [MO 2160/2-1], Supported by National Institute of Health (NIH) grants AI061093, AI071087, AI082713, and AI095848 (to E.M.) and German Research Foundation grant MO 2160/2-1 (to H.M.).

Primary immunodeficiency diseases (PIDs) are a genetically heterogeneous group of disorders that affect distinct components of the innate and adaptive immune system, such as neutrophils, macrophages, dendritic cells, complement proteins, natural killer cells, and T and B lymphocytes. The study of these diseases has provided essential insights into the functioning of the immune system. More than 120 distinct genes have been identified, whose abnormalities account for more than 150 different forms of PID. The complexity of the genetic,immunologic, and clinical features of PID has prompted the need for their classification, with the ultimate goal of facilitating diagnosis and treatment. To serve this goal, an international committee of experts has met every 2 years since 1970. In its last meeting in Jackson Hole, Wyo, after 3 days of intense scientific presentations and discussions, the committee has updated the classification of PID, as reported in this article.

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.

We evaluated a baby boy with severe combined immunodeficiency (SCID) and X-linked nephrogenic diabetes insipidus (NDI). This patient had less than 10% CD3+ T cells, almost all of which were positive for CD4 and CD45RO. Genetic studies demonstrated a 34.4 kb deletion at Xq28 which included AVPR2, the gene responsible for NDI; ARHGAP4, a hematopoietic specific gene encoding a GTPase-activating protein; and a highly conserved segment of DNA between ARHGAP4 and ARD1A, a gene involved in the response to hypoxia. Other patients with NDI, but without immunodeficiency, have had deletions that remove all ARHGAP4 except exon 1; however, no other patients have had deletions of the highly conserved intragenic region between ARHGAP4 and ARD1A. X chromosome inactivation studies, done on sorted cells from the mother and grandmother of the patient, carriers of the deletion, demonstrated exclusive use of the non-mutant X chromosome as the active X in CD4 and CD8 T cells. Surprisingly, NK cells, monocytes and neutrophils from these women demonstrated preferential use of the mutant X chromosome as the active X. These results are consistent with an X-linked form of SCID, due to the loss of regulatory elements that control the response to hypoxia in hematopoietic cells.

In recent years several rare autosomal recessive disorders that result in antibody deficiency have been reported. Some antibody deficiencies are part of a more broadly expressed systemic disorder or part of an immunodeficiency that affects T cells and/or NK cells as well as B cells. The possibility of immunodeficiency should be considered in any patient who is hospitalized for a major infection requiring intravenous therapy. Most patients with X-linked agammaglobulinemia (XLA) develop recurrent or persistent infections in the first 4 to 8 months of life, and the majority are recognized to have immunodeficiency at less than 3 years of age. Defects in μ heavy chain account for about one-third of the patients with autosomal recessive agammaglobulinemia. Mutation detection is the most practical way of making a definitive diagnosis. Single gene defects of the immune system may have features in common with common variable immunodeficiency (CVID). Recent studies suggest that analysis of B-cell phenotype may be useful in identifying patients with CVID who have more severe disease. Either whole blood or density gradient-separated mononuclear cells can be used to examine lymphocyte cell surface markers. A variety of techniques can be used to provide mutation detection. The advantages and disadvantages of some techniques are listed in this chapter. A detailed history of past infections, a careful family history, and a physical examination that focuses on sites typically involved in antibody deficiencies should come before any laboratory tests.

No clear genotype/phenotype correlations have been established in patients with X-linked agammaglobulinemia (XLA). To determine if the specific mutation in Btk might be one of the factors that influences the severity of disease or if polymorphic variants in Tec, a cytoplasmic tyrosine kinase that might substitute for Btk, could contribute to the clinical phenotype, we examined the age at diagnosis, the percentage of peripheral blood B cells and the plasma IgM in a large group of patients with XLA. The results demonstrated that polymorphic variants in Tec were not correlated with phenotypic markers; however, the specific mutation in Btk did influence disease severity. Mutations that conceivably allow the production of some Btk, amino acid substitutions or splice defects that occur at conserved but not invariant sites in the splice consensus sequence were associated with older age at diagnosis, a higher percentage of B cells in the peripheral circulation and higher concentrations of plasma IgM.

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.

Murine models of immunodeficiency were used to evaluate strategies that might allow B cell engraftment in patients with X-linked agammaglobulinemia. Mice with defects in Btk or mu heavy chain were given 2.5 x 10(6) bone marrow cells from wild-type congenic donors. In the absence of any preparative regimen or immunosuppression, Btk-deficient mice on the CBA background developed normal concentrations of serum IgM and IgG3 by 12 weeks posttransplant. By contrast, mu heavy chain-deficient mice on the C57BL/6 background required some immunosuppression to achieve engraftment. Treatment of these mice with anti-T-cell antibodies 2 and 4 days prior to transplant resulted in normal concentrations of serum immunoglobulins by 6 weeks posttransplant. These pretreated mice had only 10% of the normal number of splenic B cells and they had no evidence of donor T cell engraftment. These results suggest that myelotoxic drugs may not be needed to achieve B cell engraftment in B-cell-deficient subjects.

B-cell linker (BLNK) protein is a non-redundant adaptor molecule in the signaling pathway activated by (pre) B-cell antigen receptor signals. We present two siblings with a homozygous deleterious frameshift mutation in BLNK, resulting in a block of B cell development in the bone marrow at the preB1 to preB2 stage, absence of circulating B cells and agammaglobulinemia. This is the first description of an enteroviral infection associated arthritis and dermatitis in a patient with BLNK deficiency.

B cells play a central role in the pathogenesis of many autoimmune diseases. Therefore, understanding the mechanisms that regulate B-cell tolerance in humans is important for the development of new therapeutic strategies. Patients with monogenic diseases provide rare opportunities to study the impact of specific gene mutations on the regulation of human B cell tolerance. By this, we could show that alterations in B-cell receptor and Toll-like receptor (TLR) signaling pathways result in defective central B-cell tolerance.

Many patients with clinical and laboratory evidence of primary immunodeficiency do not have a gene specific diagnosis. The use of next generation sequencing, particularly whole exome sequencing, has given us an extraordinarily powerful tool to identify the disease-causing genes in some of these patients. At least 34 new gene defects have been identified in the last 4 years. These findings document the striking heterogeneity of the phenotype in patients with mutations in the same gene. In some cases this can be attributed to loss-of-function mutations in some patients, but gain-of-function mutations in others. In addition, the surprisingly high frequency of autosomal dominant immunodeficiencies with variable penetrance, and de novo mutations in disorders with a severe phenotype has been unmasked.

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.

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.

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.

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.

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.

Although it is expressed very transiently and at very low cell surface density, the pre-BCR plays a critical role in early B cell development ([1][1], [2][2]). The proteins that are required to assemble the pre-BCR, the signal transduction molecules Igα and Igβ, and the components of the surrogate

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.

Patients with genetic defects in B-cell development provide an unusual opportunity to dissect the requirements for normal B-cell maturation. It is striking that all of the known genetic defects that result in a failure of B-cell development involve signaling through the pre-B-cell receptor (pre-BCR). Approximately 85% of affected patients are males with mutations in the X chromosome-encoded cytoplasmic tyrosine kinase Btk. Preliminary experiments using stem cell transplants and retroviral-mediated gene therapy in Btk-deficient mice suggest that it may be relatively easy to correct serum immunoglobulins but harder to correct antibody production to T-cell-independent antigens in this disorder. About 3-6% of patients with defects in B-cell development have deletions or critical base pair substitutions in the mu constant region gene. Patients with defects in Igalpha, lambda5 and B-cell linker protein (BLNK) have also been described. All of these patients have a block at the pro-B to pre-B-cell transition. Defects in Btk, lambda5 and BLNK result in a more severe phenotype in the human compared to the mouse. These findings suggest that requirements for signaling through the pre-BCR are more stringent in the human compared to the mouse. Possible explanations for this observation are discussed.

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.

1The identification of many genes responsible for primary immunodeficiencies has provided us with a new perspective to evaluate these disorders. It has clarified the clinical and laboratory findings that are most consistently associated with a specific gene, demonstrated the spectrum of clinical severity seen in a particular disorder, and shown that not all of the patients with identical clinical and laboratory findings have mutations in the same gene. This new information allows us to reevaluate the criteria that we use to make diagnoses. The diagnostic criteria are meant to establish simple, objective, and clear guidelines that ensure that different physicians and scientists are using the same definitions when they include patients in research studies. These criteria may also help physicians formulate a diagnosis in patients with abnormalities of the immune system. The diagnostic criteria are divided into three categories: definitive, probable, and possible. To guard against the inclusion of patients who have polymorphic variants in the genes associated with immunodeficiency and to specify the clinical or laboratory finding that is most consistently abnormal in a particular disorder, the patient must fulfill an inclusion criterion that is characteristic of the disorder. Patients with a definitive diagnosis are assumed to have a greater than 98% probability that in 20 years they will still be given the same diagnosis. Mutation detection is the most reliable method of making a diagnosis. In some disorders the absence of the specific mRNA or protein is diagnostic. In others, the mRNA and/or protein may be only transiently expressed or may be produced at very low levels, or clinically useful assays may not yet have been developed. The clinical and laboratory findings in several of the X-linked immunodeficiencies are sufficiently distinctive that these findings, when coupled with a family history of disease that is specific to X-linked inheritance, can be used to make a definitive diagnosis. In families with a known mutation in a particular gene, mutation detection can be used to provide a definitive diagnosis in a newborn or fetus. Patients with a probable diagnosis are those with all of the clinical and laboratory characteristics of a particular disorder but who do not have a documented abnormality in the gene, the mRNA, or the protein that is known to be abnormal in the disorder. They are

Mutation detection for X-linked agammaglobulinemia (XLA) has revealed the heterogeneity of the clinical phenotype of patients with defects in Bruton's tyrosine kinase (Btk), the gene that is abnormal in XLA. Over 50% of patients with mutations in Btk have no family history of the disease because their cases are the first manifestation of a new mutation in their family. In 10% to 20% of patients, the serum immunoglobulins are higher than expected or the onset of disease is delayed; however, a marked reduction in B-cell numbers is consistent in all patients. Mutation detection has also shown that not all patients with presumed XLA have mutations in Btk. Mutations in mu heavy chain, and other components of the pre-B cell receptor complex, including lambda 5/14.1, cause a disorder that is clinically identical to XLA. Although new strategies for therapy are not yet available, the groundwork is being laid for cell or gene therapy.

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 xidcells 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 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.

Surrogate light chain, which escorts the mu heavy chain to the cell surface, is a critical component of the pre-B cell receptor complex. The two proteins that comprise the surrogate light chain, VpreB and λ5/14.1, contain both unique regions and Ig-like domains. The unique regions have been postulated to function in the assembly of the surrogate light chain. However, by using transient transfection of COS7 cells, we show that deletion of the unique regions of both proteins did not inhibit the assembly of surrogate light chain. Instead,in vivofolding studies showed that the unique region of λ5/14.1 acts as an intramolecular chaperone by preventing the folding of this protein when it is expressed in the absence of its partner, VpreB. The Ig domains of both λ5/14.1 and VpreB are atypical. The one in VpreB lacks one of the canonical β strands whereas the one in λ5/14.1 has an extra β strand. Deletion of the extra β strand in λ5/14.1 completely abrogated the formation of the surrogate light chain, demonstrating that complementation of the incomplete Ig domain in VpreB by the extra β strand in λ5/14.1 was necessary and sufficient for the folding and assembly of these proteins. Our studies reveal two novel mechanisms for regulating surrogate light chain formation: (i) the presence of an intramolecular chaperone that prevents folding of the unassembled subunit but that remains part of the mature assembled protein, and (ii) splitting an Ig domain between two proteins to control their folding and assembly.

X-linked agammaglobulinemia (XLA) is an immunodeficiency caused by mutations in the gene coding for Bruton agammaglobulinemia tyrosine kinase (BTK). A database (BTKbase) of BTK mutations lists 544 mutation entries from 471 unrelated families showing 341 unique molecular events. In addition to mutations, a number of variants or polymorphisms have been found. Mutations in all the five domains of BTK cause the disease, the single most common event being missense mutations. Most mutations lead to truncation of the enzyme. The mutations appear almost uniformly throughout the molecule. About one-third of point mutations affect CpG sites, which usually code for arginine residues. The putative structural implications of all the missense mutations are provided in the database. BTKbase is available at http://www.uta.fi/imt/bioinfo.

A susceptibility gene in the MHC class III region may underlie the defective B-cell differentiation in familial IgA deficiency and common variable immunodeficiency. Mutations in Bruton's tyrosine kinase, immunoglobulin heavy chain and δ5/14.1 surrogate light chain loci disrupt B-cell development to cause profound antibody deficiency. Mutational, biochemical and transgenic studies offer insight into the function of these and other ‘antibody deficiency genes’.

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.

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.

The present uncertainty of which live viral or bacterial vaccines can be given to immunodeficient patients and the growing neglect of societal adherence to routine immunizations has prompted the Medical Advisory Committee of the Immune Deficiency Foundation to issue recommendations based on published literature and the collective experience of the committee members. These recommendations address the concern for immunodeficient patients acquiring infections from healthy subjects who have not been immunized or who are shedding live vaccine-derived viral or bacterial organisms. Such transmission of infectious agents can occur within the hospital, clinic, or home or at any public gathering. Collectively, we define this type of transmission as close-contact spread of infectious disease that is particularly relevant in patients with impaired immunity who might have an infection when exposed to subjects carrying vaccine-preventable infectious diseases or who have recently received a live vaccine. Immunodeficient patients who have received therapeutic hematopoietic stem transplantation are also at risk during the time when immune reconstitution is incomplete or while they are receiving immunosuppressive agents to prevent or treat graft-versus-host disease. This review recommends the general education of what is known about vaccine-preventable or vaccine-derived diseases being spread to immunodeficient patients at risk for close-contact spread of infection and describes the relative risks for a child with severe immunodeficiency. The review also recommends a balance between the need to protect vulnerable subjects and their social needs to integrate into society, attend school, and benefit from peer education. © 2013 American Academy of Allergy, Asthma & Immunology.

Genetic mutations cause primary immunodeficiencies (PIDs), which predispose to infections. Here we describe Activated PI3K-δ Syndrome (APDS), a PID associated with a dominant gain-of-function mutation E1021K in the p110δ protein, the catalytic subunit of phosphoinositide 3-kinase δ (PI3Kδ), encoded by the PIK3CD gene. We found E1021K in 17 patients from seven unrelated families, but not among 3,346 healthy subjects. APDS was characterized by recurrent respiratory infections, progressive airway damage, lymphopenia, increased circulating transitional B cells, increased IgM and reduced IgG2 levels in serum and impaired vaccine responses. The E1021K mutation enhanced membrane association and kinase activity of p110δ. Patient-derived lymphocytes had increased levels of phosphatidylinositol 3,4,5-trisphosphate and phosphorylated AKT protein and were prone to activation-induced cell death. Selective p110δ inhibitors IC87114 and GS-1101 reduced the activity of the mutant enzyme in vitro, suggesting a therapeutic approach for patients with APDS.

The number of genetically defined Primary Immunodeficiency Diseases (PID) has increased exponentially, especially in the past decade. The biennial classification published by the IUIS PID expert committee is therefore quickly expanding, providing valuable information regarding the disease-causing genotypes, the immunological anomalies, and the associated clinical features of PIDs. These are grouped in eight, somewhat overlapping, categories of immune dysfunction. However, based on this immunological classification, the diagnosis of a specific PID from the clinician's observation of an individual clinical and/or immunological phenotype remains difficult, especially for non-PID specialists. The purpose of this work is to suggest a phenotypic classification that forms the basis for diagnostic trees, leading the physician to particular groups of PIDs, starting from clinical features and combining routine immunological investigations along the way. We present 8 colored diagnostic figures that correspond to the 8 PID groups in the IUIS Classification, including all the PIDs cited in the 2011 update of the IUIS classification and most of those reported since. © 2013 Springer Science+Business Media New York.

X-linked agammaglobulinemia (XLA) is usually considered a disorder of B cell development; however, the gene that is defective in XLA encodes a cytoplasmic tyrosine kinase called Btk, that is expressed throughout myeloid as well as B cell differentiation. A review of medical records of patients in whom mutations in Btk had been identified indicated that 13 of 50 patients (26%) had experienced episodes of profound neutropenia. In 12 of the 13 patients, neutropenia was part of the acute illness that precipitated an evaluation for immunodeficiency. These boys were more likely to be less than a year of age at the time of diagnosis and they were less likely to have a family history of immunodeficiency. Neutropenia was associated with staphylococcal or pseudomonas sepsis in 6 of the patients. The duration of neutropenia was variable but was often more than 1 week. Neutropenia was not seen in any patient with XLA receiving intravenous gammaglobulin. Although neutropenia was not associated with any specific mutation in Btk, most of the alterations in this gene in the patients with XLA and neutropenia resulted in the absence of Btk protein or in amino acid substitutions in sites thought to be critical to Btk function. Btk may not be required for neutrophil production under normal circumstances; however, it may play a role in the response to stress.