Novel insertions of Bruton tyrosine kinase in patients with X-linked agammaglobulinemia

Mutations in the gene encoding Bruton tyrosine kinase (BTK) result in X-linked agammaglobulinemia (XLA), an immunodeficiency of antibody defect. By using base excision sequence scanning method (BESS) followed by direct sequencing we found in seven unrelated families with a classical XLA phenotype various mutations including six novel mutations (g.64512_64513insC, c.108_109insG, c.1700_1701insACTACAG, g.51375_51376GC>TG, g.63991_63992insGGTAGAAAAAA, c.1956_1957insCA) and a previously known silent polymorphism (c.2031C>T). Except for two mutations, the alterations affect the kinase domain. There was exceptionally high proportion of insertions in the cohort. Frameshift insertion was found altogether in five patients, three of which are on introns, one in upstream region, and one in exon 18 leading to frameshift mutation and truncation of the protein. In the intron 4 there is a substitution of two bases. Carrier detection was performed in four families. In one case the mutation was found to be de novo.     

Identification of mutations of Bruton's tyrosine kinase gene (BTK) in Brazilian patients with X-linked agammaglobulinemia

Mutations in the Bruton tyrosine kinase (BTK) gene are responsible for X-linked agammaglobulinemia (XLA), which is characterized by recurrent bacterial infections, profound hypogammaglobulinemia, and decreased numbers of mature B cells in the peripheral blood. We evaluated 17 male Brazilian patients from 13 unrelated families who showed markedly reduced numbers of blood B cells and hypogammaglobulinemia. BTK gene analysis detected mutations in 10 of the 13 presumed XLA families. Seven mutations (Q196X, G613D, R28L, 251-273del, Q234X, H364P, and R13X) had been reported previously, whereas the remaining three mutations (M501T, IVS15+1G>C, and IVS14+1G>A) were novel. Mutation IVS15+1G>C occurred in a splice donor site and caused exons 15 and 16 to be skipped, and IVS14+1G>A might cause exon 14 to be skipped. Flow cytometry revealed deficient expression of BTK protein in 10 of the 13 families. This is the first report of the diagnosis of XLA by analysis of mutations of the BTK gene in Brazilian patients.     

Identification of the bruton tyrosine kinase (BTK) gene mutations in 20 Australian families with X-linked agammaglobulinemia (XLA)

X-linked agammaglobulinemia (XLA) is an immunodeficiency caused by mutations in the Bruton tyrosine kinase (BTK) gene. Twenty Australian patients with an XLA phenotype, from 15 unrelated families, were found to have 14 mutations. Five of the mutations were previously described c.83G>A (p.R28H), c.862C>T (p.R288W), c.904G>A (p.R302G), c.1535T>C (p.L512P), c.700C>T (p.Q234X), while nine novel mutations were identified: four missense c.82C>A (p.R28S), c.494G>A (p.C165Y), c.464G>A (p.C155Y), c.1750G>A (p.G584E), one deletion c.142_144delAGAAGA (p.R48_G50del), and four splice site mutations c.241-2A>G, c.839+4A>G, c.1350-2A>G, c.1566+1G>A. Carrier analysis was performed in 10 mothers and 11 female relatives. The results of this study further support the notion that molecular genetic testing represents an important tool for definitive and early diagnosis of XLA and may allow accurate carrier status and prenatal diagnosis.     

Clinical, immunological and molecular characteristics of 37 Iranian patients with X-linked agammaglobulinemia

BACKGROUND: X-linked agammaglobulinemia (XLA) is a hereditary immunodeficiency characterized by an early onset of recurrent bacterial infections, a profound deficiency of all immunoglobulin isotypes and a markedly reduced number of peripheral B lymphocytes. Eighty-five percent of the patients with this phenotype have mutations in Bruton's tyrosine kinase (BTK) gene. METHODS: To provide an informative outlook of clinical and immunological manifestations of XLA in Iran, 37 Iranian male patients with an age range of 1-34 years, followed over a period of 25 years, were studied. Twenty-four of the 37 patients were screened for BTK gene mutation using PCR-SSCP followed by direct sequencing. BTK protein expression assay was done by flow cytometry in 9 families. RESULTS: All patients first presented with infectious diseases, the most common of which were respiratory tract infections. Eighteen different mutations were identified, 13 of which were novel: IVS1+5G>C, 1896G>A, 349delA, 1618C>T, 1783T>C, 2084A>G, 1346delT, 1351delGAG, 587A>G, IVS14-1G>A, IVS3+2T>C, 1482G>A, 1975C>A. CONCLUSION: The fact that we found a great number of novel mutations in a relatively limited number of patients underlines the heterogeneity of BTK mutations in the Iranian population. The large number of new mutations indicates that extended studies in this region would be rewarding.     

Delayed diagnosis in X-linked agammaglobulinemia and its relationship to the occurrence of mutations in BTK non-kinase domains

Background: X-linked agammaglobulinemia (XLA) is characterized by the absence of immunoglobulin and B cells. Patients suffer from recurrent bacterial infections from early childhood, and require lifelong immunoglobulin replacement therapy. Mutations in BTK (Bruton’s Tyrosine Kinase) are associated with this phenotype. Some patients that present XLA do not show typical clinical symptoms, resulting in delayed diagnosis due to the lack of a severe phenotype. This study presents a report of five XLA patients from four different families and attempts to determine a relationship between delayed diagnosis and the occurrence of BTK mutations.

Methods: Samples from patients with antibody deficiency were analyzed to determine BTK expression, immunophenotyping and mutation analysis. Clinical and laboratory data was analyzed and presented for each patient.

Results: Most patients presented here showed atypical clinical and laboratory data for XLA, including normal IgM, IgG, or IgA levels. Most patients expressed detectable BTK protein. Sequencing of BTK showed that these patients harbored missense mutations in the pleckstrin homology and Src-homology-2 domains. When it was compared to public databases, BTK sequencing exhibited a new change, along with three other previously reported changes.

Conclusions: Delayed diagnosis and atypical manifestations in XLA might be related to mutation type and BTK expression.     

Challenges in investigating patients with isolated decreased serum IgM: The SIMcal study

The clinical consequences of isolated decreased serum immunoglobulin (Ig)M are not sufficiently known. Therefore, it is difficult to determine the clinical policy following such a finding. Only few reported IgM‐deficient patients fulfil the European Society for Immunodeficiencies (ESID) diagnostic criteria for selective IgM deficiency (true sIgMdef), or their diagnosis is uncertain due to insufficient laboratory data (possible sIgMdef). Decreased serum IgM is often incidentally found in asymptomatic adults. The objective of our study was to further characterize true sIgMdef and to compare the European data collected through the ESID Registry community (tertiary centres) to our previously published Dutch cohort (secondary centre). Fifteen centres (12 countries) participated with 98 patients. Patients were excluded if serum IgM was only determined once (n = 14), had normalized (n = 8), or if they also had other immunological abnormalities (n = 15). Ten patients (5 adults) completely fulfilled the ESID criteria for true sIgMdef. Age‐matched cut‐off values varied widely between centres; when using the ESID diagnostic protocol reference values, only six patients (five adults) had true sIgMdef. Because of these small numbers, further analyses were performed in patients with true or possible sIgMdef (13 adults, 48 children). Respiratory infections were commonly reported at presentation (adults 54%, children 60%). Symptomatic adults had lower serum IgM levels (mean 0.27 g/L, 95% CI 0.22‐0.31) than those without symptoms (mean 0.33 g/L, 95% CI 0.30‐0.36; P = 0.02). To be able to explore the clinical consequences of true sIgMdef, we should fully analyse and accurately describe those patients in whom a decreased serum IgM is found.     

T cell phenotypes in patients with common variable immunodeficiency disorders: associations with clinical phenotypes in comparison with other groups with recurrent infections

Common variable immunodeficiency disorders (CVID) are a group of heterogeneous conditions that have in common primary failure of B cell function, although numerous T cell abnormalities have been described, including reduced proliferative response and reduced regulatory T cells. This study compared the T cell phenotype of CVID patients subdivided into clinical phenotypes as well as patients with partial antibody deficiencies [immunoglobulin (Ig)G subclass deficiency and selective IgA deficiency], X-linked agammaglobulinaemia (XLA) and healthy and disease controls. Absolute numbers of T cell subpopulations were measured by four-colour flow cytometry: naive T cells, central and effector memory and terminally differentiated (TEM) T cells, using CD45RA and CCR7 expression. Early, intermediate and late differentiation status of T cells was measured by CD27/CD28 expression. Putative follicular T cells, recent thymic emigrants and regulatory T cells were also assessed. Significant reduction in naive CD4 T cells, with reduced total CD4 and recent thymic emigrant numbers, was observed in CVID patients, most pronounced in those with autoimmune cytopenias or polyclonal lymphoproliferation. These findings suggest a lack of replenishment by new thymically derived cells. CD8 naive T cells were reduced in CVID patients, most significantly in the autoimmune cytopenia subgroup. There was a reduction in early differentiated CD4 and CD8 T cells and increased CD8 TEM in the CVID patients, particularly autoimmune cytopenia and polyclonal lymphoproliferation subgroups, suggesting a more activated T cell phenotype, due perhaps to an antigen-driven process. XLA patients had significantly reduced putative follicular T cells, which may depend on B cells for survival, while no significant alterations were observed in the T cells of those with IgG subclass deficiency or selective IgA deficiency.     

Bruton tyrosine kinase gene mutations in Turkish patients with presumed X‐linked agammaglobulinemia

X‐linked agammglobulinemia (XLA) is a ptototypical humoral immunodeficiency caused by mutations in the gene coding for Bruton tyrosine kinase (BTK). The genetic defect in XLA impairs early B cell development resulting in marked reduction of mature B cells in the blood. Studies from different countries have demonstrated that approximately 90% of males with presumed XLA bear mutations in BTK. In this study, we report for the first time the occurrence of BTK mutations in Turkey. We performed mutational analysis of the BTK gene in 16 Turkish male patients from 13 separate families with presumed XLA based on abnormally low peripheral blood B‐cell numbers (lt; 1%), hypogammaglobulinemia, and recurrent bacterial infections. We found that in nine of the 13 families (69%) a Btk mutation caused XLA. Two of the mutations were previously described, but seven novel mutations were identified: two missense (Y39C, G584R), one nonsense (Q343X), and 4 deletions (1800‐1821del, 1843‐1847del, 1288‐1292del, 291del) resulting in frameshift and premature stop codon. By contrast, no mutations in the BTK gene were identified in the other 4 families. A consanguinity in three of these families raises the possibility that mutations in other autosomal genes which affect early B cell development may contribute to their phenotype resembling XLA. Hum Mutat 18:356, 2001. © 2001 Wiley‐Liss, Inc.     

Clinical,immunological and genomic characteristics of children with X-linked agammaglobulinemia from Kerala,South India

X-linked agammaglobulinemia (XLA) is an X-linked recessive primary immunodeficiency disorder caused due to a pathogenic variant in the Bruton tyrosine (BTK) gene with an incidence of 1:379,000 live births and 1:190,000 male births. Patients affected with XLA present with recurrent infections of the gastrointestinal and respiratory tracts. Here we report the first case series of 17 XLA patients of 10 South Indian families with a wide spectrum of clinical and genetic features. In our cohort, patients presented mainly with recurrent pneumonia, gastrointestinal infection, otitis media, pyoderma, abscesses, empyema, arthritis, and osteomyelitis. Using next-generation and Sanger sequencing we have identified 10 unique pathogenic and likely pathogenic variants in 17 patients. This encompasses three nonsynonymous, two stop-gain, two frameshifts, two structural, and one splicing variant, out of which two of them are novel. Based on the type of variant, patients had variable clinical features and treatment responses. We have also evaluated Btk protein expression for six patients in comparison to the healthy individuals and determined mosaic Btk expression patterns in four mothers. We have also performed family screening in 6 families using Sanger sequencing and identified 19 carriers for the variant. The diagnosis for the patients led to the proper treatment i.e. 15 patients were on intravenous immunoglobulin (IVIG) and the other two had successful hematopoietic stem cell transplantation (HSCT). Unfortunately, two of our patients died due to sepsis, while on IVIG. We envision the present study could help in better understanding of patients with XLA and help in family screening and prenatal diagnosis. To the best of our knowledge, this is the largest case series of patients affected with XLA from South India.     

Epstein-Barr virus-induced lymphoblastoid cell lines derived from the peripheral blood of patients with X-linked agammaglobulinemia can secrete IgM

We have established lymphoblastoid cell lines (LCL) from the peripheral blood of three individuals with X-linked agammaglobulinemia as well as three of their immunodeficient first-degree relatives. Lines could be induced with Epstein-Barr virus only when T lymphocytes were depleted from total mononuclear leukocytes. The LCLs derived from XLA patients expressed characteristics of IgM-secreting plasmacytes, including intense cytoplasmic fluorescence after staining with anti-, easily detectable amounts of IgM in culture supernatants, and radiolabeled IgM with both heavy and light chains in culture media and cell lysates. The cell lines induced from blood of the first-degree relatives of these patients were more varied. They often exhibited multiple isotypes for both heavy and light chains in different cells or expressed a B-lymphocyte phenotype (easily detectable surface Ig but no Ig secretion). These studies suggest that B-cell precursors are present in peripheral blood of individuals with classical XLA. Differentiation of such cells to mature cells of the B lineage can be accomplished using Epstein-Barr virus after removal of T cells.     

Clinical,immunological, and molecular analysis in a large cohort of patients with X-linked agammaglobulinemia: an Italian multicenter study

A questionnaire-based retrospective clinical and immunological survey was conducted in 73 males with a definite diagnosis of X-linked agammaglobulinemia based on BTK sequence analysis. Forty-four were sporadic and 29 familial cases. At December 2000, the patients' ages ranged from 2 to 33 years; mean age at diagnosis and mean duration of follow-up were 3.5 and 10 years respectively. After the mid-1980s all but 2 were on intravenous immunoglobulin (IVIG) substitution therapy, with residual IgG >500 mg/dl in 94% of the patients at the time of enrollment. Respiratory infections were the most frequent manifestation both prior to diagnosis and over follow-up. Chronic lung disease (CLD) was present in 24 patients, in 15 already at diagnosis and in 9 more by 2000. The cumulative risk to present at diagnosis with CLD increased from 0.17 to 0.40 and 0.78 when the diagnosis was made at the ages of 5, 10, and 15 years respectively. For the 9 patients who developed CLD during follow-up, the duration of follow-up, rather than age at diagnosis; previous administration of intramuscular immunoglobulin; and residual IgG levels had a significant effect on the development of CLD. Chronic sinusitis was present in 35 patients (48%), in 15 already at diagnosis and in 20 by 2000. Sistemic infections such as sepsis and meningitis/meningoencephalitis decreased over follow-up, probably due to optimal protection provided by high circulating IgG levels reached with IVIG.     

Clinical immunology review series: an approach to the patient with recurrent infections in childhood

Recurrent or persistent infection is the major manifestation of primary immunodeficiency, which also results in atypical infection with opportunistic organisms. Young children are also vulnerable to infection and recurrent infection is common. While most children with recurrent infection have a normal immunity, it is important to recognize the child with an underlying primary immunodeficiency and investigate and treat appropriately and yet not over investigate normal children. Prompt, accurate diagnosis directs the most appropriate treatment, and early and judicious use of prophylactic antibiotics and replacement immunoglobulin can prevent significant end organ damage and improve long-term outlook and quality of life. This paper describes important presenting features of primary immunodeficiency and indicates when further investigation is warranted.     

Unimpaired activation of c-Jun NH2-terminal kinase (JNK) 1 upon CD40 stimulation in B cells of patients with X-linked agammaglobulinemia

X-linked agammaglobulinemia (XLA) is caused by mutations in the gene encoding the cytoplasmic Brutons tyrosine kinase (Btk). Btk has been shown to play an essential role in the development of B1 (CD5⁺) and conventional circulating mature B cells (B2) in mouse and man. It has been shown in earlier studies that Btk is involved in both the BCR- and CD40-mediated signaling pathways. In this study, we analyzed the responsiveness of Epstein-Barr virus (EBV) transformed B cells from nine XLA patients to CD40 stimulation, particularly the CD40 induced activation of c-Jun N-terminal kinase (JNK). In eight XLA patients the JNK activation was unimpaired and in one case JNK could not be activated by anti-CD40 stimulation. Btk protein expression was detectable by Western blotting in six cases, in one case Btk expression was drastically reduced, and in three cases no Btk expression could be observed. Btk kinase activity was found in three cases and it was reduced in one and not detectable in five cases. Furthermore, in one female patient with an agammaglobulinemia, Btk expression and function as well as JNK activation by CD40 stimulation was unimpaired. Our findings demonstrate that JNK activation via the CD40 signaling pathway is intact in EBV-transformed B cells of most if not all XLA patients, independent of the mutation and its effect on Btk expression and kinase activity. We suggest that Btk is not necessary for the activation of JNK upon CD40 stimulation, at least in the B cell subpopulation we had studied. We cannot exclude that these B cells belong to a leaky B-cell subpopulation in which the CD40 signaling pathway has become independent of Btk function.     

Detection of a novel mutation in the SRC homology domain 2 (SH2) of Bruton's tyrosine kinase and direct female carrier evaluation in a family with X-linked agammaglobulinemia

X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency disease with a block in differentiation from pre-B to B cells resulting in a selective defect in the humoral immune response. Affected males have very low concentrations of serum immunoglobulins leading predominantly to recurrent bacterial infections beginning at age 6 to 18 months. The gene responsible for XLA was identified recently to encode a cytoplasmatic tyrosine kinase (Bruton's tyrosine kinase, BTK). We have analyzed the BTK gene in a large family in which two brothers presented with the severe phenotype of XLA. Genomic DNA of affected boys and from healthy relatives was amplified by PCR with primers specific for the putative promoter region and for all 19 exons, including flanking intron boundaries, and subsequently screened for mutations using single strand conformation polymorphism (SSCP) analysis. Altered single strand band patterns were found using primers specific for exon 10, 15, and 18. Direct cycle-sequencing of these BTK segments detected two known polymorphisms in intron 14 and in exon 18. Sequencing of exon 10 from two boys with XLA demonstrated a novel point mutation in the SH2 domain of BTK. Direct identification of healthy female carriers in three generations was performed by amplification mutagenesis using PCR with a modified first primer. This method can easily be applied also to prenatal diagnosis. © 1996 Wiley-Liss, Inc.     

Clinical and immunological evaluation of patients with mild IgG1 deficiency.

Serum IgG subclass concentrations were determined in patients visiting, the pulmonology out-patient clinic with chronic respiratory tract problems. A total of 24 patients with a serum IgG1 concentration < 4.9 g/l (i.e. below the reference range) and normal values for IgG2, IgM and IgA were included. Patients with a selective IgG1 deficiency were vaccinated with a 23-valent pneumococcal polysaccharide vaccine. There were nine patients with a poor antibody response to pneumococcal capsular polysaccharide antigens. Responsiveness to protein antigens was intact in all patients. Patients with pneumonia showed a significantly lower anti-polysaccharide response in the IgG2 subclass than patients without pneumonia. Patients with recurrent sinusitis showed a significantly lower response in the IgA isotype after vaccination with pneumococcal polysaccharide vaccine compared with non-sinusitis patients. It can be concluded that patients with recurrent sinopulmonary infections and a mild IgG1 subclass deficiency have an impaired IgG1 anti-polysaccharide response, which can extend to decreased IgG2 and IgA anti-polysaccharide responses.     

Clinical,genetic and immunological characteristics of 40 Chinese patients with CD40 ligand deficiency

CD40 ligand (CD40L) deficiency is a rare but life‐threatening primary immunodeficiency caused by mutations in the CD40L gene. Here, we investigated a cohort of 40 genetically diagnosed CD40L‐deficient patients from the Chinese mainland, analysed their clinical and genetic data, and examined CD40L expression, the proportion of T cell subsets, B cell subsets and T follicular helper (Tfh) cells. The aim was to provide a complete picture of CD40L deficiency. Initial presentations of the patient cohort mainly involved recurrent fever (47.5%) and sinopulmonary infection (42.5%). Life‐threatening infections (42.5%), caused by various pathogens, were the most serious threats faced by CD40L‐deficient patients, while neutropenia (57.5%) remained the most common complication. Opportunistic infections, including Pneumocystis carinii pneumonia and invasive fungal disease associated with Talaromyces marneffei, were also common in the cohort. In addition, seven patients (17.5%) suffered BCGitis/BCGosis, which is a major problem facing a planned immunization programme in China. It was intriguing that reduced IgM levels were observed in 12.5% of patients, while normal or elevated IgA levels were shown in 47.5% of patients. Thirty‐seven unique mutations were identified in 40 patients; of these, 10 were novel. Furthermore, we observed a lower percentage of NK cells, Tfh cells, and central memory CD4⁺ T cells, and an extremely small class‐switched memory B cell population, in CD40L‐deficient patients. Patients who underwent hematopoietic stem cell transplantation experienced better disease remission. Taken together, our data establish the largest database about CD40L deficiency in China and provide genetic, immunologic and clinical information about Chinese CD40L‐deficient patients.     

Clinical and genetic features of the patients with X‐Linked agammaglobulinemia from Turkey: Single‐centre experience

X‐linked agammaglobulinemia is a primary immunodeficiency disorder resulting from BTK gene mutations. There are many studies in the literature suggesting contradictory ideas about phenotype‐genotype correlation. The aim of this study was to identify the mutations and clinical findings of patients with XLA in Turkey, to determine long‐term complications related to the disease and to analyse the phenotype‐genotype correlation. Thirty‐two patients with XLA diagnosed between 1985 and 2016 in Pediatric Immunology Department of Hacettepe University Ihsan Dogramaci Children's Hospital were investigated. A clinical survey including clinical features of the patients was completed, and thirty‐two patients from 26 different families were included in the study. Getting early diagnosis and regular assessment with imaging techniques seem to be the most important issues for improving the health status of the patients with XLA . Early molecular analysis gives chance for definitive diagnosis and genetic counselling, but not for predicting the clinical severity and prognosis.     

Autoimmunity in common variable immunodeficiency: epidemiology,pathophysiology and management

Introduction: Common variable immunodeficiency (CVID) comprises a large heterogeneous group of patients with primary antibody deficiency.

Areas covered: The affected patients are characterized by increased susceptibility to infections and low levels of serum immunoglobulin. However, enteropathy, granulomatous organ infiltrates, malignancy, inflammatory and autoimmune conditions are also prevalent. The concomitance of immunodeficiency and autoimmunity appears to be paradoxical and creates difficulties in the management of autoimmune complications affecting these patients.

Expert commentary: The management of autoimmunity in patients with CVID requires special considerations because dysregulation and dysfunctions of the immune system along with persistent inflammation impair the process of diagnosis and treatment.