Characterization of Bruton's tyrosine kinase mutations in Mexican patients with X-linked agammaglobulinemia

X-linked agammaglobulinemia (XLA) is a humoral primary immunodeficiency in which affected patients have very low levels of peripheral B cells and a profound deficiency of all immunoglobulin isotypes. Mutations in the gene encoding for Bruton's tyrosine kinase (Btk) are responsible for most of the agammaglobulinemia. In this work, 14 Btk mutations responsible of causing XLA are described; eight of which are novel and six are mutations previously reported. Seven of the mutations were due to deletions and insertions of exons and introns, respectively, which suggest splicing defects. The others were missense mutations, five of which affect arginine residues and have been described, and two new which affect leucine and glutamine residues (L111P and E605G). Most of these mutations were located at the kinase domain of Btk and, less frequently, they were found in PH and SH2 domains. Protein expression was also affected since most of the patients did not express or express very low Btk.     

Characterization of germline mutations of the gene encoding Bruton's tyrosine kinase in families with X-linked agammaglobulinemia

Bruton's tyrosine kinase (Btk) has been identified as the protein responsible for the primary immunodeficiency X-linked agammaglobulinemia (XLA) and has been described as a new member of Srcrelated cytoplasmic protein tyrosine kinases. We have recently characterized the structure of the entire gene encoding Btk and developed a polymerase chain reaction (PCR)-based assay to detect germline mutations within it. In this report we describe six mutations, five of which are novel, of the Btk gene in patients with XLA and demonstrate the inheritance pattern of the defect within the families of the affected individuals. The mutations found include two nonsense and two missense mutations, a single base deletion at an intron acceptor splice site, and a 16-bp insertion. A single Strand conformation polymorphism was also found in the 5′ end of intron 8 with the same assay. This technique has provided a powerful tool for direct analysis of the Btk gene for the diagnosis of XLA and carrier detection. The identification of new mutations may eventually reveal the role of Btk in the signaling pathways involved in B-cell development. © 1995 Wiley-Liss, Inc.     

Bruton's tyrosine kinase mutations in 8 Chinese families with X-linked agammaglobulinemia

Bruton's tyrosine kinase (BTK) is involved in B-cell development. Mutation of BTK results in X-linked agammaglobulinemia (XLA). BTK is expressed in most haemopoietic lineages except mature T cells and plasma cells. We identified six novel and two known mutations of BTK in 11 Chinese XLA patients from 8 families. Family 1 had a novel point mutation at the start codon (135G-->T) in exon 2. Family 2 had known mutation of single A insertion in a stretch of 7 A residues (341-347insA) recognized as mutation hotspot in exon 3. Family 3 had a novel point mutation in exon 11 (1074A-->G) which led to aberrant splicing. Family 4 had known mutation in exon 19 (2053C-->T) in CpG mutation hotspot. The novel mutation of family 5 was an A deleted in a run of three As (1017-1019delA) in exon 10. In family 6, exons 2 and 3 were lost in BTK mRNA, a novel deletion. Family 7 had a novel substitution in exon 2 (227T-->C) which led to change of a conserved leucine to serine. Family 8 had a novel point mutation at beginning of intron 14 (IVS14+ 6 T-->G) resulting in aberrant splicing. Hum Mutat 15:385, 2000.     

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.     

Mutation pattern in the Bruton's tyrosine kinase gene in 26 unrelated patients with X-linked agammaglobulinemia

Mutation pattern was characterized in the Bruton's tyrosine kinase gene (BTK) in 26 patients with X-linked agammaglobulinemia, the first described immunoglobulin deficiency, and was related to BTK expression. A total of 24 different mutations were identified. Most BTK mutations were found to result in premature termination of the translation product. Mutations were detected in most BTK exons with a predominance of frameshift and nonsense mutations in the 5′ end of the gene and missense mutations in its 3′ part, corresponding to the catalytic domain of the enzyme. Nonsense and frameshift mutations were associated with diminished levels of BTK mRNA expression, except for a frameshift mutation in exon 17 and two nonsense mutations in exon 2, indicating that these cases are not confined to penultimate exons. One amino acid substitution (R28H) was found in the pleckstrin homology domain's residue, which is mutated in mice bearing the X-linked immunodeficiency phenotype; another substitution (R307G) was identified in the src homology domain 2. All remaining amino acid substitutions were found in the catalytic domain of Btk. Hum Mutat 9:418–425, 1997. © 1997 Wiley-Liss, Inc.     

Identification of nine novel mutations in the Bruton's tyrosine kinase gene in X-linked agammaglobulinaemia patients

Mutations in the Bruton's tyrosine kinase (BTK ) gene are responsible for X-linked Agammaglobulinemia (XLA), an immunodeficiency caused by a block in B cell differentiation. Non Isotopic RNAse Cleavage Assay (NIRCA), followed by sequencing was used to screen for BTK mutations in 11 Italian XLA patients. Nine novel mutations were identified: 6 missense (Y39S, L512P, L512Q, R544G, S578Y, E589K), one non-sense (Q260X), one frameshift (1599-1602del GCGC) and one in-frame insertion (2037-2038insTTTTAG), that represents the first case of premature stop codon introduction in the BTK coding frame. These data support the high molecular heterogeneity of BTK gene in XLA disease and provide new insight to the diagnosis and to the role of BTK domain in XLA and in B cell signal transduction and development. Hum Mutat 15:117, 2000.     

Identification of mutations in the Bruton's tyrosine kinase gene,including a novel genomic rearrangements resulting in large deletion,in Korean X-linked agammaglobulinemia patients

Mutations in the Bruton's tyrosine kinase (BTK) gene are responsible for X-linked agammaglobulinemia (XLA). We identified BTK mutations in six patients with presumed XLA from unrelated Korean families. Four out of six mutations were novel: two missense mutations (P565T, C154Y), a point mutation in a splicing donor site (IVS11+1G>A), and a large deletion (a 6.1-kb deletion including BTK exons 11–18). The large deletion, identified by long-distance PCR, revealed Alu-Alu mediated recombination extended from an Alu sequence in intron 10 to another Alu sequence in intron 18, spanning a distance of 6.1 kb. The two known mutations consisted of one missense (G462D) mutation, and a point mutation in a splicing acceptor site (IVS7−9A>G). This study suggests that large genomic rearrangements involving Alu repeats are few but an important component of the spectrum of BTK mutations.     

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.     

DNA-based mutation analysis of Bruton's tyrosine kinase gene in patients with X-linked agammaglobulinaemia

The identification of the BTK (Bruton's tyrosine kinase) genedefective in human immunoglobulln deficiency X-linked agammaglobulinaemla(XLA) and characterlsation of BTK exon–intron boundarleshas now allowed the analysis of mutations and polymorphismsat the level of genomic DNA. Using Southern blot analysis andthe polymerase chain reaction single strand conformation polymorphism(PCR–SSCP) assay, amplifying all 19 exons and the putativepromoter region with a single annealling temperature, mutationshave been identified in 19 out of 24 unrelated patients diagnosedas having XLA. Apart from a large deletion involving exon 19,nine missense (F25S, R288W, I370M, M509V, R525P, N526K, R562W,A582V and G594R), two nonsense (E277X and R525X), five frameshiftand two splice site mutations have been found affecting mostcoding exons and all major enzyme domains. No mutations or polymorphismswere detected in the putative promoter region. A single nucleotidedeletion located in the last exon, resulting in a truncationof the eight C-terminal residues of Btk and a typical XLA phenotype,indicates structural and/or functional importance of Btk helixI In the catalytic domain. Although allelic heterogeneity atthe BTK locus may partly explain clinical variability In familleswith XLA, compensatory and redundant mechanisms involved inB-cell development must play a role in the phenotypic diversityof the disease.     

BTK: 22 novel and 25 recurrent mutations in European patients with X-linked agammaglobulinemia

X linked agammaglobulinemia (XLA) is an immunodeficiency disease caused by mutations in the gene coding for Bruton's agammaglobulinemia tyrosine kinase (BTK), that is involved in signal transduction pathways regulating survival, activation, proliferation, and differentiation of B lineage lymphoid cells. XLA is a primary immunodeficiency disorder characterized by lack of mature, circulating B lymphocytes, and recurrent infections. Using Single Strand Conformation Polymorphism (SSCP) followed by direct sequencing we investigated 57 patients with XLA phenotype, with or without a positive family history, from 52 unrelated families enrolled in the Italian XLA Multicenter Clinical Study. We have identified 25 recurrent mutations, 22 novel mutations including one large deletion comprising the coding sequence from exon 11 to 18. Among the mutations identified, three were detected in different unrelated families, whereas all the others were private mutations.     

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.     

Bruton tyrosine kinase gene mutations in Argentina

The block in differentiation from pro-B to pre-B cells results in a selective defect in the humoral immune response characteristic of human X-linked agammaglobulinemia (XLA). Mutations of Bruton tyrosine kinase (BTK) gene have been identified as the cause of XLA. Mutation detection is the most reliable method for making a definitive diagnosis, except when clinical and laboratory findings are distinctive and coupled with history of X-linked inheritance. To provide a definitive diagnosis to 40 families incorporated in the Argentinian Primary Immunodeficiencies Registry we analysed the BTK gene by SSCP analysis as screening method for XLA, followed by direct sequencing. The molecular defect was localized in 45 patients from 34 unrelated families. From the 34 independent mutations identified, 16 were previously undescribed, 31 were unique mutations, 22 were exonic single nucleotide changes (16 missense and 6 nonsense) and four intronic mutations. Because five families had clinical, immunological and inheritance data sufficient for a definitive diagnosis, our study allowed 37 patients from 29 families previously categorized probable/ possible XLA, have now definitive diagnosis leading to appropriate genetic counseling.     

Signalling of Bruton's tyrosine kinase, Btk

Bruton's tyrosine kinase, which is encoded by the BTK gene, is a cytoplasmic protein tyrosine kinase (PTK) crucial for B-cell development and differentiation. It belongs to the Tec family of PTKs containing several domains that are characteristic of signalling molecules. In humans, mutations that disrupt the function of this gene lead to the classical XLA syndrome (X-linked agammaglobulinaemia), a primary immunodeficiency mainly characterized by lack of mature B cells as well as low levels of immunoglobulins. In contrast, animal models of this disease such as the xid mice display profoundly milder XLA phenotype. BTK phosphorylation and activation in response to engagement of the B-cell receptor (BCR) by antigen is a dynamic process whereby a variety of proteins interact with each other and recruit signalling molecules resulting in a physiological response such as B-cell proliferation and antibody production. The main players, however, that participate in the intracellular downstream cascade have not yet been identified and are therefore under intense scrutiny in several laboratories. This review discusses certain aspects of BTK activation following receptor stimulation by agonists and how this event is translated into the biochemical signals within the cell that eventually lead to nuclear responses.     

Clinical potential of targeting Bruton's tyrosine kinase

Targeting Bruton's tyrosine kinase (BTK) with a small-molecule inhibitor may be useful in treatment of BTK-expressing malignancies because of the antiapoptotic function of BTK in cancer cells. Furthermore, BTK inhibitors also exhibit antithrombotic properties, which may be desirable in the context of the increased risk of thromboembolic complications in cancer patients. This review will focus on the role of BTK in drug resistance in cancer, thromboembolism, and various pathologic immune responses, such as graft-versus-host disease. The therapeutic potential of targeting BTK is illustrated by discussion of the biologic activity profile of the rationally designed BTK inhibitor LFM-A13.     

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.     

Mutations of the human BTK gene coding for bruton tyrosine kinase in X-linked agammaglobulinemia

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.     

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.     

Expression of Bruton's tyrosine kinase in B lymphoblastoid cell lines from X-linked agammaglobulinaemia patients

X-linked agammaglobulinaemia (XLA) is an immunodeficiency caused by mutations in Bruton's tyrosine kinase (Btk) and is characterized by an almost complete arrest of B cell development. We analysed expression of Btk in B lymphoblastoid cell lines (BLCL) derived from four unrelated XLA patients. In one patient, with a 3.5 kb genomic deletion encompassing the first (untranslated) exon, mRNA levels and in vitro kinase activities were very low. The patient manifested a mild phenotype with a delayed onset of the disease. Another mutation, in which the intron 3 donor splice site is lost, was also associated with very low mRNA levels and an absence of detectable Btk protein. Patients with this mutation showed extensive heterogeneity of the immunological phenotype. In the BLCL of a third patient, with an Arg₂₈₈ substitution in the SH2 domain, the mutation did not appear to affect the expression level, nor to abrogate in vitro phosphorylation activity. In the BLCL of the fourth patient, with an Arg₂₈ mutation in the PH domain, tyrosine kinase activity in BTK precipitates appeared to be decreased compared with control BLCL.