BTK mutations in patients with X-linked agammaglobulinemia: lack of correlation between presence of peripheral B lymphocytes and specific mutations

X-linked agammaglobulinemia (XLA) is a human antibody deficiency that results from mutation of the tyrosine kinase btk. We tested the hypothesis that XLA patients who varied from the classic phenotype of XLA by presence of normal or near normal number of peripheral B lymphocytes would have a set of mutations of BTK that is different from the mutations found in patients without peripheral B lymphocytes. The mutations of BTK we found in two patients with normal numbers of peripheral B lymphocytes have been previously identified in patients without peripheral B lymphocytes. A third patient, without peripheral B cells, was found to express normal levels of wild type btk. Exmination of the mutations of the BTK gene in patients in the BTKbase who were identified as having peripheral B lymphocytes found that these same mutations, or mutations of the same protein domains, were also present in patients identified as lacking peripheral B lymphocytes. Analysis of mutations in BTK has previously led to the conclusion that severity of disease in XLA cannot be predicted from the specific mutation of BTK. The results of this study suggest that whether an XLA patient will develop peripheral B lymphocytes cannot be predicted from the specific mutation of BTK.     

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.     

B cell repertoire in patients with a novel BTK mutation: expanding the spectrum of atypical X-linked agammaglobulinemia

Immunologic Research - X-linked agammaglobulinemia (XLA) is caused by mutations in the Bruton tyrosine kinase) BTK) gene. Affected patients have severely reduced amounts of circulating B cells....     

De novo mutation in the BTK gene of atypical X-linked agammaglobulinemia in a patient with recurrent pyoderma.

BACKGROUND: X-linked agammaglobulinemia (XLA), characterized by a profound deficiency of all immunoglobulins and the absence of mature B cells, is caused by mutations in the gene encoding Bruton tyrosine kinase (BTK). Most patients have recurrent sinopulmonary infection. Infections usually occur in multiple locations across time, but single infection may be limited to one anatomic location. OBJECTIVES: To report a case of atypical XLA with recurrent pyoderma and to observe the immunologic changes in the patient in 10 years. METHODS: Immunologic investigations, skin wound culture, and molecular study with DNA sequencing were performed. RESULTS: The patient was originally diagnosed as having common variable immunodeficiency disease because of the presence of circulating B cells (CD19+ B cells: 7%) at 11 years old. On further evaluation at the age of 20 years, flow cytometric analysis of lymphocytes showed only 0.4% B cells. The molecular study with DNA sequencing of the patient showed a point mutation in complementary DNA 1630 A>G(p.R544G) in the BTK gene, indicating that the patient has XLA. The mutation analysis of the BTK gene revealed a normal DNA sequence in the other family members. CONCLUSIONS: This case is an important example of a possible presentation of XLA with a predominant skin manifestation, and it demonstrates that maintaining a high level of clinical suspicion is essential for the diagnosis of XLA in a child with recurrent pyoderma.     

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.     

Characterization of novel Bruton's tyrosine kinase gene mutations in Central European patients with agammaglobulinemia

X-linked agammaglobulinemia (XLA) is an immunodeficiency disorder caused by mutations in the gene coding for Bruton's tyrosine kinase (BTK). In this study we investigated 10 male patients with XLA-compatible phenotype (agammaglobulinemia and undetectable B cells in peripheral blood) from 9 unrelated Central European families. We identified seven different mutations, six of which were novel. One previously described point mutation caused a premature stop codon (p.C464X), two point mutations resulted in amino acid exchanges (p.W588R; p.G419E), and two point mutations affected splice sites (c.305-1G>A; c.391+1G>A). We further detected one deletion (c.1921_1927del CGTCCCA) and one large duplication. The duplication resulted from Alu element-induced unequal homologous recombination, which was only detectable by extended analysis of cDNA, while direct sequencing of genomic DNA gave a false negative result. Western blot analysis revealed that the patients with the p.W588R and the p.G419E amino acid substitutions, respectively, produced full length BTK, but in clearly diminished amounts. The patient with the 7bp deletion expressed low amounts of protein which might represent truncated BTK. All other genomic alterations resulted in complete loss of BTK protein. In two patients from unrelated families BTK protein expression was normal and no Btk gene mutation was detected. The results of this study further substantiate the importance of using elaborate molecular analysis with different detection techniques to obtain an explicit molecular diagnosis in patients with suspected XLA.     

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.     

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.     

X-linked spondyloepiphyseal dysplasia tarda: Novel and recurrent mutations in 13 European families

X-linked spondyloepiphyseal dysplasia tarda is a skeletal dysplasia mainly affecting the vertebrae and epiphyses and commonly associated with the early development of degenerative joint disease. Radiographically the disorder is characterized by a typical hump-shaped deformity of the vertebral bodies. SEDT is caused by mutations in SEDL located on Xp22.12-p22.31. To further elucidate the spectrum of underlying variations we performed a screening of all 6 exons of SEDL within 13 European SEDT families and identified 6 new (c.99delC, c.183_184delGA, c.236-5_236-8delATTA, c.325delT, c.345_346delTG, c.94-?_423+?del) and 9 previously reported mutations (c.1-?_93+?del, c.93+5G>A, c.157_158delAT, c.210G>A, c.236-9_236-12delTTAA, c.267_275delAAGAC, c.324-4_324-10delTCTTTCCinsAA). The recurrent splice site alteration c.93+5G>A (formerly described as IVS3+5G>A) was detected in 3 unrelated families. Two patients were carrying 2 changes in the allele. In one case, a novel variation in exon 4 (c.99delC) was associated with several nucleotide deletions in intron 4 (c.236-5_236-8delATTA), and in the second case we identified a previously reported transition c.210G>A and a novel deletion in exon 6 (c.325delT). All sequence variations identified are either deletions of complete exons or predicted to result in a premature stop codon or to lead into splicing defects and are associated with a loss of considerable parts of the sedlin protein.     

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.     

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.     

Genomic organization of the Btk gene and exon scanning for mutations in patients with X-linked agammaglobulinemia

The defective gene responsible for the recessively inheritedimmunodeficiency X-linked agammaglobulinemia (XLA) has beenshown to encode a cytoplasmic protein tyrosine kinase of theSrc family designated Btk (Bruton's tyrosine kinase). To facilitatethe search for germline mutations of the Btk gene, we have characterizedits genomic structure. Eighteen introns were positioned withinthe approximately 37 kb gene. Each of the exon/intron boundarieswere defined and sequenced, and all but two conform to consensussequences. We have utilized the genomic organization of Btkand the intervening sequence data to design an assay for amplifyingeach of the 19 exons from XLA patient DNA for single strandconformation polymorphism (SSCP) analysis. By using this methodwe have identified mutations in 12 of 14 unrelated affectedmales: seven different base substitutions and two small deletions.Two of the mutations described in exon 15 of the kinase domainwere found in more than one patient and may represent a mutationhot spot. Exon scanning has proven to be a valuable method foridentifying the patient mutations in genomic DNA without theuse of cDNA. The mutations are easily confirmed with directsequencing of the amplified exons. This approach will greatlybenefit XLA family studies involving carrier detection and prenataldiagnosis. In addition, the mutations identified may revealresidues involved in the specific protein interactions necessaryin the B-cell developmental pathway, of which Btk is an integralcomponent.     

Stem cell transplants for patients with X-linked agammaglobulinemia

Six young patients with X-linked agammaglobulinemia and proven mutations in Btk were treated with cord blood or bone marrow transplants from HLA-matched siblings. Complete blood counts, serum chemistries, serum immunoglobulin concentrations, lymphocyte cell surface markers, and physical findings were evaluated at 3- to 5-day intervals for the first 2 weeks after transplant and then every 3 to 6 months. The first three patients were not given any preparative regimen or antirejection drugs and at 24 to 42 months posttransplant these patients have shown no benefit or harm related to the transplants. The second three patients were not given a preparative regimen but were treated with cyclosporine A (70 days) and mycophenolate mophetil (28 days) after transplant. Two of these patients have developed normal sized, nontender cervical lymph nodes 3 to 12 months after transplant but none of the three patients have shown an increase in serum IgM or an increase in the number of peripheral blood B cells. It is likely that successful engraftment will require more aggressive immunosupressive medications.     

X-linked agammaglobulinemia: a survey of 33 Iranian patients

In order to determine the clinical and laboratory features of X-linked agammaglobulinemia, the records of 33 male patients with XLA were reviewed during 22 years (1980-2002) in the Iranian referral center of primary immunodeficiency disorders. The patients' ages ranged from 20 to 360 months (median 113 months). The median age at the onset of the disease was 8 months and the median age of diagnosis was 48 months, with a median diagnosis delay of 33 months. Almost all of the patients presented common infectious diseases, which were: pneumonia, otitis, diarrhea, sinusitis, and arthritis. During the course of illness, infections in the respiratory tract, gastrointestinal tract, central nervous system, and musculoskeletal system were seen in 93.9%, 75.8%, 33.3%, and 21.2% of XLA patients, respectively. The most common complications of these patients were chronic infections in 75.8% of them, including: chronic otitis media, chronic sinusitis, chronic diarrhea, and bronchiectasis.     

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.     

Ten novel ORF15 mutations confirm mutational hot spot in the RPGR gene in European patients with X-linked retinitis pigmentosa

RGPR was the first gene found to be mutated in XLRP, the subtype of RP displaying the most severe form of retinal degeneration with partial or complete blindness in the third or fourth decade of life. Despite the RP3 locus on Xp21.1 accounting for 60-90% of XLRP, only 10-20% of identified RPGR mutations were reported in earlier analyses. This discrepancy appeared to be resolved when Vervoort et al. identified a mutational hot spot in a new purine-rich 3' exon (ORF15) that accounted for 60% of their XLRP patients [Vervoort et al., 2000]. In our mutation screening of 37 unrelated European XLRP patients we identified two recently described deletions and 10 novel mutations in exon ORF15 of RPGR, 4 of which were nonsense and 6 frameshift mutations. The latter included one duplication and 5 deletion mutations, all of which lead to a downstream premature termination. No mutations were detected in the additionally screened new exon ORF14. The data reported here, together with previous findings, document a significant clustering of mutations as well as polymorphisms in ORF15 of RPGR. In our unselected XLRP patient population, ORF15 mutations constitute 32% of cases, a finding that contradicts the results of Vervoort and coworkers [Vervoort et al., 2000] but is in agreement with a more recent study on North American XLRP patients [Breuer et al., 2002]. The observed prevalence is sufficient to justify an initial mutation screening of ORF15 in the genetically heterogeneous group of XLRP.