BTKbase, mutation database for X-linked agammaglobulinemia (XLA).

X-linked agammaglobulinemia (XLA) is an immunodeficiency caused by mutations in the gene coding for Bruton's agammaglobulinemia tyrosine kinase (BTK). A database (BTKbase) of BTK mutations has been compiled and the recent update lists 463 mutation entries from 406 unrelated families showing 303 unique molecular events. In addition to mutations, the database also lists variants or polymorphisms. Each patient is given a unique patient identity number (PIN). Information is included regarding the phenotype including symptoms. Mutations in all the five domains of BTK have been noticed to cause the disease, the most common event being missense mutations. The mutations appear almost uniformly throughout the molecule and frequently affect CpG sites that code for arginine residues. The putative structural implications of all the missense mutations are given in the database. The improved version of the registry having a number of new features is available at http://www. helsinki.fi/science/signal/btkbase.html     

BTKbase, mutation database for X-linked agammaglobulinemia (XLA).

X-linked agammaglobulinemia (XLA) is an immunodeficiency caused by mutations in the gene coding for Bruton's agammaglobulinemia tyrosine kinase (BTK). A database (BTKbase) of BTK mutations has been compiled and the recent update lists 225 entries from 189 unrelated families showing 148 unique molecular events. Each patient is given a unique patient identity number (PIN). Information is included regarding the phenotype including symptoms. Mutations in all the five domains of BTK have been noticed to cause the disease, the most common event being missense mutations. The mutations appear almost uniformly throughout the molecule and frequently affect CpG sites forming arginine residues. A decreased frequency of missense mutations was found in the TH, SH3 and upper lobe of the kinase domain. The putative structural implications of all the missense mutations are given in the database.     

X-linked agammaglobulinemia (XLA): A genetic tyrosine kinase (Btk) disease

X-linked agammaglobulinemia is a heritable immunodeficiency disease caused by a differentiation abnormality, resulting in the virtual absence of B Iymphocytes and plasma cells. The affected gene encodes a cytoplasmic protein tyrosine kinase, Bruton's agammaglobulinemia tyrosine kinase, designated Btk. Btk and the other family members, Tec, Itk and Bmx, contain five regions, four of which are common structural and functional modules that are found in other signaling proteins. Mutations affect all domains of the gene, but amino acid substitutions seem to be confined to certain regions. More than 150 unique mutations have been identified and are collected in a mutation database, BTKbase. Here we discuss the three-dimensional structural implications of such mutations and their putative functional role. Of special interest are mutations affecting the pleckstrin homology domain, as Btk is the only disease-associated protein so far reported to carry mutations in this particular module.     

Toll-like receptors pathway in common variable immune deficiency (CVID) and X-linked agammaglobulinemia (XLA)

Common variable immunodeficiency (CVID) and X-linked agammaglobulinemia (XLA) are two major humoral immunodeficiencies, causing a high rate of early age mortality in children. In order to identifiy the possible factors involved in the pathogenesis of CVID and XLA, recent studies have focused on Toll-like receptors (TLRs) and demonstrate the defects in different TLR pathways in immune cells of CVID and XLA patients. Herein, we measured TLR-4 and TLR-9 RNA levels and consequently TNF-α and IFN-α production in peripheral blood mononuclear cells (PBMCs) of patients with CVID and XLA. Contrary to healthy individuals, TLR-9 expression was not significantly increased after ligand stimulation, whereas ligand-induced TLR-4 expression was not significantly different from that in healthy control PBMCs. Lipopolysaccharide (LPS)-stimulated TNF-α production was significantly reduced in patients compared to controls, whereas IFN-α production was increased in all groups after CpG stimulation without any significant inter-group difference. Our data suggest that defects in TLR-9 activated pathways may be a result of the decreased TLR-9 expression, although TLR-9 is not the only modulator of IFN-α production in these patients. On the other hand, impaired signaling in TLR-4 activated pathways which results in significant reduction in TNF-α production are not related to a defect in TLR-4 expression.     

X-chromosome inactivation and mutation pattern in the Bruton's tyrosine kinase gene in patients with X-linked agammaglobulinemia. Italian XLA Collaborative Group

BACKGROUND: The diagnosis of X-linked agammaglobulinemia (XLA) is not always clearcut. Not all XLA conform to the classic phenotype and less than 50% of affected boys have a family history of immunodeficiency. Mutations in the gene for Bruton's tyrosine kinase (BTK) are responsible for the majority of agammaglobulinemia cases. However, a certain proportion of patients may have mutations involving other genes, although they show with an XLA phenotype. We performed BTK gene mutation analysis in 37 males with presumed XLA and analyzed the pattern of X-chromosome inactivation (XCI) in 31 mothers to evaluate the relevance of these approaches to diagnosis and genetic counseling. MATERIALS AND METHODS: Twenty affected males with a sporadic occurrence and 17 familial cases belonging to nine families were enrolled within the framework of the Italian Multicenter Clinical Study on XLA. We used non-isotopic RNase cleavage assay (NIRCA), followed by cDNA sequence determination to screen for BTK mutations and X-chromosome inactivation analysis for carrier detection. RESULTS: Using the cDNA-based approach, the identification of BTK gene abnormalities confirmed the clinical diagnosis of XLA in 31 of 37 affected infants. Missense was the most frequent mutational event and the kinase domain was mostly involved. In addition, nine novel mutations were identified. In sporadic cases, BTK gene abnormalities were identified in 9 out of 10 patients whose mothers had a nonrandom pattern of XCI and in 5 out of 6 patients whose mother had a random pattern of XCI. With the exception of one family, all patients with a familial occurrence and born to mothers with a nonrandom pattern of XCI had mutations of the BTK gene. CONCLUSIONS: Our findings indicate that in sporadic cases BTK gene sequencing is the only reliable tool for a definitive diagnosis of XLA and support XCI as the first diagnostic tool in the mothers of affected males in multiple generations. Furthermore, our molecular analysis confirms that 10-20% of BTK-unaltered patients have disorders caused by defects in other genes.     

Close linkage of probe p212 (DXS178) to X-linked agammaglobulinemia

Summary Segregation analysis was performed in three families affected in X-linked agammaglobulinemia (XLA) with five polymorphic DNA probes linked to the disease locus. In agreement with previous studies, no recombination was observed with either pXG12 (DXS94) or S21 (DXS17). Segregation analysis was also performed with a marker, p212 (DXS178), which has been shown to be closely linked to pXG12 in normal families. No cross-over with XLA was observed in these three families and in five additional families previously analyzed with DXS17 and DXS94 (z = 5.92 at = 0). These data provide evidence against genetic heterogeneity in XLA and indicate the value of probe p212 for carrier detection and prenatal diagnosis of XLA. We were able to estimate the carrier status of six females (out of six) in the three previously unreported families.     

Evidence for male X chromosomal mosaicism in X-linked agammaglobulinemia

Summary X-Linked agammaglobulinemia (XLA) is a severe antibody deficiency disease in man, resulting from an arrest in differentiation of pre-B cells. XLA is recessive: female carriers do not exhibit antibody deficiency, but manifest an exclusive inactivation of the XLA-carrying X chromosome in all peripheral blood B lymphocytes. An exclusive inactivation of the paternal X chromosome in the B lymphocytes of all daugthers thers of a male who had no agammalobulineamia demonstrated that the XLA defect can originate from healthy males. These males are X chromosomal mosaics. X-Chromosomal RFLP segregation analyses in other XLA pedigrees suggest a frequent introduction of XLA by healthy males. This implies that XLA often originates from mitotic errors, either at postmeiotic or early postzygotic stages.     

Application of carrier testing to genetic counseling for X-linked agammaglobulinemia.

Bruton X-linked agammaglobulinemia (XLA) is a phenotypically recessive genetic disorder of B lymphocyte development. Female carriers of XLA, although asymptomatic, have a characteristic B cell lineage-specific skewing of the pattern of X inactivation. Skewing apparently results from defective growth and maturation of B cell precursors bearing a mutant active X chromosome. In this study, carrier status was tested in 58 women from 22 families referred with a history of agammaglobulinemia. Primary carrier analysis to examine patterns of X inactivation in CD19+ peripheral blood cells (B lymphocytes) was conducted using quantitative PCR at the androgen-receptor locus. Obligate carriers of XLA demonstrated > 95% skewing of X inactivation in peripheral blood CD19+ cells but not in CD19- cells. Carrier status for mothers of isolated affected males could be assessed in 10 of 11 families: 7 women showed skewing, and 3 did not. Five carriers were found in six families in which there were no living affected males. Among all those tested, one individual's carrier status was considered to be indeterminate and five women were noninformative for the carrier test. Results obtained by the carrier test were congruent with linkage analysis (where applicable) using the RFLPs DXS178 and DXS94 and two newly developed polymorphic microsatellite markers, DXS178CA and DXS101AAT. Refinements in techniques for primary carrier testing and genetic mapping of XLA now make possible an ordered approach to diagnosis, prenatal diagnosis, and genetic counseling.     

The genomic structure of human BTK,the defective gene in X-linked agammaglobulinemia

It has recently been demonstrated that mutations in the gene for Bruton's tyrosine kinase (BTK) are responsible for X-linked agammaglobulinemia. Southern blot analysis and sequencing of cDNA were used to document deletions, insertions, and single base pair substitutions. To facilitate analysis ofBTK regulation and to permit the development of assays that could be used to screen genomic DNA for mutations inBTK, we determined the genomic organization of this gene. Subcloning of a cosmid and a yeast artificial chromosome showed thatBTK is divided into 19 exons spanning 37 kilobases of genomic DNA. Analysis of the region 5' to the first untranslated exon revealed no consensus TATAA or CAAT boxes; however, three retinoic acid binding sites were identified in this region. Comparison of the structure ofBTK with that of other nonreceptor tyrosine kinases, includingSRC,FES, andCSK, demonstrated a lack of conservation of exon borders. Information obtained in this study will contribute to our understanding of the evolution of nonreceptor tyrosine kinases. It will also be useful in diagnostic studies, including carrier detection, and in studies directed towards gene therapy or gene replacement.     

A novel mutation (Cys145-->Stop) in Bruton's tyrosine kinase is associated with newly diagnosed X-linked agammaglobulinemia in a 51-year-old male.

BACKGROUND: X-linked agammaglobulinemia (XLA) is a severe, life-threatening disease characterized by failure of B cell differentiation and antibody production and is associated with mutations in Bruton's tyrosine kinase (Btk). The proband in this study is a 51-year-old male presenting with chronic nasal congestion, recurrent sinusitis, sporadic pneumonia, and pronounced B cell deficiency. A family history suggestive of an X-linked immunodeficiency disease was noted. MATERIALS AND METHODS: cDNA was synthesized from mRNA prepared from peripheral blood mononuclear leukocytes. Btk cDNA amplified by polymerase chain reaction (PCR) was subjected to both manual and automated DNA sequencing. A DNA sequence corresponding to exons 6 and 7 of Btk was amplified from genomic DNA. Western blot analysis employed both polyclonal and monoclonal antibodies to Btk and reaction patterns were obtained both by chemiluminescence and an in vitro kinase assay. RESULTS: A mutation (Cys145-->Stop) was identified in Btk cDNA and was confirmed in amplified exon 6 of genomic DNA from both the proband and an affected nephew. Neither Btk nor a truncated peptide was detected in Western blot analyses of peripheral blood mononuclear cell lysates. CONCLUSIONS: The C145A mutation reported here is novel. This family study is extraordinary in that affected male members who did not undergo aggressive medical management either succumbed to complications in early life or survived into later life. The proband is the oldest de novo diagnosed patient with XLA reported to date.     

Diagnostic DNA testing for X-linked ocular albinism (OA1) with a hierarchical mutation screening protocol

Albinism is a group of inherited conditions in which affected individuals have less than normal pigment in the eyes, skin, and hair compared to others of the same race and ethnic background. The prevalence of all types of albinism in the United States is estimated at 1 in 20,000, based on poor epidemiological data. X-linked Nettleship-Falls ocular albinism (XLOA, OA1) affects approximately 1/150,000 males in the population. XLOA effects reduce visual acuity and nystagmus, result in a mild skin and hair phenotype, and occur mostly in XY males. Female carriers of XLOA have normal visual acuity, but often show iris punctate transillumination and a classic pattern of mosaic retinal pigmentation, coarse and grainy in the macula and becoming increasingly reticular into the periphery of the retinal pigment epithelium. Studies of OA1 have shown linkage of a single gene to markers at Xp22.3-p22.2. About 48% of the reported mutations in the OA1 gene are intragenic deletions and about 43% are point mutations. We present a hierarchical strategy for mutation screening for diagnostic testing for OA1 that comprises two tiers: first, multiplex PCR to detect intragenic deletions in the OA1 gene with denaturing high-performance liquid chromatography (dHPLC), and, second, heteroduplex analysis with dHPLC to scan for mutations, with subsequent sequencing of variants to confirm putative mutations in the OA1 gene. Prenatal diagnosis can be provided for families when the mutation has been firmly identified. We have validated this procedure with positive controls that were identified in patients by Southern blot, single-stranded conformation polymorphism (SSCP), and sequencing. In this hierarchical strategy, these procedures have an analytical sensitivity of > 99%.     

Ecto-5'-nucleotidase activity in lymphoblastoid cell lines derived from heterozygotes for congenital X-linked agammaglobulinemia

Lymphoblastoid cell lines were established from female relatives of patients with congenital X-linked-agammaglobulinemia by Epstein-Barr virus transformation of their peripheral B lymphocytes. Cell lines derived from presumed carriers were characterized by low ecto-5'-nucleotidase activity and a reduced percentage of surface immunoglobulin-bearing cells. Measurement of ecto-5'-nucleotidase activity in newly established lymphoblastoid cell lines may provide a means for the identification of heterozygotes for congenital X-linked agammaglobulinemia.     

Identification of a closely linked DNA marker, DXS178, to further refine the X-linked agammaglobulinemia locus

X-linked agammaglobulinemia (XLA) is an inherited recessive disorder in which the primary defect is not known and the gene product has yet to be identified. Utilizing genetic linkage analysis, we previously localized the XLA gene to the map region of Xq21.3-Xq22 with DNA markers DXS3 and DXS17. In this study, further mapping was performed with two additional DNA probes, DXS94 and DXS178, by means of multipoint analysis of 20 families in which XLA is segregating. Thirteen of these families had been previously analyzed with DXS3 and DXS17. Three crossovers were detected with DXS94 and no recombinations were found between DXS178 and the XLA locus in 9 informative families. Our results show that XLA is closely linked to DXS178 with a two-point lod score of 4.82 and a multipoint lod score of 10.24. Thus, the most likely gene order is DXS3-(XLA,DXS178)-DXS94-DXS17, with the confidence interval for location of XLA lying entirely between DXS3 and DXS94. In 2 of these families, we identified recombinants with DXS17, a locus with which recombination had not previously been detected by others in as many as 40 meiotic events. Furthermore, DXS178 is informative in both of these families and does not show recombination with the disease locus. Therefore, our results indicate that DXS178 is linked tightly to the XLA gene.     

Stability and peptide binding specificity of Btk SH2 domain: molecular basis for X-linked agammaglobulinemia

X-linked agammaglobulinemia (XLA) is caused by mutations in the Bruton's tyrosine kinase (Btk). The absence of functional Btk leads to failure of B-cell development that incapacitates antibody production in XLA patients leading to recurrent bacterial infections. Btk SH2 domain is essential for phospholipase C-gamma phosphorylation, and mutations in this domain were shown to cause XLA. Recently, the B-cell linker protein (BLNK) was found to interact with the SH2 domain of Btk, and this association is required for the activation of phospholipase C-gamma. However, the molecular basis for the interaction between the Btk SH2 domain and BLNK and the cause of XLA remain unclear. To understand the role of Btk in B-cell development, we have determined the stability and peptide binding affinity of the Btk SH2 domain. Our results indicate that both the structure and stability of Btk SH2 domain closely resemble with other SH2 domains, and it binds with phosphopeptides in the order pYEEI > pYDEP > pYMEM > pYLDL > pYIIP. We expressed the R288Q, R288W, L295P, R307G, R307T, Y334S, Y361C, L369F, and 1370M mutants of the Btk SH2 domain identified from XLA patients and measured their binding affinity with the phosphopeptides. Our studies revealed that mutation of R288 and R307 located in the phosphotyrosine binding site resulted in a more than 200-fold decrease in the peptide binding compared to L295, Y334, Y361, L369, and 1370 mutations in the pY + 3 hydrophobic binding pocket (approximately 3- to 17-folds). Furthermore, mutation of the Tyr residue at the betaD5 position reverses the binding order of Btk SH2 domain to pYIIP > pYLDL > pYDEP > pYMEM > pYEEI. This altered binding behavior of mutant Btk SH2 domain likely leads to XLA.     

Mutations of the Btk gene in 12 unrelated families with X-linked agammaglobulinemia in Japan

Alterations of the Bruton's tyrosine kinase(Btk) gene are responsible for X-linked agammaglobulinemia (XLA). Although mutations in various regions were reported mainly in the Caucasian population, correlation between the locations of mutation and the clinical phenotypes remains unclear. We report 12 abnormalities of theBtk gene found in 12 unrelated families out of 14 XLA families in Japan and their clinical features. We utilized Southern blotting and single-strand conformation polymorphism (SSCP) analysis. Gene rearrangement in the kinase domain was identified in two patients by Southern blotting. Seven point mutations, two small deletions, and one small insertion were detected by SSCP and sequencing. The SSCP analysis also provided information about the carriers in these families. We found some clinical heterogeneity in the affected family members with the same gene mutation. Moreover, there is considerable inconsistency between the locations of gene aberrations and the immunological phenotypes. Some patients with a nonsense mutation, which may result in the lack of kinase domain, have detectable B cells and immunoglobulins. These identified alterations will provide valuable clues to theBtk protein function and the pathogenesis of XLA.