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

Abstract

The defective gene responsible for the recessively inherited immunodeficiency X-linked agammaglobulinemia (XLA) has been shown to encode a cytoplasmic protein tyrosine kinase of the Src family designated Btk (Bruton's tyrosine kinase). To facilitate the search for germline mutations of the Btk gene, we have characterized its genomic structure. Eighteen introns were positioned within the approximately 37 kb gene. Each of the exon/intron boundaries were defined and sequenced, and all but two conform to consensus sequences. We have utilized the genomic organization of Btk and the intervening sequence data to design an assay for amplifying each of the 19 exons from XLA patient DNA for single strand conformation polymorphism (SSCP) analysis. By using this method we have identified mutations in 12 of 14 unrelated affected males: seven different base substitutions and two small deletions. Two of the mutations described in exon 15 of the kinase domain were found in more than one patient and may represent a mutation hot spot. Exon scanning has proven to be a valuable method for identifying the patient mutations in genomic DNA without the use of cDNA. The mutations are easily confirmed with direct sequencing of the amplified exons. This approach will greatly benefit XLA family studies involving carrier detection and prenatal diagnosis. In addition, the mutations identified may reveal residues involved in the specific protein interactions necessary in the B-cell developmental pathway, of which Btk is an integral component.

Stability and peptide binding specificity of Btk SH2 domain: Molecular basis for X‐linked agammaglobulinemia

“…Mutations in the Btk SH2 domain were found in several patient families with XLA~Bradley et al, 1994;de Weers et al, 1994;Hagemann et al, 1994;Saffran et al, 1994;Vorechovsky et al, 1995;Schuster et al, 1996;Vihinen et al, 1996;Conly et al, 1998!. We expressed several Btk SH2 domains with point mutations identified from XLA patients and measured their binding with the pYEEI peptide~Table 2!.…”

Characterization of Mutations, Including a Novel Regulatory Defect in the First Intron, in Bruton’s Tyrosine Kinase Gene from Seven Korean X-Linked Agammaglobulinemia Families

“…Btk protein is a member of the Tec family of protein tyrosine kinases (Tec kinases), which includes Itk, Tec, Bmx, and Txk. Btk is composed of five distinct structural domains: the pleckstrin homology (PH), Tec homology (TH), Src homology (SH) 3, SH2, and catalytic kinase (SH1) domains (12)(13)(14)(15)(16). In vivo and in vitro studies indicate that Btk protein is essential for B cell survival, cell cycle progression, and proliferation in response to B cell Ag receptor stimulation (17).…”

Characterisation and chromosome mapping of the human non receptor tyrosine kinase gene, brk

“…AT consensus for the 5' and 3' ends of introns (Mount, 1982) (Table 1). As BRK shows the highest degree of sequence similarity to the SRC family of non-receptor PTKs the genomic structure of brk was ®rst compared to that of the src family and then, in addition, to the known genomic structures of two other non-receptor PTK families, csk/matk (Brauninger et al, 1993;Avraham et al, 1995) and btk/txk (Hagemann et al, 1994;Ohta et al, 1994Ohta et al, , 1996, which have similar protein structures. brk has 2 exon boundaries that are conserved with src family members.…”