title: 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.
abstract: In this report, we describe seven mutations, including a novel single base pair substitution in intron 1, of the Bruton's tyrosine kinase (Btk) gene found in 12 Korean patients with X-linked agammaglobulinemia. Various mutations, including three novel genetic alterations, were discovered using single-strand conformation polymorphism analysis and direct DNA sequencing. The effect of the intron 1 point mutation (intron 1 +5G-->A) was further evaluated using reporter constructs. Using luciferase assay experiments, we showed that the transcriptional activity of the mutant was significantly lower than in normal counterparts, indicating that the intronic mutation was functional. In addition, DNase I footprinting analysis showed that a single protected region spanning the position +3 to +15 bp hybridized with a mutant-specific probe, but not with a wild-type probe. EMSA indicated that a distinct nuclear protein has the ability to bind the mutant oligonucleotides to produce a new DNA-protein complex. We also observed decreased expression of Btk proteins in monocytes of patients having the point mutation in intron 1. Taken together with the functional analysis, our results strongly suggest the existence of a novel cis-acting element, which might be involved in the down-regulation of Btk gene transcription. Precise definition of the regulatory defect in the Btk intron 1 may provide valuable clues toward elucidating the pathogenesis of X-linked agammaglobulinemia.
authors.affiliation: Department of Microbiology, College of Medicine, Chungnam National University, 6 Munhwa-dong, Chung-ku, Taejon 301-131, Korea. hayoungj@cnu.ac.kr
authors.forename: E K
authors.initials: EK
authors.lastname: Jo
authors.forename: H
authors.initials: H
authors.lastname: Kanegane
authors.forename: S
authors.initials: S
authors.lastname: Nonoyama
authors.forename: S
authors.initials: S
authors.lastname: Tsukada
authors.forename: J H
authors.initials: JH
authors.lastname: Lee
authors.forename: K
authors.initials: K
authors.lastname: Lim
authors.forename: M
authors.initials: M
authors.lastname: Shong
authors.forename: C H
authors.initials: CH
authors.lastname: Song
authors.forename: H J
authors.initials: HJ
authors.lastname: Kim
authors.forename: J K
authors.initials: JK
authors.lastname: Park
authors.forename: T
authors.initials: T
authors.lastname: Miyawaki
mesh_terms: D000293:Adolescent; D000328:Adult; D000077329:Agammaglobulinaemia Tyrosine Kinase; D000361:Agammaglobulinemia / Q000175:diagnosis / Q000235:genetics*; D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000737:chemistry; D005434:Flow Cytometry; D017930:Genes, Reporter; D008040:Genetic Linkage; D006801:Humans; D007223:Infant; D007438:Introns; D007723:Korea; D008297:Male; D009000:Monocytes / Q000378:metabolism; D009154:Mutation; D010375:Pedigree; D017354:Point Mutation; D011505:Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014960:X Chromosome
chemical_list: D004268:DNA-Binding Proteins; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; C000625949:BTK protein, human; D003850:Deoxyribonuclease I