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Characterization of Mutations, Including a Novel Regulatory Defect in the First Intron, in Bruton’s Tyrosine Kinase Gene from Seven Korean X-Linked Agammaglobulinemia Families¹

Eun-Kyeong Jo^2,*, Hirokazu Kanegane^¶, Shigeaki Nonoyama^||, Satoshi Tsukada^#, Jae-Ho Lee, Kyu Lim, Minho Shong, Chang-Hwa Song^*, Hwa-Jung Kim^*, Jeong-Kyu Park^* and Toshio Miyawaki^¶

Departments of ^* Microbiology, Pediatrics, Biochemistry, and Internal Medicine, College of Medicine, Chungnam National University, Taejon, Korea; ^¶ Department of Pediatrics, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Toyama, Japan; ^|| Department of Pediatrics, School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan; and ^# Department of Molecular Medicine, Osaka University Medical School, Osaka, Japan

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 +5GA) 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.

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