| Co-Investigator(Kenkyū-buntansha) |
KAJIYAMA Hiroshi Tokyo Women's Medical Univ., Medicine, instructor, 医学部, 助手
KOSEKI Yumi Tokyo Women's Medical Univ., Medicine, instructor, 医学部, 助手 (30266837)
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| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2002: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2001: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
Amyloid deposit in patients with AA-amyloidosis is derived from serum amyloid A (SAA). For Japanese patients with rheumatoid arthritis, SAA1.3 (SAA1γ) haplotype was shown to be a risk factor for developing AA-amyloidosis. We have analyzed 5'-flanking region of SAA1 gene and found 3 new SNPs, -61 C/G, -13 C/T, and -2 G/A, in the promotor. We have demonstrated that the -13T is associated with a higher risk of AA-amyloidosis than SAA1γ and is in linkage disequilibrium with SAA1γ.
To address whether -13T/C is related to change in transcriptional activity of SAA1, dual luciferase reporter gene assay with promotor haplotypes of SAA1 gene has performed. In this study, four haplotypes consisted of -61C ;-13C ;-2G, G-C-G, G-C-A, and C-T-G, were amplified, subcloned into pGL-3 vector, and transfected into HepG2 cells. At 12 hours after transfection, the cells were stimulated with IL-1β (10ng/ml) and IL-6 (10ng/ml) and harvested in the lysis. The supernatants were assayed for luciferase activity using a Luminometer. In the luciferase reporter gene assay, we confirmed that the relative luciferase activity of the -13T-containing promoter was higher than that of the -13C-containing promoters. Electrophoretic mobility shift assay using biotinylated SAA1 promoters containing the -13T or -13C, showed that a much greater amount of nuclear proteins were bound to the -13T probe than the -13C probe in extracts from HepG2 cells stimulated with IL-1β and IL-6.
These results indicated that -13T SNP at the SAA1 promoter correlates to the amyloidogenicity as a result of increased transcriptional activity compared to the -13C-containing promoter.
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