Genetic Trait for Progression of Chronic Renal Disease

慢性肾病进展的遗传特征

• 批准号: 09671181
• 负责人: YOSHIDA Hiroaki
• 金额: $ 1.79万
• 依托单位: The Jikei University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09671181/
• 关键词: ANGIOTENSIN CONVERTING ENZYME; GENE POLYMORPHISM; TRANSCRIPTION; NEGATIVE REGULATORY ELEMENT; QUANTITATIVE TRAIT LOCUS; アンジオテンシン変換酵素; 慢性腎不全関連遺伝子; reporter gene assay; gel-shift assay

Recent studies by us and others have demonstrated that the homozygote of the D allele (DD) of the ACE insertion/deletion (l/D) polymorphism is a potential risk factor for poor prognosis in slowly progressive renal diseases. The ACE I/D polymorphism has been reported to have a significant association with the systemic and local levels of ACE.Recent segregation-linkage analyses suggest that one of quantitative trait loci might be the I/D locus itself or a locus in close proximity to the ACE I/D locus. The I allele of the ACE gene has a 287 bp fragment (insert) within intron l6 of the gene, which is lacking in the D allele. While our sequence analysis has revealed that the insert has a motif highly homologous to negative regulatory element (NRE) of a renin gene (NRE like sequence), the functional significance of the l/D locus remains to be determined. In this study, we tested the functionality of the insert using a reporter gene analysis.A reporter gene firefly luciferase (LUC), which was … More expressed from a truncated SV4O promoter (pLuc) fused to a DNA fragment of the intron 16 with the insert (pLuc-I), was transected into human JEG-3 cells. As a control, the reporter gene fused to a DNA fragment of the intron 16 without the insert (pLuc-D) was used. Cells were co-transected with co-reporter vector having renilla LUC gene with HSV-tk promoter. Cells transected transiently with these vectors were harvest for LUC assay 48 hr after transfection. Measured firefly LUC activity was normalized by renilla LUC activity. Our results demonstrated that luciferase gene expression in cells which transected pLuc-I was significantly lower than that in pLuc-D, indicating that the insert supressed the expression of luciferase reporter gene. Following analyses using gel-shift assay found protein complexes that bind to a oligo nucleotide with the NRE like sequence. We made mutant oligo nucleotides of the NRE like sequence and found a mutant oligo does not bind a protein complex. Then, pLuc-I with the mutation was made by site directed mutagenesis. The effects of the insert on the luciferase reporter gene expression was decreased in the pluc-I with the mutation on the NRE like sequence in the insert.These results therefore indicate that the insert located in the intron 16 of the ACE gene significantly suppresses the expression of the reporter gene. These results are consistent with the possibility that the ACE I/D locus per se has a functional significance for controlling the ACE level. Less

我们和其他人最近的研究表明，ACE 插入/缺失 (l/D) 多态性的 D 等位基因 (DD) 纯合子是缓慢进展性肾病预后不良的潜在危险因素。据报道与 ACE 的系统和局部水平具有显着相关性。最近的分离连锁分析表明数量性状基因座之一可能是 I/D 基因座本身或靠近 ACE 的基因座ACE 基因的 I 等位基因在该基因的内含子 16 内有一个 287 bp 的片段（插入片段），而我们的序列分析表明该插入片段具有与 D 等位基因高度同源的基序。肾素基因的负调控元件（NRE）（NRE 样序列），l/D 基因座的功能意义仍有待确定。在本研究中，我们使用报告基因分析测试了插入物的功能。报告基因。萤火虫荧光素酶 (LUC) 由截短的 SV4O 启动子 (pLuc) 表达，该启动子与带有插入片段 (pLuc-I) 的内含子 16 的 DNA 片段融合，被转染到人 JEG-3 细胞中作为对照。使用与无插入片段的内含子16的DNA片段融合的报告基因(pLuc-D)，将细胞与具有海肾LUC的共报告载体共横切。转染后48小时收获用这些载体瞬时转染的细胞用于LUC测定。我们的结果表明，转染pLuc-I的细胞中的荧光素酶基因表达显着。低于 pLuc-D 中的值，表明插入物抑制了荧光素酶报告基因的表达。使用凝胶迁移测定进行分析后发现蛋白质复合物与寡核苷酸结合。我们制备了NRE样序列的突变寡核苷酸，并发现突变寡核苷酸不结合蛋白质复合物，然后通过定点诱变制备了具有突变的pLuc-I。由于插入片段中NRE样序列的突变，荧光素酶报告基因表达在pluc-I中降低。因此，这些结果表明位于ACE基因内含子16的插入片段显着抑制报告基因的表达。结果与 ACE I/D 基因座本身对于控制 ACE 水平具有功能意义的可能性一致。

项目成果

吉田裕明: "糖尿病性腎症の発症・進展に関わる遺伝因子" 糖尿病. 41. 3-5 (1998)

Hiroaki Yoshida：“糖尿病肾病发生和进展的遗传因素”糖尿病。41. 3-5 (1998)。

Yoshida H: "Polymorphism of renin-angiotensin system genes in progressive IgA nephropathy" Nephrology. 3(supl). S719-S723 (1997)

Yoshida H：“进行性 IgA 肾病中肾素-血管紧张素系统基因的多态性”肾脏病学。

Yoshida H: "Functional significance of the ACE I/D locus for controlling the ACE gene expression (abstract)" J Am Soc Nephrol. 8. 633A (1997)

Yoshida H：“ACE I/D 位点对于控制 ACE 基因表达的功能意义（摘要）”J Am Soc Nephrol。

Yoshida H: "Genetic factor in Diabetic nephropathy. "Pathogenesis in incidence and progression of diabetic nephropathy" (IN JAPANESE)" J Japan Diabetic Soc. 41. 3-5 (1998)

吉田 H：“糖尿病肾病的遗传因素。“糖尿病肾病发病和进展的发病机制”（日语）”日本糖尿病协会杂志。

Yoshida H: "ACE gene Polymorphism and chronic renal diseases." Experimental Nephrology. in press (1998)

Yoshida H：“ACE基因多态性与慢性肾脏疾病。”