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

我们等人最近的研究表明，血管紧张素转换酶基因插入/缺失(L/D)多态性的D等位基因(DD)纯合子是慢性进展性肾病预后不良的潜在危险因素。ACEI/D多态与ACEs的系统和局部水平密切相关。最近的分离-连锁分析表明，一个数量性状座位可能是该I/D基因座本身或与ACEI/D基因座非常接近的一个基因座。血管紧张素转换酶基因的I等位基因在该基因的第16内含子内有一个287个碱基的片段(插入)，这是D等位基因所缺少的。虽然我们的序列分析显示该插入片段与肾素基因的负调控元件(NRE)具有高度同源性的基序(NRE类似序列)，但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来测试插入的功能。报告基因萤火虫荧光素酶(Luc)，即…将截短的SV4O启动子(PLuc)通过插入片段(pLuc-I)与内含子16的DNA片段融合后，更多地表达于人JEG-3细胞。作为对照，使用报告基因与内含子16的DNA片段融合而不插入(pLuc-D)。细胞与带有HSV-tk启动子的肾区LUC基因的共报告载体共横切。用这些载体瞬时横切的细胞在48小时后收集细胞进行LUC检测。测得的萤火虫LUC活动用肾LUC活动归一化。我们的结果表明，在pLuc-I中，荧光素酶基因的表达显著低于pLuc-D中的荧光素酶基因的表达，表明该插入片段抑制了荧光素酶报告基因的表达。在使用凝胶移位分析之后，发现了与具有NRE样序列的寡核苷酸结合的蛋白质复合体。我们制作了NRE样序列的突变寡核苷酸，发现突变的寡核苷酸不与蛋白质复合体结合。然后，用定点诱变的方法对突变后的pLuc-I进行突变。随着插入片段中NRE类序列的突变，插入片段对荧光素酶报告基因表达的影响在Pluc-I中降低，这表明位于ACE基因第16内含子的插入片段显著抑制了报告基因的表达。这些结果与ACE I/D位点本身对控制ACE水平具有功能意义的可能性是一致的。较少

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: "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。”

国枝武彦: "糖尿病性腎症-遺伝因子-" 医学のあゆみ. 184:. 847-850 (1998)

Takehiko Kunieda：“糖尿病肾病 - 遗传因素”医学史 184：847-850（1998）。