Development and application of DNA tip for the diagnosis of atherogenic hypertriglyceridemia

DNA探针诊断动脉粥样硬化性高甘油三酯血症的开发及应用

• 批准号: 12670384
• 负责人: IKEDA Yasuyuki
• 金额: $ 2.43万
• 依托单位: NATIONAL CARDIOVASCULAR CENTER RESEARCH INSTITUTE
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670384/
• 关键词: Lipoprotein lipase; Hypertriglyceridemia; type IV hyperlipoproteinemia; Insulin resistance; Heart disease; Gene analysis; 高トリグリセライド血症

Mild hypertriglyceridemia (type IV hyperlipoproteinemia) is frequently identified in Japanese and is thought to be one of risk factors for heart disease. Mild hypertriglyceridemia results from superimposition of environmental factors, such as high alcohol intake and a hyperinsulinemic state, on a genetic state of a heterozygous mutation in the lipoprotein lipase (LPL) gene. Identification of heterozygote LPL gene mutations as an early determination of etiology underlying mild hypertriglyceridemia, therefore, is important for preventing the development of hypertriglyceridemia and subsequent development of heart disease by getting the patient to change to a more healthful lifestyle. Identification and collection of various LPL gene mutations are essential for the development ofDNA diagnostic tip for analysis ofLPL gene mutations. Results obtained at this (Research Project are listed bellow.(1) We developed an ELISA for the quantification of human LPL mass using two kinds of monoclonal an … More tibodies raised against human LPL, and improved method for direct DNA sequencing of the human LPL gene using an Auto DNA sequencer.(2) We newly identified LPL gene mutations, such as G105R, F270L, G154V and a T-to-C transition in the invariant GT at position +2 of the 5' donor splice site (5'-dss) ofintron 8 (Int8/5'-dss/t (+2) c) from Japanese subjects with mild hypertriglyceridemia. Also, we identified a mutation of D204E reported elsewhere. In missense mutations ofG105R, F270L, G154V and D204E, all mutants LPL expressed in COS-1 cells were catalytically inactive. Subjects with heterozygous LPL deficiency (carriers) are prone to develop type IV hyperlipoproteinemia when complicated with factors such as a hyperinsulinemic state and/or a high alcohol intake, which stimulate triglyceride synthesis in the liver, while carriers are normolipidemic provided that they do not have factors leading to hypertriglyceridemia.(3) So far, we have collected 15 mutations of the LPL gene by our Research projects including this project. Among 15 mutations, we have succeeded in identifying five mutations of Try-61-Stop, Asp-204-Glu, Ala-221-del, Ala-261 -Thr and Trp-382-Stop using Invader assay system (DNA diagnostic chip). Less

轻度高脂血症（IV型高脂蛋白血症）在日本经常被发现，被认为是心脏病的危险因素之一。轻度高脂血症是由于环境因素（如高酒精摄入和高胰岛素血症状态）与脂蛋白脂酶（LPL）基因杂合突变的遗传状态叠加所致。因此，鉴定杂合子LPL基因突变作为早期确定轻度高脂血症病因的基础，对于通过使患者改变为更健康的生活方式来预防高脂血症的发展和随后的心脏病的发展是重要的。LPL基因突变的鉴定和收集是研制LPL基因突变DNA诊断探针的基础。本研究项目取得的成果如下。(1)我们用两种单克隆抗体建立了一种定量人LPL质量的ELISA方法， ...更多信息 针对人LPL产生的抗体，以及使用自动DNA测序仪对人LPL基因进行直接DNA测序的改进方法。(2)我们在日本轻度高脂血症患者中发现了LPL基因突变，如G105 R、F270 L、G154 V和内含子8 5 '-dss/t（+2）c的+2位不变GT的T → C转换。此外，我们还发现了一个D204 E突变。在G105 R、F270 L、G154 V和D204 E的错义突变体中，在COS-1细胞中表达的所有突变体LPL均无催化活性。杂合型LPL缺乏的受试者（携带者）在并发高胰岛素血症状态和/或高酒精摄入等因素时易于发生IV型高脂蛋白血症，这些因素刺激肝脏中的甘油三酯合成，而携带者是正常脂血症的，前提是他们没有导致高脂蛋白血症的因素。(3)到目前为止，我们已经收集了15个LPL基因的突变，我们的研究项目，包括本项目。在这15个突变中，我们成功地鉴定了Try-61-Stop，Asp-204-Glu，Ala-221-del，Ala-261 -Thr和Trp-382-Stop这5个突变。少

项目成果

A. Mori: "Improved method for direct DNA sequencing of the human lipoprotein lipase gene using an auto DNA sequencer"Clinical Biochemistry. 33. 323-327 (2000)

A. Mori：“使用自动 DNA 测序仪对人脂蛋白脂肪酶基因进行直接 DNA 测序的改进方法”《临床生物化学》。

M. Nishimura: "Development and evaluation of a direct sandwich enzymelinked immunosorbent assay for the quantificaiton of human hepatic triglyceride lipase mass in human plasma"J. Immunological Methods. 235. 41-51 (2000)

M. Nishimura：“用于定量人血浆中人肝甘油三酯脂肪酶质量的直接夹心酶联免疫吸附测定法的开发和评估”J。

池田 康行: "Hypercholesterolemia, familial"先天異常症候群辞典(上巻). 59. 828-831 (2001)

Yasuyuki Ikeda：“家族性高胆固醇血症”先天性异常综合症词典（第 1 卷）。 828-831 (2001)。

A. Takagi: "A newly identified lipoprotein lipase (LPL) gene mutationj (F270L) in a Japanese patient with familial LPL deficiency"Biochim. Biophys. Acta. 1502. 433-446 (2000)

A. Takagi：“在一名患有家族性 LPL 缺乏症的日本患者中新发现的脂蛋白脂肪酶 (LPL) 基因突变 j (F270L)”Biochim。

Y.Ikeda: "A family-based study of hyperinsulinemia and hypertriglyceridemia in heterozygous lipoprotein lipase deficiency"Clinica Chimica Acta. 316. 179-185 (2002)

Y.Ikeda：“杂合脂蛋白脂肪酶缺乏症中高胰岛素血症和高甘油三酯血症的家庭研究”《临床化学学报》。