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

轻度高甘油三酯血症(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。轻度高甘油三酯血症是由于环境因素的叠加，如高酒精摄入量和高胰岛素状态，导致脂蛋白脂酶(LPL)基因杂合突变的遗传状态。因此，识别LPL杂合子基因突变作为早期确定轻度高甘油三酯血症的病因，对于通过让患者改变更健康的生活方式来预防高甘油三酯血症的发展和随后的心脏病发展具有重要意义。鉴定和收集各种LPL基因突变对于开发LPL基因突变分析的DNA诊断提示是必不可少的。本研究的结果如下：(1)我们建立了用两种单抗和…定量测定人LPL质量的ELISA法(2)我们在日本轻度高甘油三酯血症患者中发现了LPL基因的突变，如G105R、F270L、G154V，以及8号外显子5‘-DSS(Int8/5’-Dss/t(+2)c)5‘供体剪接点+2位不变GT的T-C转换。此外，我们还发现了其他地方报道的D204E突变。在G105R、F270L、G154V和D204E错义突变中，在COS-1细胞中表达的所有突变体LPL都是催化失活的。杂合子LPL缺乏的受试者(携带者)在合并高胰岛素状态和/或高酒精摄入等刺激肝脏甘油三酯合成的因素时，容易发生IV型高脂蛋白血症，而携带者只要没有导致高甘油三酯血症的因素，就是正常血脂。(3)到目前为止，我们通过包括本项目在内的研究项目收集了LPL基因的15个突变。在15个突变中，我们利用入侵者检测系统(DNA诊断芯片)成功地鉴定了5个突变：Try-61-Stop、Asp-204-Glu、Ala-221-del、Ala-261-Thr和Trp-382-Stop。较少

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