Diagnosis of osteoporosis by analysis of vitamin D receptor gene

维生素D受体基因分析诊断骨质疏松症

基本信息

批准号：
09672365
负责人：
ARAKAWA Hidetoshi
金额：
$ 1.86万
依托单位：
Showa University
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 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09672365/
关键词：
Osteoporosis Vitamin D receptor ELISA Bioluminescence SSCP Capillary electrophoresis RFLP DNA polymorphism

项目摘要

Osteoporosis is a major public health problem which affects quality of life and raises costs for health care providers. Although an osteoporotic fracture is caused by multiple factors, the risk of osteoporotic fracture in later life is determined by the peak bone density achieved in early adulthood in relation to age and menopause-related bone loss. It is reported that common allelic variation in the vitamin D receptor locus (VDR) can be used to predict bone turnover and bone mineral density. Japanese premenopausal women, homozygous subjects in whom the ApaI in intron 8 and TaqI site in exon 9 is present (AA,tt) were found to have lower BMD than those with the (aa, TT) genotype respectively. Analysis of VDR gene polymorphism is generally done by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and single strand DNA confirmational polymorphism. These traditional techniques are not suitable for routine clinical analysis because of problems of rapidity, reproducibility and use of electrophoresis gel separation (time consuming). Recently, capillary electrophoresis (CE) is used for analysis of RFLP and single strand DNA conformation polymorphism technique (SSCP). Further, enzyme linked immunosorbent assays (ELISA) combined with RFLP and hybridization using a DNA probe are used for analysis of PCR products. These methods have enabled the fast detection and quantification of PCR products. At present, a more sensitive and simpler method for DNA diagnosis is required. In the research reported here, we developed a sensitive and rapid PCR-RFLP ELISA using acetate kinase (AK) and firefly luciferase as a detection system and SSCP analysis by CE with laser-induced fluorescence detection for VDR plymorphism analysis. DNA polymorphism types of VDR could be clearly determined by these methods. These system is suitable for determining simultaneously DNA polymorphisms of a large number of samples.

骨质疏松症是一个主要的公共卫生问题，它会影响生活质量并提高医疗保健提供者的成本。尽管骨质疏松性骨折是由多种因素引起的，但以后生命中骨质疏松性骨折的风险取决于成年早期与年龄和更年期相关的骨质流失相关的峰值骨密度。据报道，维生素D受体基因座（VDR）中常见的等位基因变异可用于预测骨骼更新和骨矿物质密度。日本的绝经前妇女，纯合的受试者，其中内含子8和外显子中的Taqi位点（AA，TT）的BMD分别低于（AA，TT）基因型的BMD。 VDR基因多态性的分析通常是通过聚合酶链反应限制片段长度多态性（PCR-RFLP）和单链DNA DNA确认多态性来完成的。这些传统技术不适合常规临床分析，因为快速性，可重复性和电泳凝胶分离的使用（耗时）。最近，毛细管电泳（CE）用于分析RFLP和单链DNA构象多态性技术（SSCP）。此外，将酶连接的免疫吸附测定法（ELISA）与RFLP结合使用，并使用DNA探针进行杂交用于分析PCR产物。这些方法使PCR产品的快速检测和量化。目前，需要一种更灵敏，更简单的DNA诊断方法。在此处报道的研究中，我们使用乙酸激酶（AK）和萤火虫荧光素酶作为检测系统开发了一种敏感的PCR-RFLP ELISA，并通过激光诱导的荧光检测进行了VDR plymorphismismiss分析，作为检测系统和SSCP分析。 VDR的DNA多态性类型可以由这些方法明确确定。这些系统适合确定大量样品的同时DNA多态性。