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，影响生活质量并增加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，但晚年骨质疏松性骨折的风险取决于成年早期达到的峰值骨密度与年龄和绝经期相关骨丢失的关系。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转换和骨矿物质密度。日本绝经前女性，内含子 8 中存在 ApaI 和外显子 9 中存在 TaqI 位点（AA，tt）的纯合受试者的 BMD 分别低于具有（aa，TT）基因型的受试者。 VDR基因多态性分析一般采用聚合酶链式反应限制性片段长度多态性(PCR-RFLP)和单链DNA确认多态性进行。由于快速性、重现性和使用电泳凝胶分离（耗时）等问题，这些传统技术不适合常规临床分析。最近，毛细管电泳（CE）被用于RFLP和单链DNA构象多态性技术（SSCP）的分析。此外，酶联免疫吸附测定（ELISA）与 RFLP 和使用 DNA 探针的杂交相结合，用于 PCR 产物的分析。这些方法使得 PCR 产物的快速检测和定量成为可能。目前，需要一种更灵敏、更简单的DNA诊断方法。在此报告的研究中，我们开发了一种灵敏、快速的 PCR-RFLP ELISA，使用醋酸激酶 (AK) 和萤火虫荧光素酶作为检测系统，并通过 CE 进行 SSCP 分析，并结合激光诱导荧光检测进行 VDR 多态性分析。通过这些方法可以明确判断VDR的DNA多态性类型。该系统适用于同时测定大量样品的DNA多态性。