Diagnosis of osteoporosis by analysis of vitamin D receptor gene

骨质疏松症是一个主要的公共卫生问题，它影响生活质量，增加医疗保健提供者的费用。虽然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是由成年早期达到的骨密度峰值决定的，这与年龄和更年期相关的骨质流失有关。据报道，维生素D受体位点（VDR）的常见等位基因变异可用于预测骨转换和骨矿物质密度。日本绝经前妇女，8内含子ApaI和9外显子TaqI位点存在的纯合子受试者（AA,tt）的骨密度分别低于基因型（AA,tt）的受试者。VDR基因多态性分析一般采用聚合酶链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。这些传统的技术由于快速、重复性和使用电泳凝胶分离（耗时）等问题而不适合常规临床分析。近年来，毛细管电泳（CE）和单链DNA构象多态性技术（SSCP）被用于RFLP分析。此外，酶联免疫吸附法（ELISA）结合RFLP和DNA探针杂交用于PCR产物的分析。这些方法实现了PCR产物的快速检测和定量。目前需要一种更灵敏、更简单的DNA诊断方法。在本文报道的研究中，我们以醋酸激酶（AK）和萤火虫荧光素酶为检测系统，建立了一种灵敏、快速的PCR-RFLP酶联免疫吸附试验，采用CE和激光诱导荧光检测进行SSCP分析，用于VDR多态性分析。通过这些方法可以清楚地确定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。

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.