DNA tip analysis for the polymorphisms of osteoporosis

为了分析骨质疏松症的基因多态性，我们建立了基于FRET的多区域多态性聚合酶链反应。此外，我们还鉴定了人类维生素D受体基因启动子的多态性。1,25-羟基维生素D3 （1,25(OH)2D3）的主要生理活性是调节小肠内钙的吸收，而维生素D受体（VDR）的水平是这一调节的重要因素。我们证明了尾端相关的同源结构域Cdx-2在人类VDR基因的肠道特异性转录中起重要作用。在本项目中，在VDR基因启动子Cdx-2结合位点的核心序列中发现了多态性。在261例VDR基因型多态性的日本女性中，48例为Cdx-A基因型（腺嘌呤相对于人类VDR基因转录起始位点为-3731 nt）， 82例为Cdx-G基因型（鸟嘌呤为-3731 nt）， 131例为Cdx-A/G基因型（杂合子）。在日本绝经后妇女中，Cdx-G纯合子的腰椎骨密度（BMD）比Cdx-A纯合子低12% （P<0.05）。电泳凝胶迁移迁移实验表明，Cdx-G等位基因的寡核苷酸与Cdx-2的结合明显低于Cdx-A等位基因。带有Cdx-G等位基因的VDR启动子的转录活性降低到Cdx-A等位基因的70%。此外，在单纯疱疹病毒胸苷激酶启动子中，与G等位基因结合的Cdx-2元件的转录活性明显低于A等位基因。因此，VDR基因Cdx-2结合位点的多态性（cdx多态性）会影响VDR在小肠中的表达。此外，这种多态性可能会调节绝经后日本妇女的骨密度。

To analyze the gene polymorphism for osteroprosis, we developed FRET based polymerase chain reaction for multiple region of the polymorphism. In addition, we identified the polymorphism of the human vitamin D receptor gene promoter. The major physiological activity of 1,25-hydroxyvitamin D3 (1,25(OH)2D3) is the regulation of calcium absorption in the small intestine, and the level of vitamin D receptor (VDR) is an important factor in this regulation. We demonstrated that the caudal-related homeodomain Cdx-2 played an important role in the intestine-specific transcription of the human VDR gene. In the present project, the polymorphism was identified in the core sequence in the Cdx-2 binding site in the VDR gene promoter. In 261 Japanese women with genotyped VDR polymorphisms, 48 were genotype Cdx-A (adenine at -3731 nt relative to the transcription start site of human VDR gene), 82 were genotype Cdx-G (guanine at -3731 nt), 131 were genotype Cdx-A/G (heterozygote). In postmenopausal Japanese women, the bone mineral density (BMD) in the lumbar spine (L2-4) with the Cdx-G homozygote was 12% lower than that with the Cdx-A homozygote (P<0.05). In electrophoretic gel mobility shift assay, the oligonucleotide with Cdx-G allele markedly decreased the binding to Cdx-2 compared with that in the Cdx-A allele. The transcriptional activity of the VDR promoter with Cdx-G allele was decreased to 70% of the Cdx-A allele. In addition, in the herpes simplex virus thymidine kinase promoter, the Cdx-2 binding element with the G allele showed significantly lower transcriptional activity than that of the A allele. Thus, the polymorphism in the Cdx-2 binding site of the VDR gene (Cdx-polymorphism) would affect the expression of VDR in the small intestine. In addition, this polymorphism may modulate BMD in postmenopausal Japanese women.