DNA Laboratory, Department of Applied Biomedical Science, Faculty of Health Sciences Department of Pathology, Faculty of Medicine and Surgery, University of Malta, Msida, Malta.
J Mol Endocrinol. 2011 Nov 21;47(3):327-33. doi: 10.1530/JME-11-0067. Print 2011 Dec.
Polymorphisms within the TNFRSF11B gene have been studied and associated with osteoporosis and fracture risk. Osteoprotegerin (OPG), the product of this gene, is a key negative regulator of osteoclastogenesis and is secreted by osteoblasts/stromal cells. A previous study in Maltese postmenopausal women showed positive association of low bone mineral density (BMD) with a polymorphism found within the promoter region of this gene (C950T). In this study, direct DNA sequencing revealed 12 variants with polymorphisms C950T, G1181C and rs4876869 observed to be in strong linkage disequilibrium. The constructed haplotype T-G-T was found to increase the risk for a low BMD, while C-G-T and C-C-C have a protective role; thus, we investigated the functional role of both C950T and rs4876869 in vitro. The promoter region, including the C950T alleles, was amplified by PCR, cloned into pGL3 enhancer vector and transfected into HeLa, COS-7 and RAW264.7 cell lines. After incubation, luciferase activity was measured. The T/C (rs4876869) change was tested for its possible effect on pre-mRNA splicing, using an exon-trapping vector. A statistical significant difference in gene expression was observed between the alleles for T950C, with the T allele showing a lower luciferase expression in all cell lines (P<0.01). For rs4876869, exon skipping was observed for the C allele, while only one transcript harbouring the whole exon was observed for the T allele. Our findings suggest that the T-G-T haplotype might be increasing the risk for osteoporosis due to lower quantities of the full OPG transcript being expressed resulting in a higher bone resorption.
TNFRSF11B 基因内的多态性已被研究，并与骨质疏松症和骨折风险相关。该基因的产物骨保护素（OPG）是破骨细胞生成的关键负调节剂，由成骨细胞/基质细胞分泌。先前在马耳他绝经后妇女中的一项研究表明，该基因启动子区域内发现的一种多态性与低骨密度（BMD）呈正相关（C950T）。在这项研究中，直接 DNA 测序显示了 12 种变体，其中 C950T、G1181C 和 rs4876869 观察到存在强连锁不平衡。构建的单体型 T-G-T 被发现增加了低 BMD 的风险，而 C-G-T 和 C-C-C 具有保护作用；因此，我们在体外研究了 C950T 和 rs4876869 的功能作用。通过 PCR 扩增包括 C950T 等位基因在内的启动子区域，将其克隆到 pGL3 增强子载体中，并转染到 HeLa、COS-7 和 RAW264.7 细胞系中。孵育后，测量荧光素酶活性。使用外显子捕获载体测试 T/C（rs4876869）变化对前体 mRNA 剪接的可能影响。在所有细胞系中，T950C 等位基因的基因表达观察到统计学上显著差异，T 等位基因的荧光素酶表达较低（P<0.01）。对于 rs4876869，C 等位基因观察到外显子跳跃，而 T 等位基因仅观察到一个含有整个外显子的转录本。我们的研究结果表明，T-G-T 单体型可能会增加骨质疏松症的风险，因为表达的完整 OPG 转录本数量较少，导致骨吸收增加。
