Panach Layla, Pineda Begoña, Mifsut Damián, Tarín Juan J, Cano Antonio, García-Pérez Miguel Ángel
Research Foundation, Institute of Health Research INCLIVA, 46010 Valencia, Spain.
Orthopedic Surgery and Traumatology, Clinic Hospital, Institute of Health Research INCLIVA, 46010 Valencia, Spain.
Bone. 2016 Feb;83:94-103. doi: 10.1016/j.bone.2015.11.002. Epub 2015 Nov 8.
Compelling data are revealing that the CD40/CD40L system is involved in bone metabolism. Furthermore, we have previously demonstrated that polymorphisms in both genes are associated with bone phenotypes. The aim of this study is to further characterize this association and to identify the causal functional mechanism. We conducted an association study of BMD with 15 SNPs in CD40/CD40L genes in a population of 779 women. In addition, we assessed the functionality of this association through the study of the allele-dependent expression of CD40 and CD40L in peripheral blood leukocytes (PBLs) and in human osteoblasts (OBs) obtained from bone explants by qPCR and by sequencing. When an allelic imbalance (AI) was detected, studies on allele-dependent in vitro transcription rate and on CpG methylation in the gene promoter were also performed. Our results confirm the genetic association between SNP rs116535 (T>C) of CD40L gene with LS-BMD. Regarding CD40 gene, two SNPs showed nominal P-values<0.05 for FN- and LS-BMD (Z-scores), although the association was not significant after correcting for multiple testing. Homozygous TT women for SNP rs1883832 (C>T) of CD40 gene showed a trend to have lower levels of OPG (Q-value=0.059), especially when women of BMD-quartile ends were selected (P<0.05). Regarding functionality, we detected an AI for rs1883832 with the C allele the most expressed in OBs and in PBLs. Since the rs116535 of CD40L gene did not show AI, it was not further analyzed. Finally, we described a differential methylation of CpGs in the CD40 promoter among women of high in comparison to low BMD. Our results suggest that the CD40/CD40L system plays a role in regulating BMD. Effectively, our data suggest that a decreased production of OPG could be the cause of the lower BMD observed in TT women for rs1883832 of the CD40 gene and that the degree of methylation of CpGs in the CD40 promoter could contribute to the acquisition of BMD. One possibility that deserves further study is whether the degree of methylation of the CD40 gene affects the level of CD40 expression and, consequently, the level of OPG.
有力的数据表明，CD40/CD40L系统参与骨代谢。此外，我们之前已经证明这两个基因的多态性与骨表型相关。本研究的目的是进一步表征这种关联并确定因果功能机制。我们在779名女性群体中对CD40/CD40L基因中的15个单核苷酸多态性（SNP）与骨密度（BMD）进行了关联研究。此外，我们通过定量聚合酶链反应（qPCR）和测序研究外周血白细胞（PBL）和从骨外植体获得的人成骨细胞（OB）中CD40和CD40L的等位基因依赖性表达，评估这种关联的功能。当检测到等位基因失衡（AI）时，还进行了关于等位基因依赖性体外转录率和基因启动子中CpG甲基化的研究。我们的结果证实了CD40L基因的SNP rs116535（T>C）与腰椎骨密度（LS-BMD）之间的遗传关联。关于CD40基因，两个SNP在股骨颈骨密度（FN-BMD）和LS-BMD方面显示出名义P值<0.05（Z分数），尽管在多重检验校正后这种关联并不显著。CD40基因的SNP rs1883832（C>T）的纯合子TT女性显示出骨保护素（OPG）水平较低的趋势（Q值=0.059），特别是当选择BMD四分位数两端的女性时（P<0.05）。关于功能，我们检测到rs1883832存在AI，其中C等位基因在OB和PBL中表达最多。由于CD40L基因的rs116535未显示AI，因此未进一步分析。最后，我们描述了高BMD与低BMD女性中CD40启动子中CpG的差异甲基化。我们的结果表明，CD40/CD40L系统在调节BMD中起作用。实际上，我们的数据表明，OPG产生减少可能是CD40基因的rs1883832的TT女性中观察到的较低BMD的原因，并且CD40启动子中CpG的甲基化程度可能有助于获得BMD。一个值得进一步研究的可能性是CD40基因的甲基化程度是否会影响CD40表达水平，进而影响OPG水平。
