骨质疏松症易感基因BDNF的遗传学关联分析及功能研究

为探索脑源性神经营养因子BDNF基因与中国汉族人群骨密度(bone mineral density, BMD)的关系,解析该基因调节骨质疏松症的功能机制,进而为中国汉族人群的骨质疏松症的防治提供依据。本研究从我国陕西地区征集了1300例汉族样本,并测量髋部/脊椎骨密度。选取BDNF基因上的14个标签SNPs进行基因分型,与1300例样本BMD进行关联分析,发现8个SNPs与髋部/脊椎BMD显著关联(P < 0.05)。其中,SNP rs16917237同时与髋部和脊椎骨密度关联,经Bonferroni校正后仍表现出显著性(0.05/14 = 0.0036)。整合连锁不平衡和单体型分析、表观功能注释、表达数量基因座分析、代谢通路分析进一步探索BDNF基因调节骨质疏松症的机制。构建小鼠前成骨细胞系(MC3T3-E1)人骨形态形成蛋白(rhBMP-2)诱导分化模型,利用小干扰RNA(siRNA)敲除BDNF。结果显示：14个SNPs位于同一单体型内;rs16917237在成骨细胞中表现出较强的激活型组蛋白H3K4me1、H3K4me3、H3K27ac修饰信号以及P300结合信号,表明其在成骨细胞中可能具有调控活性;rs16917237在11个组织中均能显著影响BDNF基因表达;BDNF基因位于与成骨细胞增殖分化相关的MAPK通路中;BDNF敲低能够显著降低MAPK通路中与成骨分化相关的CREB 基因mRNA和蛋白表达水平,提示其可能通过调控CREB表达进而影响成骨分化。生物信息学分析和功能实验结果一致,表明BDNF基因可能是影响骨质疏松症的重要功能基因。

Genetics association study and functional analysis on osteoporosis susceptibility gene BDNF

To explore the relationship between brain-derived neurotrophic factor (BDNF) gene and bone mineral density (BMD) in Chinese Han population, we performed association analysis of 14 tag SNPs on BDNF gene with hip/spine BMD in 1300 Han Chinese samples from Shaanxi Province. We found that 8 of the 14 SNPs were significantly associated with hip or spine BMD (P < 0.05). Moreover, the SNP rs16917237 was significantly associated with both hip and spine BMD, with significant Bonferroni correlation (P value 0.05/14 = 0.0036) in hip BMD. To further explore the regulatory mechanism of BDNF gene in osteoporosis, we further performed a set of data analyses, including linkage disequilibrium and haplotype analysis, epigenetic annotation, expression quantitative trait locus (eQTL) analysis and metabolic pathway analysis. Further, we have established a mouse pre-osteoblasts differentiation cell model (MC3T3-E1) by recombination human bone morphogenetic protein (rh-BMP2) induction. siRNA- mediated knock down of BDNF in this cell model showed that all 14 SNPs are in the same haplotype block. Strong signals of active histone H3K4me1, H3K4me3, H3K27ac modifications and P300 binding were observed in osteoblasts, in the region surrounding the most significant SNP rs16917237, suggesting that this SNP might have a regulatory function in osteoblasts. Furthermore, analysis of genotype data of rs16917237 and BDNF expression in multiple tissues from GTEx showed that rs16917237 SNP could significantly affect the expression of BDNF in 11 tissues. Through analysis of the various BDNF pathways, we showed that BDNF participates in the MAPK pathway, which is a vital and well-established pathway affecting osteoblasts proliferation and differentiation. siRNA knock down of BDNF significantly decreased the mRNA and protein levels of CREB, which is important in the MAPK pathway in osteoblast differentiation. These findings suggest that BDNF might affect osteoblast differentiation via regulation of CREB expression. In conclusion, our results from combined genetic association and functional analyses show that BDNF is a vital osteoporosis susceptibility gene, which can affect BMD not only in Chinese Han but also likely in other populations.