【作者】 于娜；

【导师】 蔡际群；

【作者基本信息】 中国医科大学 ， 药理学， 2009， 博士

【摘要】 前言骨质疏松症是最常见的老年性疾病,骨质疏松症的严重后果是骨质疏松性骨折。随着人口老龄化日趋明显,骨质疏松症以及其所导致的严重后果骨质疏松性骨折已成为一个社会性的健康问题,备受老年病学者的关注,并引起了各国政府的高度重视。骨质疏松症是以骨量减少,骨的微观结构退化为特征,致使骨的脆性增加,并易于发生骨折的全身性骨骼疾病。低骨密度(Bone Mineral Density, BMD)是骨质疏松性骨折(Osteoporotic Fracture, OF)的重要危险因子。骨密度是一种由多基因和环境因素共同决定的复杂性状,它有很强的遗传性,其遗传率高达50%-90%。目前,大多数的研究以骨密度作为骨质疏松性骨折的替代研究表型运用于遗传学和临床等方面的研究。然而,流行病学调查已经证实骨密度在不同的人群和种群中存在较大的异质性,因此若运用BMD来评估种族间的骨折风险差异,往往有失精准。在临床上或是遗传研究中,正确的运用合适的表型指标,对于准确诊断疾病和研究疾病的发病机理都是极为重要的。因此,一个良好的可以准确评估种族间骨折风险差异的指标急待发掘。股骨颈压缩强度指数(Compression strength index, CSI)是独立于BMD的OF风险因子,CSI用于测量个体对于作用于股骨颈主轴上面相当于体重重量的压力的抵抗能力。此指数提供大量骨强度相关信息,进而更有助于对股骨颈骨折风险的评估。该指数的遗传性易很高。本文主要目的是：比较评估骨折风险的新复合指标——CSI与BMD和股骨颈宽度(Femoral Neck Width, FNW)两个普遍应用的骨学表型指标在预测骨折风险中的差异；以CSI作为骨质疏松性骨折的替代表性,运用全基因组关联研究和全基因组连锁研究的方法,找出与CSI变量相关的骨折相关基因和区域,并研究相关基因和区域在骨质疏松和骨折风险中的可能潜在意义。实验材料和方法1、材料此次试验研究,以来自于中国人群和高加索人群的两个特大样本作为实验对象。研究志愿者一部分是来自美国中部内布拉斯加州奥马哈市周边和密苏里州堪萨斯城周边的高加索起源白人(Caucasian Whites),研究从一个大的样本库中挑选出所有符合本研究要求的个体,该部分样本即具有相关的表型指标信息、骨折信息史又具有完整的遗传信息。本人亲自参与该部分数据库的构建,其中包括数据的采集、样本的制备和最后的统计分析。研究志愿者另一部分是来自中国西安和湖南的中国汉人(Chinese Han),研究也是从一个大的样本库中挑选出所有符合本研究要求的个体,该部分样本仅具有相关的表型指标而不具有相关的遗传信息。这部分数据的采集工作由合作单位西安交通大学和湖南师范大学的相关部门完成,本人仅负责该部分数据的统计与分析。研究使用美国Hologic公司生产的Quantitative Digital Radiography (QDR)系列1000,2000+或4500型双能X线骨密度仪来测量股骨颈骨密度和骨的投照面积,单位分别是g/cm2和cm2。本研究的全基因组SNP分型采用Affymetrix公司的500K芯片套装完成,实验工作由合作方美国范德堡大学(Vanderbilt University)的Vanderbilt Microarray SharedResource (http://array.mc.vanderbilt.edu/)完成。连锁分析的基因分型由Marshfield医学中心完成,每个样本均采用410个微卫星标记进行基因分型,微卫星标记的平均群体杂合度为0.75,相邻标记间的平均距离为8.9cM。2.方法一般的统计分析运用SPSS统计分析软件包完成(SPSS Corp, Chicago, IL, USA);关联分析由HelixTree软件完成；连锁分析由SOLOR (Sequential Oligogenic Linkage Analysis Routines)软件完成。结果本文的主要创新之处在于本实验研究第一次就CSI特点以及其与BMD和FNW在预测股骨颈骨折风险中的差异进行研究,第一次运用CSI作为遗传学指标,运用于寻找与骨质疏松或骨折相关的基因和位点区域。结果发现：1、CSI是与FNK BMD和FNW相比,能更好的反映和预测股骨颈骨折风险的因子。首先,中国人群样本的CSI值高于高加索人群,这一发现与流行病学中对于两种族间股骨颈骨折发生率的调查结果相一致——中国人群股骨颈骨折发生率低于高加索人群。本实验研究对于CSI在反映不同种族骨折发生率中是一个极大的突破。其次,通过计算三者的比对比发现,CSI在预测骨折风险中也是一个良好的指标,特别是在校正了相关协变量之后。最后,在研究样本中,CSI随年龄的增高逐年递减,同老年人的骨折发生率较高相一致,从而进一步印证了CSI在预测股骨颈骨折风险中的全面性和正确性。2、CSI综合了BMD,股骨颈尺寸和体测指标的信息,准确衡量股骨颈承受作用于其主轴上相当于体重重量的压力的能力。而股骨颈骨折主要也是在这种力的作用下引起的, CSI将是一个既直接又可靠的股骨颈质量和强度预测指标。鉴于目前的骨质疏松学的遗传学研究主要集中以BMD为研究表型,如果将CSI运用于遗传学研究,作为遗传学表型探讨骨质疏松的遗传学规律,相信将会开辟一个崭新的领域。在本次研究中,首次运用CSI这一预测骨质疏松性骨折的新指标,对1000个高加索人群的随机样本,采用Affymetrix公司的500K芯片套装以及相关的统计学工具进行了该指标的首次全基因组关联分析。关联分析的结果是,发现了一个与骨质疏松相关的新基因HS3STl,其29个SNP位点的P值均小于0.05,两个位点(rs4916641和rs6448751)的P值最为显著,达到了全基因组的显著水平。HS3ST1基因有硫酸乙酰肝-葡萄糖胺3-氧硫转移酶的活性,硫酸乙酰肝转换的活性,以及限制乙酰胺合成速度的作用。其对于骨质疏松的影响作用还有待进一步的研究。3、在本次研究中,首次运用CSI这一预测骨质疏松性骨折的新指标对来自451个家庭的4,498个高加索人样本,使用SOLAR (Sequential Oligogenic Linkage Analysis Routines)软件对所有个体进行首次全基因组范围的多点连锁扫描。连锁分析的结果是,发现了三个连锁信号较强的染色体区域按LOD值大小依次为20p11.23、6q22.33、2q36.1。将本次的研究成果与早前的研究成果加以比对,发现有重叠的区域存在。另外在本次研究中,把“关联分析和连锁分析等的研究结果适于进一步的整合”的实验理念运用于本次研究中,简单的以染色体物理厘摩为桥梁,将本次全基因组连锁分析(Genome-Wide Linkage Studies, GWLS)结果和全基因组关联分析(Genome-Wide Association Studies, GWAS)结果相结合,并且发现了一个可能的骨质疏松相关基因ARFGEF2。而且ARFGEF2基因中有8个单核苷酸多态性(Single-Nucleotide Polymorphism, SNP)位点连锁不平衡,并且与CSI的变异高度相关。目前发现该基因与大脑皮质结构畸形以及重复感染相关,其对于骨质疏松的影响作用还有待进一步的研究。结论研究首次证明了CSI是较BMD和FNW更好的预测骨折风险的表型指标。其次,研究首次以CSI作为遗传学表型指标,进行了全基因组关联分析和全基因组连锁分析,发现了两个基因,HS3ST1和ARFGEF2,以及三个染色体区域20p11.23、6q22.33、2q36.1,可能与CSI的变异密切相关。研究结果对于在人群中预防、治疗及诊断骨质疏松症有重要的实用价值。更多还原

【Abstract】 Osteoporosis is one of the most popular old people’s diseases. Along with aging population, osteoporosis as a degenerative disease or a complication, has become a social health issue. It is getting great attention world widely. A lot of geriatrists are working in this area. The most serious result of osteoporosis is osteoporotic fracture.Osteoporosis is characterized by low bone mineral density (BMD) and deterioration of skeletal microarchitecture, leading to increased risk of fragility fracture. Low BMD is recognized as a major risk factor for osteoporotic fracture (OF). BMD is a quantitative trait determined by genetic and environmental factors, with heritability estimates ranging from 50% to 90%. A large number of studies used BMD as the major surrogate phenotype for osteoporotic fracture in the genetic studies and clinical works, et al. Many epidemiologic studies have indicated that BMD, as a major phenotype predicting fracture risk, has population- and ethnic-specificity. Therefore, studies clearly demonstrate that BMD, by itself, falls short of capturing the entire variation in fracture risk. In clinical work and genetic studies, the properly use of a surrogate is the key of disease diagnosis and studying pathogenesis. Compression strength index (CSI) is another independent risk factor for OF under strong genetic determination. Since CSI measures the capacity of withstanding compressive forces proportional to body weight along the main femoral neck axis, it may provide more information than BMD alone in assessing the risk of hip fracture.The purpose of this study is:to investigate the characteristics of compression strength index (CSI), a new parameter for measuring bone strength at the hip, in Caucasian and Chinese populations; to compare CSI with hip BMD and femoral neck width (FNW) in predicting the risk of hip fracture; by using genome-wide association analysis and genome-wide linkage analysis, looking for the fracture related gene or locus with CSI, and their potential function in osteoporosis and fracture risk.Materials and Methods1. Materials Samples:Two large samples were used in this study, including a Chinese Han sample from Xian’an province and Hu’nan province, recruited by related department in Xi’an Jiaotong University and Hunan Normal University, and a US Caucasian sample of Turopean origin, recruited by myself and some group members in University of Missouri-Kansas City.2. MethodsMeasurements:All the subjects were measured for hip BMD and FNW using the DEXA machine Hologic QDR4500. CSI was calculated as CSI=(BMD×FNW)/Weight.In the GWA study, genotyping with the Affymetrix arrays was performed at the Vanderbilt Microarray Shared Resource at Vanderbilt University Medical Center.In the linkage study, all the subjects were genotyped with 410 microsatellite markers from the Marshfield screening set 14 by Marsh Weld Center for Medical Genetics (Marshfield, WI, USA). These markers have an average population heterozygosity of 0.75 and were spaced, on the average, 8.9 cM apart.Statistical analyses:Regular ANOVA and regression analyses were conducted using the SPSS statistical package.In the linkage study, variance component linkage analyses for quantitative traits were performed using SOLAR (Sequential Oligogenic Linkage Analysis Routines).All GWA statistical analyses were performed using various statistical tests implemented in HelixTree 5.3.1 (Golden Helix, Bozeman, MT, USA).ResultsThe innovation in this study is that this is the first study investigating basic characteristics of CSI and comparing CSI with BMD and FNW in predicting hip fracture in populations of different ethnicities, and this is the first study using CSI as a surrogate to search the fracture related gene or locus.Our results showed as follows:1. An important finding in this study is that Chinese had higher CSI values than Caucasians, which is consistent with the observed lower hip fracture rates in Chinese compared to Caucasians in previous epidemiology studies. This finding may explain the discrepancy observed between BMD and hip fracture rates in Caucasian and Chinese populations. In addition, in the Chinese sample, after adjusting for covariates, CSI was the only bone phenotype that was significantly associated with the odds ratios for hip fracture. Our findings suggest that CSI may be a better predictor of incident hip fracture risk than FNK BMD or FNW. Moreover, the age-related decrease in CSI, as shown in this study in both Caucasians and Chinese, is consistent with the observation of higher hip fracture risk in the elderly.2. CSI, calculated as CSI=(BMD×FNW)/Weight, measures the ability of withstanding compressive forces proportional to body weight along the main femoral neck axis. Hip fracture is often a direct consequence of this compressive force and thus, CSI may represent a more direct and reliable measure of hip bone quality and strength than other commonly used phenotypic traits (e.g. BMD and FNW). Given the advantages of CSI in assessing risk of hip fracture and its strong genetic basis, it may serve as a useful complementary phenotype to BMD for gene identification for osteoporotic fractures at the hip. In this study, we report the first genome-wide association studies (GWAS) for CSI variation. Using Affymetrix 500 K arrays, we successfully genotyped and analyzed an independent cohort containing 1,000 homogeneous unrelated Caucasian subjects, including 501 females and 499 males. We identified a gene, HS3ST1, that was associated with CSI at the genome-wide significance level in subjects. HS3ST1 gene possesses both heparan sulfate glucosaminyl 3-O-sulfotransferase activity, anticoagulant heparan sulfate conversion activity, and is a rate limiting enzyme for synthesis of anticoagulant heparan. Our findings strongly support the importance of the HS3ST1 gene to CSI, and the function of HS3STl in osteoporosis need to be further studied.3. For a sample of pedigrees that contain 4,498 subjects from 451 families, we performed the first genome-wide linkage studies (GWLS) using 410 microsatellite markers to identify genomic regions that may contain quantitative trait loci (QTL) of CSI. The variance component linkage analysis for QTL was performed by using SOLAR. Several potentially important genomic regions (20p11.23、6q22.33、2q36.1) were identified. Some of the regions we identified are supported by earlier findings. In addition, to improve the power of WGLS, we conduct genomic convergence of WGLS results with our GWAS data here. We identified a gene, ARFGEF2, that was associated with CSI at the genome-wide significance level in subjects. Haplotype analyses using the Haploview program and our own genotype data indicated that strong LD exists among 8 single-nucleotide polymorphisms (SNPs) in ARFGEF2 gene, which form a single haplotype block. As a whole, the block is also strongly associated with CSI, with the p value approaching the genome-wide significance level. ARFGEF2 gene may relate to severe malformation of the cerebral cortex and recurrent infections, and the function of ARFGEF2 gene in osteoporosis need to be further studied.ConclusionsOur study for the first time suggests that, in comparison to BMD and FNW, CSI is a much improved parameter in predicting hip fracture, in particular when assessing fracture risk across different ethnic groups. We performed the first GWAS and GWLS for CSI variation. We identified two genes, HS3ST1 and ARFGEF2, which were associated with CSI. Our findings may have significant clinical implications.更多还原