绝经后妇女骨标志物特点及染色体6q25.1区域相关基因多态性研究
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摘要
研究背景和目的
骨质疏松症(Osteoporosis, OP)是一种以骨量低下、骨微结构破坏,导致骨脆性增加,易发生骨折为特征的全身性骨病(世界卫生组织,WHO)。随着老龄化的加剧,骨质疏松症的患病率正在逐年上升。虽然骨密度(BMD)是目前诊断骨质疏松症的最佳定量指标,但是骨标志物可以及时反映个体骨代谢的状况,对评价骨骼综合健康情况意义重大。目前临床上治疗骨质疏松的药物多为骨吸收抑制剂,既往的观点认为绝经后妇女仅仅是在绝经早期骨转换较高,此后逐渐下降,但是目前临床上不同年龄的绝经后骨质疏松妇女,使用骨吸收抑制剂都有一定效果。因此,我们希望通过测定大样本绝经后妇女的骨标志物水平,了解绝经后妇女骨转换的特点,及其与骨密度、椎体骨折的关系。另外,维生素D水平也与骨代谢密切相关,国内绝经后妇女维生素D的水平尚不清楚,故同时测定维生素D水平,为临床治疗绝经后骨质疏松提供更多的依据。
骨质疏松症的发生受遗传因素与环境因素的共同作用。遗传因素不仅对人群骨密度的变异有影响,而且还可能对骨标志物及骨折都有影响。全基因组关联研究(genome wide association study, GWAS)发现了多个新的与BMD相关联的单核苷酸多态性(single-nucleotide polymorphism, SNP)位点。我们选择了冰岛研究中所发现的与BMD强烈相关的4个SNP位点(rs1038304、rs6929137、rs4870044、rs1999805),它们均位于染色体6q25.1区域,将其在中国绝经后妇女中进行验证,旨在分析这些SNP位点在中国的情况,及其与骨密度、椎体骨折的关系。
研究方法
1.研究对象临床资料的采集：
本研究的研究对象为北京市椎体骨折研究所调查的北京市常住妇女。在北京7个地区随机整群抽样绝经1年以上的妇女2070例。收集其临床资料与生化指标,包括一般资料、既往病史、家族史、月经史等。根据纳入排除标准,最终纳入分析的研究对象为1753例。对所有研究对象,均测量骨密度、拍摄腰椎X片、检测骨标志物(βCTX,PINP)和25(OH)D3水平。
2.6q25.1区域SNP的选择：
本研究SNP的选择基于以下2个方面：①Hapmap提供的中国北京汉族人群(China Bejing Han, CBH) 6q25.1区域的遗传多态性位点信息,挑选标签SNP(tagSNP)。②基于国外GWAS发现的,与BMD相关并且在欧洲人群中已经验证的阳性位点。通过以上方法我们从中选择4个待检测的SNP位点：rs6929137(G／A)、rs1999805(T／C)、rs4870044(G／A)、rs1038304(A／G)。
3.6q25.1区域基因SNP的检测：
用TaqMan探针技术,采用AB17900仪器对rs1999805、rs4870044、rs1038304、rs6929137进行基因分型的检测。
4.维生素D与骨标志物的检测：
采用罗氏E170电化学发光仪及其配套的试剂,采用电化学发光免疫分析的方法,检测血清25(OH)D3、βCTX和P1NP水平。
5.其它生化指标的检测：
采用西门子自动检测仪及配套试剂检测肝肾功能、血钙、血磷及碱性磷酸酶。
研究结果
1.一般资料：最终纳入分析的研究对象共1753例,均为绝经1年以上的健康妇女,平均年龄为65.3岁。
2.骨标志物的特征：总体P1 NP的平均值是58.39±29.32(10.5～429.7)ng／ml总体βCTX的平均值是0.45±0.21(0.04～2.14)ng／ml,均呈正态分布。绝经早期(49～59岁)的P1NP和βCTX水平均较高,随后下降,到70-79岁年龄段最低,80岁以后又呈现出升高的趋势。股骨颈与椎体部位均分为骨质疏松、骨量减少和骨量正常这三组,组间βCTX与P1NP的水平有显著差异。βCTX水平和腰椎及股骨颈骨密度呈显著负相关,P1NP也表现出类似的情况。
3.维生素D特征：测得总体25(OH)D3的平均值为13.15±5.39(4-35.11)ng／ml,仅有0.55%的研究对象的25(OH)D3≥30ng／ml,89.9%的研究对象25(OH)D3<20ng／ml。年龄与25(OH)D3水平显著负相关。25(OH)D3在股骨颈骨质疏松、骨量减少、骨量正常组的平均值有显著差异(P=0.002)。25(OH)D3水平与βCTX的水平和P1NP均呈负相关。
4.SNP与骨质疏松症表型的关联性分析：根据研究对象的BMD水平,以及是否具有椎体骨折来分组,进行病例对照分析。将骨质疏松与骨量减少合并定义为病例组(低骨量组),以骨量正常为对照,进行病例对照分析。以发现椎体骨折为病例组,未发现椎体骨折为对照组进行病例对照分析。将骨标志物水平在第50百分位以上定义为高骨转换组,在第50百分位以下定义为对照组,进行病例对照分析。对总样本、各病例组、各对照组的SNP位点进行Hardy-Weinberg平衡检验,其P值均>0.05,均符合Hardy-Weinberg平衡,说明本研究的对象能代表总体人群。
4个SNP位点(rs1999805、rs4870044、rs1038304、rs6929137)的等位基因频率在低骨量组与正常对照组之间没有差异。4个SNP位点的等位基因频率在椎体骨折组与无椎体骨折组之间没有差异。4个SNP位点的等位基因频率在高骨转换组与低骨转换组之间没有差异。
基因型分析发现,rs103834的基因型与血清PINP水平有关(P=0.015),具有AA基因型的研究对象,其PINP水平显著高于AG或GG基因型。未发现4个SNP位点的基因型与骨密度有明显关联。未发现4个SNP位点的基因型与绝经后妇女是否椎体骨折有关联。
显隐性模型分析发现,rs1038304显性模型中P1NP的水平差异有统计学意义。未发现SNP位点基因型与骨密度或骨折有关联。单倍体分析发现,rs1038304与rs6929137位于一个连锁不平衡区域内,可构建单倍体型AG、GA与GG,未发现各单体型类型与骨密度、椎体骨折及骨标志物水平有关。
年龄配对的椎体骨质疏松组和对照组间(各314例),骨质疏松组的身高、体重及骨密度明显低于对照组。没有发现等位基因频率及基因型分布存在差异。校正身高、体重影响后,未发现各位点基因型之间椎体骨密度或股骨颈骨密度的差异存在统计学意义。未发现各位点基因型之间骨标志物水平存在差异。
结论
1.年龄是影响骨标志物的因素之一,绝经早期的骨转换最高,随着年龄增大逐渐减低,70～79岁年龄段最低,但80岁以后又有所上升,尚不能解释该现象的原因。骨标志物水平与骨密度呈显著负相关。绝大部分研究对象存在维生素D缺乏。维生素D水平与骨标志物呈负相关,监测维生素D与骨标志物水平能更好的反应骨骼的综合健康情况,有利于更好的防治骨质疏松。
2.中国北京地区绝经后女性人群中,6q25.1区域相关SNP位点rs1038304的基因型与P1 NP水平有关。未发现多态性位点rs1999805、rs4870044、rs1038304、rs6929137与骨密度及是否椎体骨折有关联。因此,推测rs1038304的基因型可能与骨形成有关,仍需更多研究加以证实。
Background and Objective
Osteoporosis is a systemic skeletal disease characterized by low bone mineral density (BMD) and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture (WHO). Nowadays, the incidence of osteoporosis is increasing yearly with the aging society. BMD is the best criterion for osteoporosis diagnosis, while bone turnover markers (BTM) reflect whole body rates of bone resorption and bone formation, and provide a dynamic assessment of the skeleton. In our opinion, bone turnover is active at early postmenopausal period, and then decreasing with age. But nowadays anti-resorption drugs are often effective in the treatment of postmenopausal osteoporosis. The problem is why the anti-resorption drugs are useful when the bone resorption is decreasing. And vitamin D plays an important role in maintaining calcium homeostasis and bone metabolism, which has close association with osteoporosis. Our study is to investigate the status of vitamin D and BTM in a large sample of postmenopausal women, so that to analyze the relationship among BTM, vitamin D, BMD and vertebral fractures.
Osteoporosis is a common disease with a strong genetic component. Genetic factors contribute to osteoporosis by influencing not only bone mineral density but also bone turnover, and fractures. GWAS have found out many new single-nucleotide polymorphism (SNP) which had significant association with BMD in Caucasians. This study aims to verify the potential associations between candidate loci and BMD, BTM in Chinese postmenopausal women, these SNPs were reported by one recent GWAS in Iceland people, including rs1038304, rs6929137, rs4870044, rs1999805.
Methods
1. Collection of clinical samples and data
2070 cases of postmenopausal women in Beijing were selected through random cluster sampling method. Clinical data was collected, including general information, medical history, familial history, menstrual history. At last 1753 cases were into the study. All study subjects were required to have DXA lumbar X-ray. And whole blood samples and serum samples were required in order to detect vitamin D and BTM, includingβCTX and PINP.
2. Selection of SNP
Selection criteria of SNPs was based on 2 aspects following:①select genetic tag SNP of China Beijing Han provided by Hapmap.②The positive SNP site from abroad study.4 SNPs were selected:rs6929137 (G/A), rs1999805 (T/C) rs4870044 (G/A), rs1038304 (A/G)
3. SNP genotyping
All the 4 SNPs were genotyped by Applied Biosystems 7900HT Fast Real-Time PCR System, which were operated by TaqMan probe technology.
4. Test of bone turnover markers and vitamin D
Serum concentrations of P1NP,β-CTX,25OHD3 were determined by a fully automated Roche electrochemiluminescence system((E170, Roche Diagnostics). The principle was electrochemiluminescence immunoassay (ECLI).
5. Test of other biochemical markers
Serum concentration of ALT, ALP, calcium and phosphorus were test by automated Siemens system.
Results
1. General data
1753 cases were at last added to the database for statistical analysis. All of them were postmenopausal women, and the mean age was 65.3 years.
2. Characteristics of BTM
The level of P1NP in the total study group was 58.39±29.32 (10.5-429.7) ng/ml, andβCTX was 0.45±0.21 (0.04-2.14)ng/ml. Both P1NP andβCTX were higher at early postmenopausal period, then both serum BTM decreased up till the age of 70-79 years, and showed a trend of increasing older than 80 years. Both BTM showed significant differences among groups of femoral neck osteoporosis, osteopenia and normal, so as in vertebral site. Negative correlation were found betweenβCTX and femoral neck BMD, similar situation were found betweenβCTX and lumbar BMD, so as P1NP.
3. Characteristics of vitamin D
The mean level of 25 (OH) D3 was 13.15±5.39 (4-35.11) ng/ml. The prevelance of 25 (OH) D3<20 and≥30ng/ml was 89.9%,0.55% respectively. Negative correlation was found between vitamin D level and age.25 (OH) D3 showed significant differences among groups of femoral neck osteoporosis, osteopenia and normal. Both BTM had negative correlation with the level of 25 (OH) D3.
4. Association analysis between SNPs and genotypes
Subjects with osteoporosis and osteopenia were defined as cases and the subjects with normal BMD as controls. Subjects with vertebral fractures were defined as cases, and the subjects without were defined as controls. Subjects with higher bone turnover markers (above the 50th percentile) were defined as cases and others were defined as controls. Hardy-Weinberg equilibrium was calculated using the chi-square test. All SNPs were in Hardy-Weinberg equilibrium either in the cases or control groups.
Compared osteoporosis/osteopenia group with control group, no association was found in allele frequencies between the case-control analyses. Compared vertebral fracture group with control group, no association was found in allele frequencies. Compared higher bone turnover group with control group, no association was found in allele frequencies.
There was significant difference in P1NP level among genotypes of rs1038304. Subjects with genotype AA had higher P1 NP level than who with genotype AG or GG (P=0.015). Compared osteoporosis/osteopenia groups with control group, the P values of genotype frequencies of each SNP were all over 0.05, which had no statistical significance. Compared vertebral fracture group with control group, no association were found in genotypes either. There was no difference of BMD and bone turnover markers within genotypes.
In dominance model, significant difference was found in P1NP level between the case and control group of rs1038304. In dominance and recessive models, there was no obvious difference of the genotype frequency of all above SNPs between the case and control group. Data analysis results calculated by Haploview 4.1 software show that rs1038304 and rs6929137 were in the same LD block, haplotype AG, GA and GG can be constructed. There was no significant difference in haplotypes frequency between case and control groups.
Matching the age between vertebral osteoporosis group and normal group, the height, weight and average BMD were significantly lower than the control group. No difference was found in allele frequencies and genotypes between the case and control group, and no association was found between genotypes and BMD. No asscioation was found between genotypes and bone turnover markers.
Conclusion
In conclusion, we found age-related changes in bone turnover markers in a large cross-sectional study of Chinese postmenopausal women from 49 to 108 years of age. Both P1NP andβCTX were higher at early menopausal period, and then both decreased up till the age of 70-79 years, and showed a trend of increasing older than 80 years. Increases in the levels of biochemical markers were correlated with low BMD. Most subjects were suffering vitamin D insufficiency, negative correlation was found between vitamin D level and bone turnover markers. So, monitoring vitamin D and bone turnover marker may be better for evaluating the comprehensive health condition of bone, which could be better for prevention and treatment of steoporosis,
Genotypes of rs1038304 in chromosome 6q25.1 region had significant association with P1NP level. So, rs1038304 may have some association with bone formation, further studies are needed. SNPs (rs1999805, rs4870044, rs1038304, rs6929137) may have no association with BMD and vertebral fracture 'in Chinese postmenopausal women.
骨质疏松症(Osteoporosis, OP)是一种以骨量低下、骨微结构破坏,导致骨脆性增加,易发生骨折为特征的全身性骨病(世界卫生组织,WHO)。随着老龄化的加剧,骨质疏松症的患病率正在逐年上升。虽然骨密度(BMD)是目前诊断骨质疏松症的最佳定量指标,但是骨标志物可以及时反映个体骨代谢的状况,对评价骨骼综合健康情况意义重大。目前临床上治疗骨质疏松的药物多为骨吸收抑制剂,既往的观点认为绝经后妇女仅仅是在绝经早期骨转换较高,此后逐渐下降,但是目前临床上不同年龄的绝经后骨质疏松妇女,使用骨吸收抑制剂都有一定效果。因此,我们希望通过测定大样本绝经后妇女的骨标志物水平,了解绝经后妇女骨转换的特点,及其与骨密度、椎体骨折的关系。另外,维生素D水平也与骨代谢密切相关,国内绝经后妇女维生素D的水平尚不清楚,故同时测定维生素D水平,为临床治疗绝经后骨质疏松提供更多的依据。
骨质疏松症的发生受遗传因素与环境因素的共同作用。遗传因素不仅对人群骨密度的变异有影响,而且还可能对骨标志物及骨折都有影响。全基因组关联研究(genome wide association study, GWAS)发现了多个新的与BMD相关联的单核苷酸多态性(single-nucleotide polymorphism, SNP)位点。我们选择了冰岛研究中所发现的与BMD强烈相关的4个SNP位点(rs1038304、rs6929137、rs4870044、rs1999805),它们均位于染色体6q25.1区域,将其在中国绝经后妇女中进行验证,旨在分析这些SNP位点在中国的情况,及其与骨密度、椎体骨折的关系。
研究方法
1.研究对象临床资料的采集：
本研究的研究对象为北京市椎体骨折研究所调查的北京市常住妇女。在北京7个地区随机整群抽样绝经1年以上的妇女2070例。收集其临床资料与生化指标,包括一般资料、既往病史、家族史、月经史等。根据纳入排除标准,最终纳入分析的研究对象为1753例。对所有研究对象,均测量骨密度、拍摄腰椎X片、检测骨标志物(βCTX,PINP)和25(OH)D3水平。
2.6q25.1区域SNP的选择：
本研究SNP的选择基于以下2个方面：①Hapmap提供的中国北京汉族人群(China Bejing Han, CBH) 6q25.1区域的遗传多态性位点信息,挑选标签SNP(tagSNP)。②基于国外GWAS发现的,与BMD相关并且在欧洲人群中已经验证的阳性位点。通过以上方法我们从中选择4个待检测的SNP位点：rs6929137(G／A)、rs1999805(T／C)、rs4870044(G／A)、rs1038304(A／G)。
3.6q25.1区域基因SNP的检测：
用TaqMan探针技术,采用AB17900仪器对rs1999805、rs4870044、rs1038304、rs6929137进行基因分型的检测。
4.维生素D与骨标志物的检测：
采用罗氏E170电化学发光仪及其配套的试剂,采用电化学发光免疫分析的方法,检测血清25(OH)D3、βCTX和P1NP水平。
5.其它生化指标的检测：
采用西门子自动检测仪及配套试剂检测肝肾功能、血钙、血磷及碱性磷酸酶。
研究结果
1.一般资料：最终纳入分析的研究对象共1753例,均为绝经1年以上的健康妇女,平均年龄为65.3岁。
2.骨标志物的特征：总体P1 NP的平均值是58.39±29.32(10.5～429.7)ng／ml总体βCTX的平均值是0.45±0.21(0.04～2.14)ng／ml,均呈正态分布。绝经早期(49～59岁)的P1NP和βCTX水平均较高,随后下降,到70-79岁年龄段最低,80岁以后又呈现出升高的趋势。股骨颈与椎体部位均分为骨质疏松、骨量减少和骨量正常这三组,组间βCTX与P1NP的水平有显著差异。βCTX水平和腰椎及股骨颈骨密度呈显著负相关,P1NP也表现出类似的情况。
3.维生素D特征：测得总体25(OH)D3的平均值为13.15±5.39(4-35.11)ng／ml,仅有0.55%的研究对象的25(OH)D3≥30ng／ml,89.9%的研究对象25(OH)D3<20ng／ml。年龄与25(OH)D3水平显著负相关。25(OH)D3在股骨颈骨质疏松、骨量减少、骨量正常组的平均值有显著差异(P=0.002)。25(OH)D3水平与βCTX的水平和P1NP均呈负相关。
4.SNP与骨质疏松症表型的关联性分析：根据研究对象的BMD水平,以及是否具有椎体骨折来分组,进行病例对照分析。将骨质疏松与骨量减少合并定义为病例组(低骨量组),以骨量正常为对照,进行病例对照分析。以发现椎体骨折为病例组,未发现椎体骨折为对照组进行病例对照分析。将骨标志物水平在第50百分位以上定义为高骨转换组,在第50百分位以下定义为对照组,进行病例对照分析。对总样本、各病例组、各对照组的SNP位点进行Hardy-Weinberg平衡检验,其P值均>0.05,均符合Hardy-Weinberg平衡,说明本研究的对象能代表总体人群。
4个SNP位点(rs1999805、rs4870044、rs1038304、rs6929137)的等位基因频率在低骨量组与正常对照组之间没有差异。4个SNP位点的等位基因频率在椎体骨折组与无椎体骨折组之间没有差异。4个SNP位点的等位基因频率在高骨转换组与低骨转换组之间没有差异。
基因型分析发现,rs103834的基因型与血清PINP水平有关(P=0.015),具有AA基因型的研究对象,其PINP水平显著高于AG或GG基因型。未发现4个SNP位点的基因型与骨密度有明显关联。未发现4个SNP位点的基因型与绝经后妇女是否椎体骨折有关联。
显隐性模型分析发现,rs1038304显性模型中P1NP的水平差异有统计学意义。未发现SNP位点基因型与骨密度或骨折有关联。单倍体分析发现,rs1038304与rs6929137位于一个连锁不平衡区域内,可构建单倍体型AG、GA与GG,未发现各单体型类型与骨密度、椎体骨折及骨标志物水平有关。
年龄配对的椎体骨质疏松组和对照组间(各314例),骨质疏松组的身高、体重及骨密度明显低于对照组。没有发现等位基因频率及基因型分布存在差异。校正身高、体重影响后,未发现各位点基因型之间椎体骨密度或股骨颈骨密度的差异存在统计学意义。未发现各位点基因型之间骨标志物水平存在差异。
结论
1.年龄是影响骨标志物的因素之一,绝经早期的骨转换最高,随着年龄增大逐渐减低,70～79岁年龄段最低,但80岁以后又有所上升,尚不能解释该现象的原因。骨标志物水平与骨密度呈显著负相关。绝大部分研究对象存在维生素D缺乏。维生素D水平与骨标志物呈负相关,监测维生素D与骨标志物水平能更好的反应骨骼的综合健康情况,有利于更好的防治骨质疏松。
2.中国北京地区绝经后女性人群中,6q25.1区域相关SNP位点rs1038304的基因型与P1 NP水平有关。未发现多态性位点rs1999805、rs4870044、rs1038304、rs6929137与骨密度及是否椎体骨折有关联。因此,推测rs1038304的基因型可能与骨形成有关,仍需更多研究加以证实。
Background and Objective
Osteoporosis is a systemic skeletal disease characterized by low bone mineral density (BMD) and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture (WHO). Nowadays, the incidence of osteoporosis is increasing yearly with the aging society. BMD is the best criterion for osteoporosis diagnosis, while bone turnover markers (BTM) reflect whole body rates of bone resorption and bone formation, and provide a dynamic assessment of the skeleton. In our opinion, bone turnover is active at early postmenopausal period, and then decreasing with age. But nowadays anti-resorption drugs are often effective in the treatment of postmenopausal osteoporosis. The problem is why the anti-resorption drugs are useful when the bone resorption is decreasing. And vitamin D plays an important role in maintaining calcium homeostasis and bone metabolism, which has close association with osteoporosis. Our study is to investigate the status of vitamin D and BTM in a large sample of postmenopausal women, so that to analyze the relationship among BTM, vitamin D, BMD and vertebral fractures.
Osteoporosis is a common disease with a strong genetic component. Genetic factors contribute to osteoporosis by influencing not only bone mineral density but also bone turnover, and fractures. GWAS have found out many new single-nucleotide polymorphism (SNP) which had significant association with BMD in Caucasians. This study aims to verify the potential associations between candidate loci and BMD, BTM in Chinese postmenopausal women, these SNPs were reported by one recent GWAS in Iceland people, including rs1038304, rs6929137, rs4870044, rs1999805.
Methods
1. Collection of clinical samples and data
2070 cases of postmenopausal women in Beijing were selected through random cluster sampling method. Clinical data was collected, including general information, medical history, familial history, menstrual history. At last 1753 cases were into the study. All study subjects were required to have DXA lumbar X-ray. And whole blood samples and serum samples were required in order to detect vitamin D and BTM, includingβCTX and PINP.
2. Selection of SNP
Selection criteria of SNPs was based on 2 aspects following:①select genetic tag SNP of China Beijing Han provided by Hapmap.②The positive SNP site from abroad study.4 SNPs were selected:rs6929137 (G/A), rs1999805 (T/C) rs4870044 (G/A), rs1038304 (A/G)
3. SNP genotyping
All the 4 SNPs were genotyped by Applied Biosystems 7900HT Fast Real-Time PCR System, which were operated by TaqMan probe technology.
4. Test of bone turnover markers and vitamin D
Serum concentrations of P1NP,β-CTX,25OHD3 were determined by a fully automated Roche electrochemiluminescence system((E170, Roche Diagnostics). The principle was electrochemiluminescence immunoassay (ECLI).
5. Test of other biochemical markers
Serum concentration of ALT, ALP, calcium and phosphorus were test by automated Siemens system.
Results
1. General data
1753 cases were at last added to the database for statistical analysis. All of them were postmenopausal women, and the mean age was 65.3 years.
2. Characteristics of BTM
The level of P1NP in the total study group was 58.39±29.32 (10.5-429.7) ng/ml, andβCTX was 0.45±0.21 (0.04-2.14)ng/ml. Both P1NP andβCTX were higher at early postmenopausal period, then both serum BTM decreased up till the age of 70-79 years, and showed a trend of increasing older than 80 years. Both BTM showed significant differences among groups of femoral neck osteoporosis, osteopenia and normal, so as in vertebral site. Negative correlation were found betweenβCTX and femoral neck BMD, similar situation were found betweenβCTX and lumbar BMD, so as P1NP.
3. Characteristics of vitamin D
The mean level of 25 (OH) D3 was 13.15±5.39 (4-35.11) ng/ml. The prevelance of 25 (OH) D3<20 and≥30ng/ml was 89.9%,0.55% respectively. Negative correlation was found between vitamin D level and age.25 (OH) D3 showed significant differences among groups of femoral neck osteoporosis, osteopenia and normal. Both BTM had negative correlation with the level of 25 (OH) D3.
4. Association analysis between SNPs and genotypes
Subjects with osteoporosis and osteopenia were defined as cases and the subjects with normal BMD as controls. Subjects with vertebral fractures were defined as cases, and the subjects without were defined as controls. Subjects with higher bone turnover markers (above the 50th percentile) were defined as cases and others were defined as controls. Hardy-Weinberg equilibrium was calculated using the chi-square test. All SNPs were in Hardy-Weinberg equilibrium either in the cases or control groups.
Compared osteoporosis/osteopenia group with control group, no association was found in allele frequencies between the case-control analyses. Compared vertebral fracture group with control group, no association was found in allele frequencies. Compared higher bone turnover group with control group, no association was found in allele frequencies.
There was significant difference in P1NP level among genotypes of rs1038304. Subjects with genotype AA had higher P1 NP level than who with genotype AG or GG (P=0.015). Compared osteoporosis/osteopenia groups with control group, the P values of genotype frequencies of each SNP were all over 0.05, which had no statistical significance. Compared vertebral fracture group with control group, no association were found in genotypes either. There was no difference of BMD and bone turnover markers within genotypes.
In dominance model, significant difference was found in P1NP level between the case and control group of rs1038304. In dominance and recessive models, there was no obvious difference of the genotype frequency of all above SNPs between the case and control group. Data analysis results calculated by Haploview 4.1 software show that rs1038304 and rs6929137 were in the same LD block, haplotype AG, GA and GG can be constructed. There was no significant difference in haplotypes frequency between case and control groups.
Matching the age between vertebral osteoporosis group and normal group, the height, weight and average BMD were significantly lower than the control group. No difference was found in allele frequencies and genotypes between the case and control group, and no association was found between genotypes and BMD. No asscioation was found between genotypes and bone turnover markers.
Conclusion
In conclusion, we found age-related changes in bone turnover markers in a large cross-sectional study of Chinese postmenopausal women from 49 to 108 years of age. Both P1NP andβCTX were higher at early menopausal period, and then both decreased up till the age of 70-79 years, and showed a trend of increasing older than 80 years. Increases in the levels of biochemical markers were correlated with low BMD. Most subjects were suffering vitamin D insufficiency, negative correlation was found between vitamin D level and bone turnover markers. So, monitoring vitamin D and bone turnover marker may be better for evaluating the comprehensive health condition of bone, which could be better for prevention and treatment of steoporosis,
Genotypes of rs1038304 in chromosome 6q25.1 region had significant association with P1NP level. So, rs1038304 may have some association with bone formation, further studies are needed. SNPs (rs1999805, rs4870044, rs1038304, rs6929137) may have no association with BMD and vertebral fracture 'in Chinese postmenopausal women.
引文
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[4]. Ray, N.F., et al., Medical expenditures for the treatment of osteoporotic fractures in the United States in 1995:report from the National Osteoporosis Foundation. J Bone Miner Res,1997.12(1):p.24-35.
[5].Cooper, C., G. Campion and L.R. Melton, Hip fractures in the elderly:a world-wide projection. Osteoporos Int,1992.2(6):p.285-9.
[6].Hannon, R. and R. Eastell, Preanalytical variability of biochemical markers of bone turnover. Osteoporos Int,2000.11 Suppl 6:p. S30-44.
[7].皮银珍等,女性骨转换指标与年龄和腰椎骨密度的关系.中华内分泌代谢杂志,2005.21(5)：第441-445页.
[8].Gerdhem, P., et al., Biochemical markers of bone metabolism and prediction of fracture in elderly women. J Bone Miner Res,2004.19(3):p.386-93.
[9]. Melton, L.R., et al., Relationship of bone turnover to bone density and fractures. J Bone Miner Res,1997.12(7):p.1083-91.
[10].Dawson-Hughes, B., et al., Estimates of optimal vitamin D status. Osteoporos Int,2005.16(7):p.713-6.
[11].夏维波,苏华与周学瀛,维生素D缺乏与骨质疏松.中华骨质疏松和骨矿盐疾病杂志,2009.2(3)：第145-154页.
[12].Nesby-O'Dell, S., et al., Hypovitaminosis D prevalence and determinants among African American and white women of reproductive age:third National Health and Nutrition Examination Survey,1988-1994. Am J Clin Nutr,2002.76(1):p.187-92.
[13].Holick, M.F., High prevalence of vitamin D inadequacy and implications for health. Mayo Clin Proc,2006.81(3):p.353-73.
[14].Bischoff-Ferrari, H.A., et al., Estimation of optimal serum concentrations of 25-hydroxyvitamin D for multiple health outcomes. Am J Clin Nutr,2006. 84(1):p.18-28.
[15].Wat, W.Z., et al., Prevalence and impact of vitamin D insufficiency in southern Chinese adults. Ann Nutr Metab,2007.51(1):p.59-64.
[16].Bischoff, H.A., et al., Muscle strength in the elderly:its relation to vitamin D metabolites. Arch Phys Med Rehabil,1999.80(1):p.54-8.
[17].Janssen, H.C., M.M. Samson and H.J. Verhaar, Vitamin D deficiency, muscle function, and falls in elderly people. Am J Clin Nutr,2002.75(4):p.611-5.
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[19].Genant, H.K., et al., Vertebral fracture assessment using a semiquantitative technique. J Bone Miner Res,1993.8(9):p.1137-48.
[20].刘红等,正常女性与年龄相关的骨转换生化指标和骨密度的关系.中华内科杂志,2004.43(11)：第805-809页.
[21].Pi, Y.Z., et al., Age-related changes in bone biochemical markers and their relationship with bone mineral density in normal Chinese women. J Bone Miner Metab,2006.24(5):p.380-5.
[22].Martinez, J., et al., Bone turnover markers in Spanish postmenopausal women:the Camargo cohort study. Clin Chim Acta,2009.409(1-2):p.70-4.
[23].Garnero, P., et al., Biochemical markers of bone turnover, endogenous hormones and the risk of fractures in postmenopausal women:the OFELY study. J Bone Miner Res,2000.15(8):p.1526-36.
[24].Delmas, P.D., et al., The use of biochemical markers of bone turnover in osteoporosis. Committee of Scientific Advisors of the International Osteoporosis Foundation. Osteoporos Int,2000.11 Suppl 6:p. S2-17.
[25].Hart, G.R., et al., Measurement of vitamin D status:background, clinical use, and methodologies. Clin Lab,2006.52(7-8):p.335-43.
[26].Kuchuk, N.O., et al., Vitamin D status, parathyroid function, bone turnover, and BMD in postmenopausal women with osteoporosis:global perspective. J Bone Miner Res,2009.24(4):p.693-701.
[27].Rucker, D., et al., Vitamin D insufficiency in a population of healthy western Canadians. CMAJ,2002.166(12):p.1517-24.
[28].Hutchinson, M.S., et al., Low serum 25-hydroxyvitamin D levels are associated with increased all-cause mortality risk in a general population:the Tromso study. Eur J Endocrinol,2010.162(5):p.935-42.
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[2].朴俊红等,中国人口状况及原发性骨质疏松症诊断标准和发生率.中国骨质疏松杂志,2002.8(1)：第1-7页.
[3].中华医学会,临床诊疗指南：骨质疏松症和骨矿盐疾病分册[M].,2006,北京：人民卫生出版社.第1-7页.
[4]. Ray, N.F., et al., Medical expenditures for the treatment of osteoporotic fractures in the United States in 1995:report from the National Osteoporosis Foundation. J Bone Miner Res,1997.12(1):p.24-35.
[5].Cooper, C., G. Campion and L.R. Melton, Hip fractures in the elderly:a world-wide projection. Osteoporos Int,1992.2(6):p.285-9.
[6].Hannon, R. and R. Eastell, Preanalytical variability of biochemical markers of bone turnover. Osteoporos Int,2000.11 Suppl 6:p. S30-44.
[7].皮银珍等,女性骨转换指标与年龄和腰椎骨密度的关系.中华内分泌代谢杂志,2005.21(5)：第441-445页.
[8].Gerdhem, P., et al., Biochemical markers of bone metabolism and prediction of fracture in elderly women. J Bone Miner Res,2004.19(3):p.386-93.
[9]. Melton, L.R., et al., Relationship of bone turnover to bone density and fractures. J Bone Miner Res,1997.12(7):p.1083-91.
[10].Dawson-Hughes, B., et al., Estimates of optimal vitamin D status. Osteoporos Int,2005.16(7):p.713-6.
[11].夏维波,苏华与周学瀛,维生素D缺乏与骨质疏松.中华骨质疏松和骨矿盐疾病杂志,2009.2(3)：第145-154页.
[12].Nesby-O'Dell, S., et al., Hypovitaminosis D prevalence and determinants among African American and white women of reproductive age:third National Health and Nutrition Examination Survey,1988-1994. Am J Clin Nutr,2002.76(1):p.187-92.
[13].Holick, M.F., High prevalence of vitamin D inadequacy and implications for health. Mayo Clin Proc,2006.81(3):p.353-73.
[14].Bischoff-Ferrari, H.A., et al., Estimation of optimal serum concentrations of 25-hydroxyvitamin D for multiple health outcomes. Am J Clin Nutr,2006. 84(1):p.18-28.
[15].Wat, W.Z., et al., Prevalence and impact of vitamin D insufficiency in southern Chinese adults. Ann Nutr Metab,2007.51(1):p.59-64.
[16].Bischoff, H.A., et al., Muscle strength in the elderly:its relation to vitamin D metabolites. Arch Phys Med Rehabil,1999.80(1):p.54-8.
[17].Janssen, H.C., M.M. Samson and H.J. Verhaar, Vitamin D deficiency, muscle function, and falls in elderly people. Am J Clin Nutr,2002.75(4):p.611-5.
[18].Cummings, S.R., et al., Endogenous hormones and the risk of hip and vertebral fractures among older women. Study of Osteoporotic Fractures Research Group. N Engl J Med,1998.339(11):p.733-8.
[19].Genant, H.K., et al., Vertebral fracture assessment using a semiquantitative technique. J Bone Miner Res,1993.8(9):p.1137-48.
[20].刘红等,正常女性与年龄相关的骨转换生化指标和骨密度的关系.中华内科杂志,2004.43(11)：第805-809页.
[21].Pi, Y.Z., et al., Age-related changes in bone biochemical markers and their relationship with bone mineral density in normal Chinese women. J Bone Miner Metab,2006.24(5):p.380-5.
[22].Martinez, J., et al., Bone turnover markers in Spanish postmenopausal women:the Camargo cohort study. Clin Chim Acta,2009.409(1-2):p.70-4.
[23].Garnero, P., et al., Biochemical markers of bone turnover, endogenous hormones and the risk of fractures in postmenopausal women:the OFELY study. J Bone Miner Res,2000.15(8):p.1526-36.
[24].Delmas, P.D., et al., The use of biochemical markers of bone turnover in osteoporosis. Committee of Scientific Advisors of the International Osteoporosis Foundation. Osteoporos Int,2000.11 Suppl 6:p. S2-17.
[25].Hart, G.R., et al., Measurement of vitamin D status:background, clinical use, and methodologies. Clin Lab,2006.52(7-8):p.335-43.
[26].Kuchuk, N.O., et al., Vitamin D status, parathyroid function, bone turnover, and BMD in postmenopausal women with osteoporosis:global perspective. J Bone Miner Res,2009.24(4):p.693-701.
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