【作者】 宋海峰；

【导师】 邱贵兴；

【作者基本信息】 中国协和医科大学 ， 骨外科学， 2008， 博士

【摘要】 研究背景:椎间盘退变性疾病(degenerative disc disease,DDD)是指由椎间盘退变(intervertebral disc degeneration,IDD)引起的以颈、腰腿疼痛为主要表现的临床症候群,包括了临床上常见的颈、腰椎间盘突出症,颈椎病,腰椎管狭窄症,退性行腰椎不稳,退性行腰椎滑脱、退行性脊柱侧凸等。通常认为,椎间盘的退变主要是由于环境因素的积累效应和衰老过程引起。重体力负荷,尤其是与职业相关的重体力负荷,一直被怀疑是椎间盘退变的主要危险因素,但始终无法确认负荷与退变之间的剂量—效应关系。流行病学对单卵双生和双卵双生的双胞胎进行研究发现,环境因素对椎间盘退变作用不明显,说明负重仅是危险因素的一部分,从而改变了传统观点。随着研究逐渐深入到分子水平,多数学者认为DDD是一种多基因遗传病。同时,已经陆续发现了一些与DDD相关的具有遗传特性的特异性基因和一些与IDD相关的基因位点。目前已基本证实维生素D受体(Vitamin D receptor,VDR)基因的Taq1和Fok1基因型与IDD的有关;其他重要的候选基因,如COL9A2基因、MMP-3基因与DDD的关系还存在争议,结果往往互相矛盾。因此,我们有必要从基因水平出发,对COL9A2基因、MMP-3基因与DDD的关系加以研究。研究目的:1、探索COL9A2基因与DDD的关联及发挥的效应;2、明确COL9A2基因与DDD不同表型的关联。3、探索MMP-3基因与DDD的关联,明确MMP-3基因与DDD不同表型的关联。研究方法:本研究采用医院为基础的病例一对照研究设计。1、研究对象根据入选和排除标准,选取2006年10月—2008年1月期间北京协和医院骨科病房和门诊确诊的123例中国北方汉族腰椎DDD患者(DDD~+)。以来自北京协和医院骨科门诊和体检中心的123例非腰椎DDD(DDD~-)为对照组,与病例组匹配。123例DDD~-对照组在年龄、性别(年龄差别≤5岁)、体重指数、种族(中国北方汉族)、劳动强度(参照中华人民共和国国家标准GB3869-83)与DDD~+病例组以1:1相匹配。所有DDD~+组与DDD~-组影像学资料完整。2.实验方法1、QIAamp DNA Blood Mini Kit试剂盒提取DNA;2、SNP位点的选择原则:3、根结合已经研究过的有功能的多态性位点及NCBI数据库和国际人类基因组单体型图计划(http://www.Hapmap.Org)提供的基因型数据,优先选择位于外显子区域或有错译突变的SNPs;其次为标签SNP,以及有文献报道可能与DDD有关的位点。4、通过对临床表现与L3-4,L4-5,L5-S1椎间盘退变在影像学上的表现分析,进行候选基因多态性与DDD的关联分析。将病例组根据性别、椎间盘退变程度、及是否合并矢状面的畸形(滑脱)和冠状面的畸形(侧凸)分为不同的亚组。5、在123例DDD~+组和123例DDD~-对照组应用超高通量的SNP分型系统—SNPstream UIT(Genotyping System)对所选SNPs进行鉴定。6、拟和优度x~2检验(Goodness-of-fit Chi-square test)分析病例、对照组基因型频率的分布是否符合H—W平衡;7.基于基因型/等位基因频率的关联分析上,应用在线软(SNIP stats)进行非条件Logistic回归模型评估位点基因型与DDD发生风险的相关程度,并计算优势比(OR)及95%可信区间(Cls);8.连锁不平衡和单倍体型分析:应用Haploview4.0软件计算对照组人群中统一基因SNPs两两间连锁不平衡统计值D’和r,并评估所有单倍体型在病例组和对照组间的分布差异。3.研究结果1、DDD~+组和DDD~-组在年龄、性别分布、体重指数无统计学差异。2、筛查2个候选基因,12个位点。3.COL9A2基因筛查的6个位点:rs6676013 rs7533552 rs12077871s12722877 rs3737820 rs209914基因型分布在DDD~-组中和DDD~+组均符合Hardy—Weinberg平衡;在病例/对照组中等位基因分布频率分别为:SNP02(rs1207781)C/T=290.24)/15(0.11);单位点分析显示该位点等位基因在病例和对照组中的分布频率统计学上存在显著差异(P=0.012)。进一步分析得知SNP02(rs1207781)等位基因C/T与DDD~+的易感性升高相关。在非条件Logistic回归分析中,经年龄和性别校正后,SNP02(rs1207781)位点基因型分布符合Codominant(OR=2.23;95%CI=1.14—4.46,p=0.033,AIC=345.4)遗传模型,dominant(OR=2.08;95%CI=1.05—4.12,p=0.032;AIC=345.7)遗传模型和Overdominant(OR=2.25;95%CI=0.97-3.63,p=0.019;AIC=344.8)遗传模型,后者最佳(AIC值较小)。这些结果说明COL9A2基因是中国汉族人群DDD发病的一个易感基因。6个位点今均不符合连锁不平衡,未形成单倍体。4.MMP—3基因筛查的6个位点:rs520540 rs645419 rs522616rs655403 rs591058 rs602128基因型分布在对照组中和病例组均符合Hardy—Weinberg平衡;在基于基因型的“病例一对照”关联分析中,我们筛到阳性位点SNP09(rs591058)和病例/对照组中基因型分布频率分别为:SNP09(rs591058)C/T=68(0.65)/49(0.40);单位点分析显示该位点基因型在病例和对照组中的分布频率统计学上存在显著差异(P=0.043),进一步分析得知SNP9基因型CT与DDD的易感性升高相关;在非条件Logistic回归分析中,经年龄、性别校正后,SNP09(rs591058)位点基因型分布符合Overdominant(OR=I.81,95%CI=1.07—3.05,P=0.025,AIC=340.6)。连锁不平衡与单倍体分析显示所有位点不符合连锁不平衡。5.各SNP位点基因型与DDD~+的中间表型的关联分析中,发现COL9A2基因的SNP2(rs12077871)的C/T型与椎间盘退变程度(MRI分级)的易感性升高有关。6.在各SNP位点基因型与DDD~+其他中间分型(性别、退变程度、合并冠状面或矢状面的畸形)的关联分析中,没有发现阳性位点。结论:1.中国汉族人群中,COL9A2基因与DDD的发生发展有关联。2.COL9A2基因rs12077871位点的基因型C/T可能是决定DDD遗传易感性的重要因素。2、COL9A2基因rs12077871位点与椎间盘退变程度有关联。4.MMP—3基因可能与DDD的发生发展有关联。更多还原

【Abstract】 BackgroundDegenerative disc disease(DDD) is a common disorders and is usually found in the neck(cervical) and lower back(lumbar) regions of the spine.DDD is a kind of syndrome that includ prolapse of intervertebral,cervical spondylosis,umbar vertebrae;unsteadiness,lumbar spinal stenosis, degenerative Scoliosis etal.A number of studies have shown an association between genetic influences and disc degeneration.A study of monozygotic twins with different environmental backgrounds research showed that disc degeneration might be explained primarily by genetic influences.Another study on a large population twin sample have shown quantitative measures of disc degeneration to have a large genetic component Indeed,a number of studies have identified specific genetic risk factors(genes) associated with DDD.Although it’s still not sure about the genetic methods of DDD,It is also likely that DDD is a complex/multifactorial disease determined by the interplay between gene(s) and the environment.As for the current methods of genetic research,usually,there are two kinds:the linkage analysis and the association analysis.The first is mainly for familial idiopathic scoliosis while the second is the kinless sufferer.At present,only the association for VDR and COL9A2 has been replicated in more than one population.Lack of replication may be related to the complexity of DDD,different criteria used for genetic studies of DDD and the size of the cohorts.This is not a unique problem to DDD but genetic association studies in general,.Therefore,for genetic association studies to be successful,one needs large sample sizes,small P values.A clear phenotype definition is an important prerequisite for genetic studies.Objects1.To examine the association between polymorphic phenotype and different candidate genes(COLA2 Gene AND MMP3 gene).2.To investigate the relationship between genotypes of SNPs and intermediate phenotype of DDD.3.Material and MethodsA hospital-based case-control design was applied in this study.A total of 123 patients with DDD(65 male,57 femal,mean age-49.80 years old),and 123 healthy controls(58 male,65 femal,mean age-45.31 years old).All of the subjects were from Peking Union Medical College Hospital.,Meanwell,,all of them were from north china ban population.Genolnic DNA was extracted from peripheral blood leukocy’ tes of each subject who had Signed informned consellt,using QIAamp DNA Blood Mini KIT.Single nucleotide polymorphisms were selected.Hardy—Weinberg equilibrium both in control and in case groups were Analyzed through Goodness-of-fit Chi-square test.Case group were classified into different phenotypes.Genotying of all selected SNF’s was done by SNPstream technology All the data of 12 SNPs with polynlorphism are analyzed by the association analvsis based on a single SNP the association analysis between phenotyepes and SNPs.Results1.COL9A23 gene and Mmp3 gene were genotyped,their polymorphisms were in Hardy-weinberg equilibrium.2.All of the polymorphisms were not in linkagedis equilibrium. 3.IN the association analysis between the genotyhpes of SNPs and case group,we got two posstive SNPs:SNP02 and SNP09.the minor gentype frequencies in cases/control were,respectively,as followly: SNP02=SNP02 C/T=29(0.24)/15(0.11),SNP09 C/T=68(0.65)/49 (0.40).The single locus analysis realved the genotype distributions of SNP2 and SNP9 were statisticslly signficantly different between case patient and control subject(P=0.012,and P=0.043).In the unconditional logistic regression analysis,after sdjustment for age and gender,SNP2 showed significant in Codominant,dominantand Overdominant model.4 In the association analysis between the genotyhpes mad the phenotype,,we got one positive SNPs(SNP02),The association analysis between the genotyhpes and other phenotype,we didi not have got any positive SNP.ConclusionGenetic variants of COL9A2 gene and MMp-3 genes are associated with DDD and may play an important role developing DDD in a Chinese Han population.更多还原