【作者】 雷署丰；

【导师】 邓红文；

【作者基本信息】 湖南师范大学 ， 生物化学与分子生物学， 2006， 博士

【摘要】 骨质疏松症是严重影响人类健康的疾病之一,它是以骨量减少,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（BMD）是骨质疏松性骨折（OF）的重要危险因子。以前,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。骨密度是一种由多基因和环境因素共同决定的复杂性状,它有很强的遗传决定,其遗传率高达50%-90%。骨大小（BS）也是一种独立于BMD的OF风险因子,并也受很强的遗传决定。本文旨在中国人群中,通过运用传统的关联分析、传递不平衡检测（TDT）等统计学方法,并通过RFLP、微卫星标记和单体型分析,检测9个重要的骨候选基因与骨质疏松相关表型之间的复杂决定关系;并且用基因芯片在高低BMD之间验证这些基因的RNA差异表达。结果表明: 1)运用QTDT程序,在由父母以及至少一个年龄在20-45岁的健康女性后代组成的402个中国核心家庭中,发现了-1997 G/T多态性与骨密度变异之间有显著的家庭内关联（p<0.05）。由-1997G/T多态性位点解释的BMD变异值在髋骨、股骨颈、转子和转子间区分别是1.6%、2.0%、1.2%和1.3%。 2)在388个核心家庭（包括双亲和至少一个健康的女性子代,总共1220个体）中,同时分析了腰椎和髋部骨密度与COL1A2基因内含子1的（GT）n多态位点、PTHR1基因P3启动子的（AAAG）n多态位点之间的群体混层、总的家庭关联、家庭内关联。结果发现M（GT）₁₂单体型和转子间区骨密度间存在显著的家庭内关联（p<0.001）。与M（GT）₁₂单体型携带者相更多还原

【Abstract】 Osteoporosis is a serious healthy disease, which 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). A large number of studies used BMD as the major surrogate phenotype for osteoporotic fracture. BMD is a quantitative trait determined by genetic and environmental factors, with heritability estimates ranging from 50% to 90%. Bone size (BS) is also another risk factor for OF independent of BMD, and also is under strong genetic determination. Using traditional association analysis, transmission disequilibrium test (TDT) and also by genotyping RFLP and microsatellite marker, the present study was performed to test the complex relationship of nine bone candidate genes with osteoporosis related phenotypes. Then microarray was performed to test the differential expression RNA between two groups with high BMD and low BMD. Our results showed as follows: 1)Using program QTDT, significant within-family association (via TDT) between the - 1997 G/T polymorphism of COL1A1 gene with BMD variation at all the hip sites were found (p < 0.05) in 1,263 subjects from 402 Chinese nuclear families, consisted of bothparents and at least one healthy female offspring aged from 20 to 45 years. The amount of BMD variation explained by the - 1997G/T polymorphism was 1.6%, 2.0%, 1.2% and 1.3% at the total hip, femoral neck, trochanter and intertrochanter, respectively. 2^Population stratification, total-family association, andrwithin-family association were performed between BMDs at the spine and hip and the (GT)n marker in the intron 1 of the COL1A& gene and the (AAAG)n marker in the P3 promoter of PTHR1 gene in 388 nuclear families composed of both parents and at least one healthy daughter with a total of 1, 220 individuals. Significant within-family association was found between the M(G1) i2 haplotype and trochanter BMD (p < 0. 001) . Individuals with this haplotype have, on average, 9. 53% lower trochanter BMD than the non-carriers. Suggestive evidence of the within-family association was detected between the (GT)i7 allele and BMD at the spine (p = 0.012), hip (p = 0.011), femoral neck (p = 0.032), trochanter (p = 0.023), and intertrochanter {p = 0. 034).3 )A total of 258 unrelated healthy Chinese men aged 50-80 years were genotyped the Msp I, Hind III, BsrB I, Sac I and BstB I markers in C0L1A2, BGP, IL-6, AHSG and PTH genes, respectively. Significant association of the AHSG gene with the spine BMD (p - 0. 006), even after adjusting for multiple testing in our study.Carriers of 1*1 and 2*2 genotypes of AHSG gene had, ■respectively, ~5. 1% and ~~8. 1% higher spine BMD than those of 1*2 genotype. For the other four genes, no evidence of association was found (p > 0.10). No significant evidence of gene-by-gene interaction was found by two-way factorial ANOVA on the BMD variation.4 population stratification, total-family association, andwithin-family association were used to test the relationship of BS (at the spine and hip) and height with Pvull and Xbal polymorphisms in the intron 1 of the gene and the Mspl and (GT)n markers in the intron 47 and intron 1 of the C0L1A2 gene in 400 Chinese nuclear families with a total of 1,256 individuals. Weak within-family association was found between the C0LlA2-ifepI {p = 0. 05) and the femoral neck BS, between the ER-a-PJ (p = 0. 04) and the intertrochanter BS, and between the COL1A2-(GT) n (p= 0. 02), COL1A2-/?(GT) n (/?= 0.009) and height. Subsequent permutation tests generally confirmed the suggestive within-family association. For the weak within-family association, the proportions of phenotypic variance accounted by the C0LlA2-ifepI, ER-a~PX, CQLlk2-(GT)n, C0L1A2-JZ?(￡#i7markers were 1.50%, 1.51%, 2.15%, and 2.43% for the corresponding phenotypes.5 population stratification, total-family association, andwithin-family association were used to test the relationship of the Apal, Eco31I, BstBl and (AAAG)n polymorphisms in the vitamin D receptor (VDR), collagen type I a 1 (C0L1A1), parathyroid hormone (PTH), and parathyroid hormone (PTH)/PTH~related peptide receptor (PTHR1) genes with the variation of bone size (BS) and ■height in 400 Chinese nuclear families with a total of 1,256 individuals. We did not detect any significant within-family association and total-family association between the VDR, COL1A1,■?PTH and PTHR1 gene polymorphisms, and the variations of BS and height in our sample.6)0n RNA level, we used microarray of affymetrix to analysis thedifferential expression of nine bone candidate genes between two?groups with extremely high and low peak bone mass (PBM). Theresults from t^test detected no significant differentialexpressions of these genes were found in the present sample.Based on the complex determination for osteoporosis relatedphenotypes, the present study represented the first effort onidentify the complex relationship of nine bone candidate genes andosteoporosis related phenotypes from two gene expression levels inChinese.更多还原