【作者】 邓飞艳；

【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】 本文对具有复杂遗传模式和常染色体显性遗传模式的两种疾病—骨质疏松症和良性家族性成年人肌阵挛癫痫—进行了基因定位与鉴定研究。关于骨质疏松症，我们运用遗传流行病学研究策略和蛋白质组学研究策略，从两个不同的层面，在中国汉族女性人群中定位和鉴定骨质疏松症的基因。1)首先，我们选用了RFLP标记MspI和MS遗传标记（GT）n，采用QTDT程序同时检测了COL1A2基因与中国人群骨密度和骨大小的关联和连锁。MspI位点和（GT）n位点存在紧密的连锁不平衡。检测到以下家庭内关联：MspI与腰椎（p=0.013）、全髋（p=0.053）、大转子（p=0.004）的骨密度，（GT）₁₇与腰椎（p=0.012）、股骨颈（p=0.032）、转子（p=0.023）的骨密度；（GT）₁₂与腰椎（p=0.009）的骨密度；M（GT）₁₂与腰椎（p=0.009）、髋部（p=0.017）、转子（p=0.001）的骨密度；（GT）₁₈与髋部（p=0.053）、股骨颈（p=0.024）的骨密度；m（GT）₁₈与股骨颈（p=0.013）的骨密度。MspI与转子间区（p=0.024）、全髋（p=0.043）的骨大小。除MspI与全髋（p＞0.10）骨大小外，以上结果均得到排列检测验证。这些结果表明COL1A2是影响中国女性骨密度和骨大小的QTL。2)其次，运用二元方差分析检测了7对基因的14对相互作用对中国女性峰值骨密度的潜在影响。发现，ERP×IL6的相互作用对全髋部（p=0.019），转子间（p=0.016）和股骨颈（p=0.019）的PBM显著。AHSG×IL6对股骨颈PBM也有作用（p=0.046）。携有GGPp的个体，全髋部，转子间和股骨颈的BMD比GGpp的携带者平均要高18.0％，19.5％和14.8％。而GGSS携带者的BMD比GGSs携带者平均要高18.8％。3)最重要的是，我们将蛋白质组学研究策略应用于复杂疾病骨质疏松症的研究。应用通过双相电泳和质谱鉴定技术，首次对中国女性高、低峰值骨密度个体的血液单核细胞进行了蛋白表达谱分析和比较，共鉴定了6个差异表达蛋白（色氨酸-天冬氨酸重复蛋白1，肌凝乳蛋白，alpha微管蛋白，ras抑制蛋白1，蛋白酶复合体亚单位5，脯氨酸4-羟化酶），后面5个差异表达蛋白得到Western Blotting验证。蛋白酶复合体亚单位5，脯氨酸4-羟化酶对骨代谢的潜在作用机制有待进一步研究阐明。色氨酸-天冬氨酸重复蛋白1，肌凝乳蛋白，alpha微管蛋白，ras抑制蛋白1与破骨作用有关，它们可能影响穿壁过程（通过调节单核细胞的黏附、变形和迁移）、影响单核细胞分化、影响骨吸收功能（调节破骨细胞足细胞器的形成、黏附和运动），最终导致骨密度的变化。关于良性家族性成年人肌阵挛癫痫，我们首次对中国一个BFAME大家系进行了临床诊断和遗传分析。通过候选区域扫描排除了国际上报道的两个基因组区域，通过全基因组扫描鉴定了一个新的基因组区域10p15，并最终将致病基因缩小到6 Mb的区间。遗传学研究结果结合临床特征初步确立中国的该家系属于一个新的BFAME亚型。本文的主要创新之处在于，1)首次运用蛋白质组学研究策略对骨质疏松症进行基因表达研究，最终鉴定了6个差异表达蛋白，并初步提出了骨质疏松症的一个发病机制。2)首次对中国的良性家族性成年人肌阵挛癫痫综合征进行基因定位研究，找到了一个新的、不同于国际上已经报道的基因组区域，初步确立了一个新的BFAME亚型。更多还原

【Abstract】 The present study aims to map and identify genes of two kinds of diseases, i.e. osteoporosis and benign familial adult myoclonus epilepey, the former with complex hetitable mode and the latter with autosomal dominant heritance mode.Regarding Osteoporosis, we utilized genetic epidemiology strategy and proteomics study strategy to map and identify the genes, on two levels i. e. DNA and protein levels, for osteoporosis in Chinese Han female populations.1) Firstly, we used a RFLP marker MspI and a microsatllite marker (GT) n in COL1A2 genes, and utilized QTDT programs to test association and linkage of this gene with bone mineral density and bone size in Chinese nuclear families. The two markers were in close linkage disequilibrium. We detected the following significant within-family association: MspI and BMD at spine (p = 0. 013), total hip(p = 0. 053), and intertrochanter(p = 0. 004), (GT) 17 and BMD at spine (p = 0. 012) , femoral neck (p = 0. 032) , and trochanter (p = 0. 023); (GT) 12 and BMD at spine (p = 0. 009); M(GT) 12and BMD at spine (p = 0. 009) , total hip (p = 0. 017) , and trochanter (p = 0. 001); (GT) 18 and BMD at total hip (p = 0. 053) , femoral neck (p = 0.024); m(GT)18 and BMD at femoral neck (p = 0. 013). MspI and bone size at intertrochanter (p = 0.024) and total hip (p = 0. 043) . Except MspI and total hip bone size, the above associations were all confirmed by permutation test. These indicated that C0L1A2 is in linkage disequilibrium with QTL underlying BMD and bone size in Chinese Han females.2) Secondly, we utilized two-way analysis of variation to test the relationship of 14 pairs of interaction among 7 genes with Chinese females’ peak bone mass. We found significant interaction effect of ERP X IL6 on PBM at total hip(P = 0. 019), intertrochanter (P = 0. 016), and femoral neck(P = 0. 019), AHSG×IL6 on femeral neck PBM(P = 0. 046) . Carriers of GGPp have an average of 18. 0%,. 19. 5% and 14. 8% higher BMD at total hip, intertrochanter, and femoral neck than carriers of GGpp. GGSS carriers have an average of 18. 8% higher femoral neck BMD than GGSs carriers.3) Most importantly, we utilized the proteomics strategy into the study of complex, disease osteoporosis. Through two dimension electrophorosis combined mass spectrometry, we compared the protein expression profiles of circulating monocytes from Chinese Han females with high vs. low PBM. A total of 96 irredundant proteins were identified. Six of them were confirmed by western blotting for their differential expression in the two samples: WDR1, gelsolin, K-alpha-1 tubulin, RSU1, PSMA5, P4HB. They might be involved in monocytes’ attachment, transendothelial movement, and differentiation into osteoclast, thus affect the osteoclastogenesis, and finally lead to BMD variationRegarding BFAME, we diagnosed and recruited a large pedigree affected with BFAME, for the first time in Chinese. Firstly, we excluded the two chromosomal.regions previously reported linked to BFAME in Japanese and Italy. Through whole geneome scan and genehunter analysis, we mapped the causative gene in a new chromosomal region 10pl5 and narrowed it within a region spanning 6.0 Mb. Combined the specific clinical features, our results indicated that the pedigree may belong to a new subtype of BFAME.The present study represents the first attempt to identify genes for osteoporosis using proteomics strategy, and we successfully identified six interesting proteins which might relative to osteoclastgeneiss, including two new factors, i.e. PSMA5 and P4HB. This study is also the first effort of exploring the specific genetic locus of BFAME in Chinese pedigree. The new genomic region identified in the Chinese pedigree indicates the existences of a third subtype of BFAME.更多还原