【作者】 唐炜；

【导师】 贺林；

【作者基本信息】 上海交通大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 互作用,常常伴有环境因素或其他未知的随机因素参与,在各种因素共同协同作用下发生。复杂疾病在普通人群中发病率较高（一般≥1%）,所以也称作常见疾病,如精神分裂症、老年黄斑、糖尿病、哮喘、心血管疾病等等,均属于复杂疾病。由于缺乏对复杂疾病的致病机制的了解,一直没有找到有效的治疗手段,因而也日益成为各国科学家们的研究热点。本论文的主要工作是采用连锁不平衡的分析方法在中国汉族人群中研究精神分裂症和老年黄斑变性这两种复杂疾病和它们的易感基因之间的相关性。精神分裂症（MIM 181500）是最严重精神疾病之一,在世界范围发病率为1%左右。双生子,家系,寄养子的研究结果,提供了强有力的证据表明精神分裂症是一种复杂遗传疾病,具有很强的遗传性。但引发精神分裂症的基因仍然不明。精神疾病的谷氨酸假说中提示,NMDA受体对精神类疾病的易感性起着重要的作用。由NOS1催化产生的一氧化氮是NMDA的二级信使,在神经系统,免疫系统和心血管系统中起着重要的调节作用。NOS1定位在12q22-24,是和许多精神疾病相关的一个易感基因。我们选择了11个位点,包括一个微卫星标记在总共1705份病例对照组样品中采用测序的手段进行了连锁不平衡分析。我们发现rs3782206这个位点即使经过校正也在全部的病例对照组样品中与精神分裂症显著关联p<0.05。而且由它和rs3837437组成的单倍型也和精神分裂症以及偏执型亚型存在显著的关联.我们的实验结果证实了NOS1是中国汉族人群中精神分裂症的易感基因,也支持了精神分裂症的谷氨酸通路假说。DISC1自从1990年在苏格兰精神病大家系中被发现后,一直是研究者的焦点候选基因。特别是最近几年研究者们陆续阐明了DISC1的通路以及通路上的蛋白。DISC1通过跟PDE4B、NDEL1、LIS1、FEZ1等蛋白的相互作用影响了神经元的迁移、细胞骨架定位和神经传导过程。DISC1和它的通路很好的支持了精神类疾病的神经发育假说并且给认知障碍的发病机制提供了可信的解释。我们选择了覆盖了整个DISC1基因的16个SNPs位点,采用TaqMan的分型手段在576份病例对照组样品中进行了分型。结果显示rs1322784、rs7514199、rs2738864、rs1000731、和rs16841582都显示出与精神分裂症的关联P<0.05。而rs1322784、rs7514199和rs2738864组成的核心单倍型也显示出了强烈的关联。同时,我们对基因表达水平在病例和对照组的外周血细胞中进行了研究,发现这些基因的表达关联程度很强, NDEL1和PDE4B在病例组中都显著的高于正常组。通过Logistic多元回归方法构建的疾病模型,发现NDEL1和DISC1能够具有73.74%的准确度将样品的疾病状态分类。我们的结果在分子遗传和表达水平上都支持DISC1作为中国人群中的精神分裂症的易感基因。老年性黄斑变性（MIM 153800）是一种随年龄增加而发病率上升并导致双眼中心视力损害,是视网膜色素上皮细胞和神经视网膜退行性变造成的一种不可逆性视力下降或丧失的疾病。最近科学家们通过全基因组连锁分析和关联分析的手段找到了致病的两个候选基因CFH和ARMS2。我们在192个病例对照样本中进行了关联分析。结果显示在中国人群中CFH的致病位点Y402H的作用并不像在高加索人群中那样显著。在中国人群中发现了一系列同义和非同义突变rs800292、rs2274700、rs3753396、rs1329428和rs1065489与疾病有显著的相关（P<0.05,OR>1.5）。而针对ARMS2的A69S我们得出了较为一致的结果,尤其在湿性老年黄斑变性中,P值达到了1.55×10^-7,OR达到了5.46。在相互作用分析当中我们也证实了它们之间是相对独立的作用于疾病模式。我们的实验结果进一步的支持了CFH和ARMS2是中国人群中老年黄斑变性的易感基因。论文的另一部分是关于ENU（乙酰基亚硝基脲）诱变小鼠模型的研究。ENU是公认的强有力的化学诱变剂,能够随机地诱导出各种表型。国际上一些研究机构通过大规模ENU诱变筛选建立了一系列突变小鼠模型家系,通过对这些小鼠模型的研究,从另一个侧面研究人类的罕见疾病和复杂疾病。我们通过ENU诱变筛查出了具有转圈表型的小鼠,通过建立小鼠家系,将突变基因定位在4号染色体上,尔后通过测序在Chd7基因的exon13上寻找到一个从精氨酸到终止子的新的突变。CHD7基因曾被发现是常染色体显性遗传病CHARGE综合症的致病基因,基于它的临床表型和Chd7突变小鼠之间存在太多的表型相似,我们相信这个ENU诱变鼠系能够成为研究CHARGE综合症发病机制的一个非常宝贵的工具。更多还原

【Abstract】 Complex diseases are conditions that are influenced by the actions of multiple genes, their interactions with each other and with the environment. The prevalence of complex diseases is higher than 1% in general population, hence also called common diseases, such as schizophrenia, age-related macular degeneration, diabetes, asthmas, cardiovascular disease, et al. Since we know little about them and lack of effective treatments, more and more scientists participate in research on pathology and etiology of these complex diseases. The main work of my thesis is about linkage disequilibrium study on susceptible genes of schizophrenia and age-related macular degeneration in Chinese population.Schizophrenia （MIM 181500） is a common severe mental illness that affects 1% of the population worldwide. Data from twin, family, and adoption studies provide strong evidence that genetic factors play a major role in schizophrenia. Though its etiology remains unknown, it is well known that schizophrenia is a kind of complex disease.N-methyl-D-aspartate （NMDA） receptors have been proposed as mediators in glutamate hypothesis of schizophrenia. NO mainly catalyzed by NOS1 is the second messenger of NMDA and plays a vital role in nervous system, immune system and cardiovascular system. NOS1 is located in 12q22-24, a linkage location that has been linked to many neuropsychiatric phenotypes. We chose 11 variants including a microsatellite in NOS1 gene and genotyped 1705 case-control samples in our Han Chinese population by directly sequencing. Among these markers, rs3782206 revealed significant association with schizophrenia in our samples, even after corrected by 10000 permutations. And its haplotype with rs3837437 also showed the association with schizophrenia and S-P subtype. Our results supported NOS1 as a susceptible gene of schizophrenia and solidified the glutamate hypothesis.DISC1 gene is disrupted by the chromosome 1 breakpoint of a balanced t （1;11） translocation that co-segregates with schizophrenia and related mood disorders in a large multi-generational Scottish pedigree. It is always the hottest candidate gene of schizophrenia. Recent research has elucidated that the DISC1 binding partners, such as ATF4, PDE4B, NDEL1, LIS1 and FEZ1, and how they interact with DISC1 to effect the neuronal migration, cytoskeletal location and neuronal signaling. The biochemical evidence supports the hypothesis of neurodevelopment of schizophrenia and provides the convincing explanation for deficit cognition performance. We selected 16 SNPs covering the whole genomic DNA of DISC1 and genotyped in 576 case-control samples by TaqMan method. Our results found that rs1322784, rs7514199, rs2738864, rs1000731 and rs16841582 showed significant deviation of allelic distribution between case and control. Furthermore, the core haplotype of rs1322784, rs7514199 and rs2738864 was associated with schizophrenia as well. Meanwhile, we conducted RT-PCR to measure the expression of DISC1 and its binding molecular on peripheral blood mononuclear cell. We found a high correlation between them, particularly the expression of NDEL1 and PDE4B which was increased in case compared to the control. Logistic multiple regression model including NDEL1 and DISC1 made 73.74% contribution to diagnosis the status of disease. Our results provided further evidence for DISC1 as the important gene in the etiology of schizophrenia in Han Chinese population.Age-related macular degeneration （AMD, MIM 153800） is an age-associated progressive disease affecting the central regions of the retina and choroid, which can lead to loss of central vision. The whole genome linkage and association studies have identified two convincing genes, CFH and ARMS2 in the etiology of AMD currently.We performed an association study on our 192 case-control Han Chinese AMD samples. As for Y402H in CFH, we did not get the significant signal in Y402H as in Caucasian. Instead, we found many other mutations as rs800292, rs2274700, rs3753396, rs1329428 and rs1065489 showing positive result in our samples （P<0.05, OR>1.5）. As for ARMS2, we gained the consistent result and P value of A69S reached to 1.55×10^-7 when we stratified samples into wet type of AMD. We evaluated the role of epistasis among these positive mutations in two genes; we observed no statistically significant non-additive interactions, which suggested these loci independently contributed to the disease. Our results provided a further support for CFH and ARMS2 as the susceptible genes to AMD in Han Chinese population.Another part of my work was about the ENU （ethylnitrosourea） mutagenesis mouse model. ENU is a synthetic compound described as the most potent mutagen in mice. Large-scale ENU mutagenesis program has provided us with a large number of new mouse mutants applied for the analysis of gene function and further for good models of some human diseases. We screened circling phenotype mice by ENU mutagenesis, mapping the related gene in chromosome 4 and sequenced a novel mutant in exon13 of mouse Chd7 gene. This novel mutant resulted in functional change from arginine to premature stop codon. It has been reported that human CHD7 gene was linked to autosome dominant disease CHARGE syndrome. On the basis of the overlap in clinical features between Chd7 mutant mice and CHARGE, we believe that these mice will be a valuable tool in further analysis of the pathology underlying the abnormalities in CHARGE syndrome.更多还原