【作者】 刘林林；

【导师】 李修义； 尉军；

【作者基本信息】 吉林大学 ， 放射医学， 2004， 博士

【摘要】 人类基因组序列提供了人类生命过程中的全部遗传信息，作为生命背景的一个特有的参照物，将大大促进下个世纪人类生物学和医学的进展。人类基因组计划是将人类的基因组DNA序列分为大小为150-200碱基片段，装载到定位重叠的细菌克隆中，力求每个克隆序列的准确率大于99.9%。这种基因序列的可参照性，特别是最近研究较多的单核苷酸多态性（SNP），为研究人类基因序列变异提供了基础。人类基因组序列是基于少数人的草图，反映人类基因组的稳定性的一面，未反映变异性的一面。有关人类的信息可归于2个方向，纵向人类基因组测序计划是绘制完成全部核苷酸序列的检测；横向则是在基因特异位点上的序列变化即SNP。SNP分型是一种十分有效的基因分析工具，能帮助我们揭示在基因中某些特殊位点和许多疾病的关系。基因组中SNP的发生率大约为1个SNP/Kb。近期广泛进行的发现定位SNP并建立SNP基因图谱的工作，可以提供一个对所有基因的完全性的描述。SNP做基因分析的优点是可以提供一套在致病基因内部或者附近的标志，同时位于基因内部的SNP更可能影响蛋白质的结构或表达水平。SNP的遗传性稳定，数量大，有希望迅速获得大量有效型SNP来做遗传风险因子分析。通过新定位的基因产物比较和已知功能蛋白的同源性分析，有助于推测并检验这些基因的生物学功能。随着对基因序列及其突变性的认识的深入，人类能够更精确地定位越来越多的常见人类复杂疾病的易感基因。如果充分识别了那些控制基因表达的序列，人类就能够对自身基因的功能有一个更详尽的理解。通过基因序列比较，我们还可以检测基因在个别组织的表达情况，通过比较不同种属之间的相应基因序列，可以识别那些在进化中高度保守的区域。这些研究都大大加速了对人类基因序列的解释和翻译。精神分裂症是一种常见的病因尚未完全阐明的精神系统疾病。发病率约为1%。多起病于青壮年，常有特殊的思维、知觉、情感和行为等多方面的障碍和精神活动的不协调。精神分裂症作为特殊的疾病，给社会和家庭带来了严重的经济和精神上的负担。因此，明确其发病机制是目前迫切需要解决的重大课题。 <WP=107>目前，通过连锁研究发现1q21-22、5q31-33、6p24-21、6q25-26、8p22-21、10p11-15、13q14-33 及22q11-13等染色体区域可能含有精神分裂症易感基因。其中第6号染色体自Kendler在1994年首次发现其短臂可能存在易感基因之后，Straub等人进一步研究证实6p24-22区与精神分裂症连锁，并发现最显著性位点分别是D6S296（P=0.00001）、D6S274（P=0.004）及D6S285（P=0.006）。2000年Wei和 Hemmings首次报道第6号染色体短臂人类主要组织相容性复合体（MHC）区域NOTCH4基因及其附近区域可能与精神分裂症密切相关。此后，许多学者对此区域做了重复性研究，也得到了类似的结果。本研究在上述研究的基础上，选择人类第6号染色体MHCⅡ、Ⅲ 类基因区为精神分裂症易感基因候选区，以中国汉族精神分裂症患者和他们的健康父母组成的核心家系为研究对象，利用连锁不平衡分析方法，在ADR、TSBP、NOTCH4和TNXB基因上构建SNP连锁不平衡图，检测该区域内是否存在精神分裂症易感位点。在技术路线上，遵循全基因组扫描染色体阳性区域→基因图→SNPs图→RFLP（限制性片段长度多态）图这样一个基本研究过程。用PCR-RFLP方法检测SNPs基因型，采用SPSS统计学软件管理和分析基因分型数据，用拟合优度卡方检验验证每个SNP基因型在抽样群体中的分布是否符合Hardy-Weinberg平衡。用EH软件分析两个SNPs之间的连锁不平衡程度。利用单倍体相对风险（HRR）和传递不平衡检验（TDT）方法分析单位点SNP数据，用Transmit软件分析由两个以上SNPs构成的单倍型数据。此外，通过临床亚组分析，证实遗传异质性的存在，通过用来源不同的核心家系重复阳性位点，排除假阳性结果，探讨决定精神分裂症的易感基因。患者均为吉林省四平市精神病院、长春市凯旋精神病院、汽车厂职工医院及北京大学第六医院的住院病人。依据ICD-10的精神分裂症诊断标准，精分症患者和他们的健康父母双亲组成的124个核心家系，共372名研究对象。以父母双亲传递给患病子女的等位基因为疾病基因，以未传递的等位基因为对照基因。在每个遗传标记位点，先分析50~80个核心家系，发现阳性位点时，用来源不同的核心家系进行重复。通过http://www.ncbi.nlm.nih.gov/、http://www.ncbi.nlm.nih.gov/SNP及http://snp.cshl.org/等人类基因组数据库，确定与疾病相关的染色体阳性区域内候选基因和含有限制性酶切位点的SNPs。1．Hardy-Weinberg平衡检验结果采用SNP Alyze2.0遗传学软件进行Hardy-Weinberg平衡检验。父母组和患者组的每个SNP基因型在抽样群体中的分布均符合Hardy-Weinberg平衡（P>0.05），说明该样品池适合遗传学分析。 <WP=108>2．SNPs与精分症的相关性分析2.1 传递不平衡检验（TDT）分析结果对父母双亲至少一方为杂合子的核心家系进行TDT检验，发现NOTCH4基因SNP108位点由父母传递给患病子女的等位基因频率偏离50%的期望值（P<0.05），说明NOTCH4基因与精分症相关联。而ADR、TSBP、TNXB基因与精分症无关联。2.2 单倍体相对风险（HRR）分析结果更多还原

【Abstract】 The sequence of Human Genome Project (HGP) includes all the informations of the course of life of human beings. It will promote the advance of biology and medicine greatly in next century as a special control of groundback of life. For Human Genome Project planning digested human gene DNA sequence into many 150-200bp fragments, and reconstructed into the germ clone and to be sure the accuracy is over 99.9%. Single nucleotide polymorphism (SNP) provides the basis of the research of variations of human gene sequence. The sequence of Human Genome Project is a draft coming from a few people and it can reflect the stable aspects of human genome, but it can’t reflect the varied aspects of human genome. SNP is a very efficient gene analyse tool and it can help us explore the relationships between some special sites and many diseases. The occurrence rate of SNP is 1SNP/kb. Mapping and constructing the SNP map can provide a complete description of all the genes. It is beneficial to elucidate the difference of phenotype and susceptibility to disease of individual person. On the basis of high density and high polymorphisms SNP map, genome scanning and association study are used to screen for more relative genes of complex diseases. It makes a great progress in mapping the susceptibility genes of polygenic disease. These studies will accelerate the pace of the description and translation of the sequence of Human Genome Project greatly.Schizophrenia is a common mental disorder with 1% of the life-time prevalence in the general population worldwide. Schizophrenia is mostly <WP=113>present special thinking, sense, feelings and behavior disorder. It brings heavy burden to society and family, so how to declare its mechanism is the most urgent important study filed. Epidemiological data demonstrate that schizophrenia is not a simple Mendelian disease, but it looks like a complex disease with a polygenic mechanism. At present, Genome-wide scanning shows the susceptibility genes linkage findings on some chromosome regions, such as 1q21-22, 5q22-23, 6p24-21, 8p22-21, 13q14-33 and 22q11-12 .Our study has focused on identifying the candidate susceptibility genes on the MHCⅡ,Ⅲ region of chromosome 6 using a family-based LD analysis. The family trios consist of healthy fathers, healthy mothers and affected offspring with schizophrenia. Technique route was followed in such a way that from positive region determined by genome scanning to gene map, and SNP-based LD map. The SNP-based LD map was constructed near the NOTCH4 gene and the TNXB gene. 8 SNPs were chosen in this region spanning 101365bp. SNPs were genotyped using PCR-based RFLP analysis. Genotyping data were put into the SPSS database. The Hardy-Weinberg (H-W) equilibrium was tested for genotype frequency distributions of SNPs using the goodness of fit test. The LD between paired SNPs was estimated with the EH program. The haplotype relative risk (HRR) test and the transmission/disequilibrium test (TDT) were applied to detect allelic association between an SNP and schizophrenia. Haplotypes consisting of two or more SNPs were tested by the program Transmit (Version2.5). To elucidate genetic heterogeneity, in addition, schizophrenic patients were sub-grouped based on their clinical symptoms and the genetic association between SNPs and clinical subgroups was then analyzed. Positive loci were replicated with different family trios to rule out the false positive results. A total of 120 family trios of Chinese Han decent, consisting of fathers, mothers and affected offspring with schizophrenia, were recruited. Patients were diagnosed as having schizophrenic illness by the ICD-10 criteria. Eight SNPs included SNP99 (rs8084), SNP100 (rs2143466), SNP101 (rs387071), SNP102 (rs367398), SNP103 (rs915894), SNP104 (rs422951), SNP108 (rs1009382) and SNP120 (rs520688). The following is the details of major results obtained in this study. <WP=114>1. The H-W equilibrium:The goodness of fit test showed that genotype frequency distributions of 8 SNPs were not deviated from the H-W equilibrium,更多还原