【作者】 李超；

【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， 2007， 博士

【摘要】 精神分裂症(schizophrenia)是最严重最常见的精神疾病之一,在全世界各个人群中普遍存在,发病率在1%左右。主要表现为特征性的感知、思维、情感和行为的障碍以及精神活动与环境的不协调。同胞对分析、家系分析以及寄生子的研究一致表明,遗传因素在发病机制中起了重要的作用。目前对于精神分裂症的发生被认为是在环境因素影响下,多个中至微效基因协同作用的结果。本论文从基因组水平上出发,研究了KIF2和PDLIM5在中国汉族人群中与精神分裂症的相关性。KIF2是动力蛋白Kinesin家族的一员,在哺乳动物的神经元生长过程中承担重要的转运膜复合体和蛋白复合体的作用,并且通过在生长锥的边缘解聚微管调节微管动力。Homma等报道Kif2a基因敲除的小鼠在海马体的CA1和CA3区域出现了不正常的松散排列;而且在海马神经元的体外培养实验中,Kif2a-/-的海马神经元细胞在缺失了KIF2蛋白的抑制侧枝生长后较野生型神经元细胞长出很多侧枝。因此,KIF2很有可能是精神分裂症的易感基因之一,具有功能候选的特性。小鼠Kif2基因的同源基因在人类基因组的5q12.1。为了检测KIF2基因与精神分裂症发病的相关性,我们根据NCBI数据库中已有的KIF2基因的信息,选择了四个常见高频率单核苷酸多态性(Single Nucleotide Polymorphism,SNP)位点,并用软件设计了PCR引物,在中国汉族人群280个核心家系样本中,用等位基因实时PCR技术试验获得了样本所选四个位点的基因型,进而根据基因型数据和家系样本的结构,用ETDT、2LD、Hapview和FBAT软件分析了KIF2基因和精神分裂症的相关性。所选四个常见SNPs位点的关联分析中没有得到任何显著性结果。然而,本研究发现一个2-SNP的核心区域(rs2289883/rs464058,G/A)单倍型分析结果与精神分裂症密切相关;此外研究发现一个拥有23.4%的频率的常见4-SNP的单倍型(T/G/A/G)与精神分裂症相关(P=0.00795)。实验结果支持了KIF2基因是精神分裂症的易感基因之一的假设。PDLIM5是一个LIM结构域的蛋白,在细胞骨架的组织,细胞系的分化,器官发育和肿瘤形成中都有重要作用。这个蛋白同时也是Enigma蛋白家族中的一员,具有典型的由N端PDZ结构域和C端的1-3个LIM结构域构成的典型特征。PDLIM5蛋白在大脑的许多区域都有表达,包括海马体,皮层,丘脑,丘脑下部,杏仁体和小脑。在海马的神经元上,PDLIM5蛋白分布在突触后,是阿尔茨海默症发病期过渡表达蛋白AD7c-NTP的同源蛋白。Iwamoto等研究者发现在精神分裂症和双向情感障碍的患者的淋巴细胞系中,PDLIM5的mRNAs的量较正常人的量偏低,但是在精神分裂症、双向情感障碍以及抑郁症的患者的脑中,PDLIM5的mRNAs的量较正常人偏高。Kato等研究者报道了PDLIM5 mRNA在精神分裂症和双向情感障碍的患者脑中表达上调。从基因组研究的角度上,PDLIM5定位于人类基因组的4q22.3的位置,这一区域已经被多个连锁研究证明是精神分裂症的阳性区域。以上蛋白功能方面和连锁方面的证据提示,PDLIM5可能是精神分裂症的易感基因之一。因此,我们根据NCBI和HapMap数据库中已有的PDLIM5基因的信息和以往的研究,选择了SNPs位点,并用软件设计了引物。接着我们在江西地区中国汉族人群的精神分裂症病例对照样本中,用单碱基延伸的试验方法获取了样本PDLIM5基因上六个常见单核苷酸多态性位点的基因型,并根据病例对照组样本的特点和基因型的结果,用COCAPHASE和Hapview等软件统计分析,根据统计结果估计精神分裂症与PDLIM5之间是否存在关联。所选的六个单核苷酸多态性位点中,rs2433322在病例组和对照组中的等位基因频率有显著性差异(P=0.000010),包含这个位点的单倍型也同样发现存在显著性差异(在邦弗朗尼校正后Global P=0.00019)。我们的结果支持了PDLIM5很可能是精神分裂症的易感基因之一的假设。更多还原

【Abstract】 Schizophrenia is a chronic, severe mental disorder that affects approximately 1% of the world’s population. The disorder is characterized by psychotic symptoms and by cognitive, affective, and psychosocial impairment. Studies of family, twin, and adoption reveal that genetic factors play an important role in the etiological complex of schizophrenia. The inheritance pattern of schizophrenia suggests that there are multiple susceptibility genes, each with individually small effects, which interact with one another and with environmental factors to influence susceptibility to the diseases. In this thesis, we investigated associations between the susceptibility genes and schizophrenia (KIF2 and PDLIM5) in the Chinese Han population using geomic techniques.KIF2 belongs to the kinesin-like protein family and regulates microtubule dynamics at the growth cone edge by depolymerizing microtubules. It plays an important role in the suppression of collateral branch extension. On the other hand, KIF2 is a motor for anterograde transport, and plays an important role in expansion at the nerve growth cone. Homma et al. in 2003 reported that Kif2a knockout mice showed many brain abnormalities, especially in the CA1 and CA3 fields of hippocampal pyramidal cells which are associated with many mental disorders. The extension of collateral growth cones is suppressed in Kif2a+/+ and Kif2a-/- cultured neuron extends more long collaterals without the suppression of Kif2a. Therefore, KIF2 might be one of the susceptible genes of schizophrenia with functional susceptibility. KIF2, the orthologue of Kif2a, maps on 5q12.1 in human genome. On the basis of that KIF2 may be a potential candidate gene for schizophrenia, we selected four common SNPs in the region of KIF2 according to the data in NCBI database and designed appropriate primers for these SNPs. Then we acquired the genotypes of four common SNPs in the sample of 280 nuclear family of the Chinese Han population using the allele specific PCR technology, and studied the association relationship between the KIF2 gene and schizophrenia using ETDT, 2LD, Hapview and FBAT softwares. None of the four markers we selected in the KIF2 gene revealed transmission distortion in the trios. However, a common two-SNP haplotype (rs2289883/rs464058, G/A) showed a significant association with schizophrenia. In addition, a four-SNP haplotype (T/G/A/G) with a frequency of 23.4%, which was identified in parental chromosomes, showed a significant association with schizophrenia (P = 0.00795). Our results demonstrate that the KIF2 gene, located at 5q12.1, is a potential schizophrenia susceptibility gene. The PDZ and LIM domain 5 protein (PDLIM5) contains one PDZ (postsynaptic density-95/discs large/zone occludens-1) domain and three LIM (Lin-11, Isl-1, and Mec-3) domains, which is also thought as enigma-homologue LIM domain (ENH) protein or LIM protein. As a member of the Enigma class of proteins, LIM domain containing protein is involved in cytoskeleton organization, cell lineage specification, organ development, and oncogenesis. The enigma-homologue LIM domain (ENH) protein was expressed in various regions of the brain, most notably in the hippocampi, cortex, thalamus, hypothalamus, amygdala, and cerebellum. In hippocampal neurons, ENH appears to be localized in presynaptic nerve terminals. PDLIM5 is also a homolog of the AD-associated neuronal thread protein (AD7c-NTP), which is over-expressed in Alzheimer disease sufferers, beginning early in the course of the disease. Iwamoto et al. compared the mRNA levels of PDLIM5 in the lymphoblastoid cell lines (LCLs) and postmortem brains of subjects with mental disorders with those of controls. They found that the mRNA level of PDLIM5 was decreased in the lymphoblastoid cells derived from patients with schizophrenia and bipolar disorders, but commonly increased in the postmortem brains of patients with schizophrenia, bipolar disorders, and major depression. Moreover, Kato et al. provided confirmation of up-regulation of the mRNA level of PDLIM5 in an independent brain sample set obtained from the Stanley Array Collection. On the other hand, PDLIM5 is located on human chromosome 4q22.3, which was suggested a hotspot region with susceptibility genes of schizophrenia by several independent linkage studies. These evidences imply that PDLIM5 is a potential susceptible gene for schizophrenia. In this work, we selected six common SNPs in the region of PDLIM5 according to the previous studies and the information of PDLIM5 in NCBI and HapMap databases and designed appropriate primers for these SNPs. Then we acquired the genotypes of the six common SNPs in the case-control samples of Jiangxi province using the single nucleotide primer extension method, and studied the association relationship between the PDLIM5 gene and schizophrenia using Hapview and COCAPHSE softwares. Among six SNPs we selected, rs2433322 showed significantly different frequencies between cases and controls (P=0.000010). Haplotypes constructed by rs2433322 were also significantly associated with schizophrenia (Global P=0.00019 even after strict Bonferroni correction). Our results provide further evidence to support PDLIM5 as a potential susceptible gene for schizophrenia.更多还原