CCKAR、DRD2、DAT和SNAPIN基因与偏执型精神分裂症的关联研究及其功能初探

发布时间：2019-05-21 09:54

【摘要】：精神分裂症是一种病因尚未完全阐明的重性精神疾病。人群终生患病率约为1%,多于青壮年发病,严重威胁人类的身心健康,给家庭和社会带来沉重负担。精神分裂症是一种典型的多基因疾病,其遗传度高达80%；但由于遗传异质性、基因间/位点间联合作用以及基因-环境相互作用等因素的存在,加剧了该病的复杂性。精神分裂症的多巴胺假说是普遍接受的假说之一,多巴胺系统功能紊乱被认为是精神分裂症发病的最终共同途径。本组前期工作对多巴胺信号转导通路相关基因筛查发现胆囊收缩素A型受体(Cholecystokinin type A receptor, CCKAR)基因与幻听相关。另一项前期工作初步发现SNAP相关蛋白(SNAP-associated protein, SNAPIN)基因与精神分裂症相关。本文以多巴胺假说为理论依据,在前期工作的基础上,应用病例-对照的研究方法,以CCKAR、多巴胺受体1-5(Dopamine receptor D1-D5, DRD1-5)基因、多巴胺转运体(Dopamine transporter, DAT)基因和SNAPIN基因为候选基因,扩大样本验证基因的功能多态性位点与中国汉族人群偏执型精神分裂症的相关性,并对部分相关基因在nRNA表达、miRNA调控及相互作用蛋白等层面进行功能学探索。多项研究显示CCKAR基因的rs1800857与精神分裂症的幻听等阳性症状相关。我们在522名偏执型精神分裂症患者和527名对照中检测了CCKAR基因5’端调控区的rs1800857等5个单核苷酸多态性位点(Single nucleotide polymorphism, SNP)。尽管未能发现rs1800857与疾病相关,但观察到rs1800857对其上游的其他4个SNPs均存在顺势调节作用,并且存在3种5-SNP单倍型G-A-G-C-T、T-G-G-G-T和T-G-G-C-C均为疾病发生的风险因素(p=2.2×10-5,p=1.1×10-6'p=1.9×10-6),说明CCKAR基因的等位基因异质性参与精神分裂症的发生。同时,数量性状分析发现5-SNP单倍型与PANSS量表中幻觉行为、猜疑／被害、敌对性和阳性症状总分显著相关。此外,我们采用real-time PCR检测了抗精神分裂症药物氟哌啶醇(Haloperidol)和氯氮平(Clozapine)对CHP212细胞系中CCKAR基因表达的影响,发现36h内氟哌啶醇使CCKAR基因的表达量出现先升高再恢复到初始表达水平的现象,而氯氮平组并未观察到明显变化。对DRD1-5和DAT基因初步筛查发现DRD2和DAT与偏执型精神分裂症相关。随后在1351名患者和1640名对照中验证了DRD2基因的rs1076560-T可提高精神分裂症的发病风险(p=0.022)。同时,在368名患者和420名对照中发现DAT基因的rs2455391与疾病相关(allelic p=0.015,genotypicp=0.018)其单倍型rs2975223(G)-rs2455391(C)在患者组中的频率显著高于对照组(p=0.0012)。 Snapin蛋白可结合SNAP-25以促进SNARE复合物的形成进而促进突触囊泡的释放,其相互作用蛋白dysbindin的编码基因DTNBP1是精神分裂症最热门的易感基因之一。在516名患者和532名对照样本中我们首次发现SNAPIN基因3’UTR的rs7345与偏执型精神分裂症相关(allelic p=0.014,genotypic p=0.003)；但体外报告基因实验并未发现rs7345位点直接影响基因表达。随后,通过生物信息学预测发现hsa-miR-30家族可能与SNAPI-3'UTR结合,经双荧光素酶报告基因方法检测发现miR-30e可以结合SNAPIN-3'UTR,并抑制报告基因的表达。在113名患者和127名对照血浆样本中发现患者组miR-30e含量较对照组明显降低(p=0.001)。之后,我们又在蛋白质相互作用层面应用串联亲和纯化(tandem affinity purification,TAP)结合质谱鉴定的方法,初步找到57种Snapin的候选相互作用蛋白。综上所述,我们不仅在遗传学水平上验证了CCKAR.DRD2.DAT和SNAPIN基因在中国汉族人群中与偏执型精神分裂症相关,而且在药物对基因表达水平、miRNA调控水平及蛋白相互作用水平初步探索了易感基因的功能。
[Abstract]:Schizophrenia is a major mental disorder that is not yet fully set forth in the cause of schizophrenia. The lifetime prevalence of the population is about 1%, more than the onset of the young and the middle-aged, the physical and mental health of the human being seriously threatened, and the heavy burden on the family and the society. Schizophrenia is a typical multi-gene disease, and its genetic degree is as high as 80%; however, due to the genetic heterogeneity, the combination of intergenic/ intersite and the gene-environment interaction, the complexity of the disease is aggravated. The dopamine hypothesis of schizophrenia is one of the most commonly accepted hypotheses, and the dysfunction of the dopamine system is thought to be the final common way of the onset of schizophrenia. In this study, we found that the gene of the type A receptor (CCKAR) of the cholecystokinin A receptor (CCKAR) and the auditory phase were found in the early stage of this group. On the other hand, SNAP-associated protein (SNAP-associated protein, SNAPIN) gene and schizophrenia On the basis of the hypothesis of dopamine, this paper, on the basis of the previous work, applied the case-control study to the candidate base of the gene of CCKAR, dopamine receptor 1-5 (Dopammine receptor D1-D5, DRD1-5), the dopamine transporter (DAT) and the SNAPIN gene. The relationship between the functional polymorphism site of the sample verification gene and the paranoid schizophrenia in the Chinese Han population was expanded, and some related genes were studied in the aspects of nRNA expression, miRNA regulation and interaction protein. The results showed that the rs1800857 of the CCKAR gene and the auditory hallucination of the schizophrenia were positive. In 522 paranoid and 527 controls, we tested 5 single-nucleosonic acid polymorphism sites, such as rs1800857 of the 5 '-end regulatory region of the CCKAR gene, S NP). Although rs1800857 was not found to be related to the disease, it was observed that rs1800857 had a homeopathic regulatory effect on the other 4 SNPs upstream of it, and there were three 5-SNP haplotype G-A-G-C-T, T-G-G-G-T and T-G-G-C-C all the risk factors for disease (p = 2.2)10-5, p = 1.1,10-6 'p = 1.9%10 -6), indicating that the allele heterogeneity of the CCKAR gene is involved in the schizophrenia Occurrence of hallucination, suspicion/ murder, hostility and positive symptoms in 5-SNP haplotype and PANSS scale were found in quantitative trait analysis. In addition, the effect of clozapine and clozapine on the expression of CCKAR gene in CHP212 cell line was detected by the real-time PCR, and the expression of CCKAR gene in 36 h was first raised and then returned to the initial expression level. And the chlorazepine group was not observed. Significant changes. Preliminary screening of DRD1-5 and DAT genes found DRD2 and DAT and paranoia The risk of the onset of schizophrenia (p = 0) was then increased in 1351 patients and in the 1640 control with the rs1076560-T of the DRD2 gene. .022). At the same time, the frequency of rs2455391 (G)-rs2455391 (C) of the DAT gene was found to be significantly higher in the patient group than in the control group (p = 0) in 368 patients and 420 controls. 0012). The Snapin protein can be combined with SNAP-25 to promote the formation of the SNARE complex and further promote the release of the bursal, and the coding gene DNBP1 of the interaction protein, dlybindin, is the most popular in the case of schizophrenia. One of the susceptible genes was found in 516 and 532 control samples for the first time that the rs7345 of the SNAPIN gene 3 'UTR was associated with a paranoid type of schizophrenia (allelic p = 0.014, genetic p = 0.003); however, the in vitro reporter assay did not find that the rs7345 site was straight The expression of the influence gene was affected. Subsequently, the hsa-miR-30 family was found to be bound to the SNAPI-3 'UTR by bioinformatics prediction, and it was found that the miR-30e could bind to the SNAPIN-3' UTR by the double luciferase reporter gene method and inhibit The expression of the reporter gene. In 113 patients and 127 control plasma samples, the content of miR-30e in the patient group was significantly lower in the control group (p = 0.001). After that, we applied tandem affinity purification (TAP) in the protein-interaction level to identify the 57 types of Snapin by means of tandem affinity purification (TAP) and mass spectrometry. To sum up, we have not only verified that the CCKAR. DRDD2. DAT and SNAPIN gene are related to the paranoid schizophrenia in the Chinese Han population, but also the level of gene expression, the level of miRNA regulation and the level of protein interaction in the Chinese Han population.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R749.3

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