【作者】 刘JIE；

【导师】 贺林；

【作者基本信息】 上海交通大学 ， 遗传学， 2011， 博士

【摘要】 复杂疾病是指由基因组中多个基因,往往还伴有环境因素以及其它未知因素的影响共同作用而导致的一类疾病的总和。常见的复杂疾病有:精神分裂症、重度抑郁症、双向情感障碍、糖尿病视网膜病变、心血管疾病等。由于目前对这类疾病的发病机制缺乏了解,因而也日益成为广大科研工作者日益关注的焦点。在本论文中,我们进行了YWHAE基因和中国汉族人群中精神分裂症、重度抑郁症及双向情感障碍的连锁不平衡分析,CTLA-4基因和中国汉族人群中精神分裂症、重度抑郁症及双向情感障碍的连锁不平衡分析。YWHAE基因定位于染色体17p13.3上,编码14-3-3ε蛋白,该蛋白属于14-3-3蛋白家族。因为14-3-3ε可能参与神经系统疾病的发生,且DISC1蛋白可以与14-3-3ε相互作用并调节其亚细胞定位,而DISC1基因已经被证实与精神疾病的发病高度相关,那么我们也有理由怀疑编码14-3-3ε的YWHAE基因与精神疾病相关联。Ikeda等的研究表明,YWHAE基因与日本人群中精神分裂症的发病高度相关。为了检验YWHAE是否和中国人群中的精神疾病相关联,我们进行了病例-对照的关联分析。我们选择了Ikeda等报道的在日本人群中与精神分裂症呈阳性关联的7个位点: rs34041110、rs7224258、rs3752826、rs11655548、rs2131431、rs1873827和rs28365859,此外还选择了4个中国人群的tagSNP:rs12452627、rs1532976、rs8064578和rs7225165。对于11个SNP的分型采用了Taqman技术,直接向ABI公司订购Taqman探针。我们对1140个精神分裂症患者、1140个重度抑郁症患者、1140个双向情感障碍患者及1140个正常对照人群进行了关联分析。病例对照分析中,我们没有重复Ikeda等报道的阳性结果,我们发现:在1140对1140的病例-对照分析中,未经过permutation校正之前有4个位点与精神分裂症的发生存在弱相关,经过10000次permutation之后,11个遗传多态性位点的数据均未显示出阳性结果。在1140对1140的病例-对照分析中,未经过permutation校正之前有3个位点与抑郁症的发生存在弱相关,经过10000次permutation之后,11个遗传多态性位点的数据均未显示出阳性结果。在1140对1140的病例-对照分析中,未经过permutation校正之前有3个位点与双向情感障碍的发生存在弱相关,经过10000次permutation之后,11个遗传多态性位点的数据均未显示出阳性结果。关联分析的重复性差是普遍存在的问题,究其原因可能是不同人群中的LD block不同造成的,而且从数据统计效力上来讲,我们的样本量已经足够检测出与精神分裂症相关联的风险位点。所以我们觉得在中国人群中,我们所得到的YWHAE并非是一个精神疾病致病的风险基因这一结论是可靠的。尽管我们未能在中国人群中重复出YWHAE与精神分裂症之间存在关联这一结论,但也给后续研究工作提供了一定的参考。关于YWHAE与精神疾病之间的关系还有待于在其他人群以及更大规模的样本中得到进一步的论证。CTLA4 (cytotoxic T lymphocyte associated antigen 4 )基因,定位于染色体2q33,包含4个外显子和3个内含子。根据现有资料,CTLA-4基因多态性具有重要的生物学意义, CTLA-4基因内或者基因附近的多态性与许多自身免疫性疾病相关。目前研究较多的主要有3个多态性位点,即启动子-318位点C/T碱基互换(-318C/T)、外显子1+49位点A/G二态性(+49G/A)和外显子4的3’未翻译区(AT)n重复序列。近年来有关精神疾病患者中枢神经系统(CNS)免疫功能的异常,已越来越引起人们的关注。在精神分裂症和其他重性精神障碍患者中,存在着感染或自身免疫活动,具体表现在细胞免疫系统(T淋巴细胞)处于激活状态;结合细胞因子和自身抗体的研究,提示某些精神分裂症患者存在自身免疫应答。鉴于CTLA-4在T细胞激活及自身免疫疾病发生中的重要作用,该基因与精神疾病的关联研究也受到关注。Jun等在韩国人群中发现CTLA-4基因+49G/A位点与精神分裂症的发病相关,但与抑郁症的发病并无显著性关联;而Amanda等的研究则表明,在高加索人群中女性精神分裂症的发病与-318 C/T位点相关,但与+49G/A位点并不相关。为了检验CTLA-4是否和中国人群中的精神疾病相关联,我们进行了病例-对照的关联分析。我们在1140个精神分裂症患者、1140个重度抑郁症患者、1140个双向情感障碍患者及1140个正常对照人群进行了关联分析。我们选择了6个SNP位点,除了目前研究较多的三个位点rs231775(A49G), rs3087243 (3’UTR A/G), rs5742909(-318C/T)外,还有3个中国人群中的tagSNP rs231777, rs231779, rs16840252。对于6个SNP的分型采用了Taqman技术,直接向ABI公司订购Taqman探针。1140对1140的病例-对照分析中,未经过permutation校正之前有3个位点与精神分裂症的发生存在相关:rs231777 (P allele = 0.0201) rs231779 (P allele = 0.0003) and rs16840252 (P allele = 0.0081, Pgenotype = 0.0117),在10000次permutation之后,仅rs231779 (P allele = 0.0010, Pgenotype = 0.0145)与精神分裂症的发生存在相关性。根据我们的结果,CTLA-4 rs231779位点多态性与中国汉族人群精神分裂症发病相关。1140对1140的病例-对照分析中,仅rs231779在10000次permutation前(P allele = 0.0006, Pgenotype = 0.0026) ,后(P allele =0.0010, Pgenotype = 0.0201),均与抑郁症的发生存在相关性。根据我们的结果,CTLA-4 rs231779位点多态性与中国汉族人抑郁症发病相关。1140对1140的病例-对照分析中,rs231777 (P allele = 0.0199), rs231779 (P allele = 0.0004, Pgenotype = 0.0018)与双向情感障碍的发生存在相关性,而在10000次permutation之后,rs231779 (P allele = 0.0008, Pgenotype = 0.0125)与双向情感障碍依然相关。根据我们的结果,CTLA-4 rs231779位点多态性与中国汉族人群双向情感障碍发病相关。综上所述,CTLA-4基因的rs231779位点多态性与中国汉族人中精神分裂症、抑郁症及双向情感障碍的发生密切相关,病例组中该位点等位基因C的频率明显高于对照组中,基因型CC的频率在病例组中也明显增高,该结果提示rs231779位点基因型CC可能在精神疾病的发生中起重要作用。我们的结果与Green等关于CACNA1C的rs1006737位点与精神分裂症、抑郁症和双向情感障碍的发生相关联的报道非常相似。而Becker于2004年提出常见变异/多个疾病(common variant/multiple diseases, CV/MD)假说认为,表型相关的一些复杂疾病,在遗传学上共享一些风险等位基因,常见变异在不同的遗传和环境因素背景下将会导致不同但相关的临床表型。比如,精神分裂症,可能与双向情感障碍以及抑郁症,有着相关的临床症状,也共享有一些致病基因。我们的研究为这一理论提供了进一步的实验证据,也为精神疾病的诊断及病因学分析提供了参考。人类Chd7基因位于染色体8q12.1,一共有38个外显子,编码了2997个氨基酸。Chd7是一种染色质重构蛋白质,属于CHD家族。CHD蛋白家族是目前已知的染色质重构复合物之一。而染色质重构是DNA修复、基因表达调控过程中的一个重要环节。近年来的研究表明,Chd7基因多个位置的突变与人类CHARGE综合症及卡尔曼氏综合征(Kallmann’s syndrome )相关。CHARGE综合症的发病率为1/8500-12000的活产儿。它的死亡率在新生儿和幼年早期很高。小鼠Chd7基因定位于4号染色体,其序列高度保守,小鼠与人之间同源性非常高。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。我们实验室在前期工作中,在ENU诱变的小鼠模型中发现了有一个系的小鼠有转圈的表型,而该表型在Chd7基因突变的小鼠中也可见,经过进一步的基因定位和测序在Chd7基因exon13中出现了一个A/G杂合子的突变,该突变将会引起exon13内密码子CGA到TGA的转变(3503C-T),即精氨酸(Arg,R)到终止子的变化,会导致翻译的提前终止,Chd7基因SNF2结构域的一部分缺失和整个的C端消失。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。本论文的另一部分工作是对突变小鼠做初步的表型分析,并利用这一模型对Chd7基因的功能做进一步的研究。基本状况分析结果发现,Chd7突变小鼠体重明显减轻,而心脏与体重的比例则明显增加,其他脏器在重量上无明显变化。X光片检查未发现突变小鼠有脊柱侧弯现象。Chd7基因表达谱分析,RT-PCR的方法检测了Chd7在不同组织中的表达情况,结果显示RNA水平有多个组织均有表达,而以肌肉、睾丸、骨髓表达最高。流式分析结果发现,在外周血中,粒细胞比例明显增加,B细胞比例明显减少而T细胞比例略有下降;而在脾脏中,B细胞比例明显下降、粒细胞比例略有升高、T细胞比例略有下降而其中CD4+T细胞无变化CD8+T细胞比例下降。本结果提示Chd7可能在T细胞、B细胞及粒细胞的增殖分化过程中发挥一定作用。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, major depresive disorder, bipolar disorder, cardiovascular disease, et al. With the increase of social pressure and the speedup of social life, people are paying more and more attention to psychiatric disorders. The first and second parts of my thesis is about linkage disequilibrium study on YWHAE and CTLA-4 genes of schizophrenia, major depresive disorder, and bipolar disorder in Chinese Han population. YWHAE is a gene encoding 14-3-3epsilon, which is highly conserved across species, from bacteria to humans, and binds to phosphoserine/phosphothreonine motifs in a sequence-specific manner.YWHAE has been reported to be associated with schizophrenia in a study based on the Japanese population. Here, we conducted a genetic association analysis between common SNPs in the YWHAE gene and psychiatric diseases including schizophrenia, major depressive disorder and bipolar disorder in Han Chinese samples (1140 schizophrenia cases, 1140 major depressive disorder cases, 1140 bipolar disorder cases and 1140 normal controls). We studied 11 SNPs, 7 of which had previously been reported as significant, in YWHAE. No association was found with schizophrenia, major depressive disorder or bipolar disorder. Considering the size of our sample sets (power>90%), our results suggest that the YWHAE does not play a major role in schizophrenia, major depressive disorder or bipolar disorder in the Han Chinese population. Ikeda M et al. identified YWHAE, as a possible susceptibility gene for schizophrenia in the Japanese population. YWHAE encodes 14-3-3epsilon, which is an interacting partner of DISC1. DISC1 has been identified as a potential susceptibility gene for major psychiatric disorders including schizophrenia, depressive disorder and bipolar disorder. Several DISC1 interactors have also been defined as independent genetic susceptibility factors for psychiatric illness. DISC1 is a hub protein in a multidimensional risk pathway for major psychiatric illness, and studies of this pathway are opening up opportunities for a better understanding of causality and possible mechanisms of intervention. As an interacting partner of DISC1, 14-3-3epsilon may also play an important role in the development of these psychiatric disorders. In 2005, Yanagi et al.’s study showed that YWHAE is associated with suicide in the Japanese population, but to date, no other studies about the relationship of YWHAE and major depressive disorder or bipolar disorder have been reported. To investigate whether the YWHAE gene plays a significant role in psychiatric diseases, we focused on the YWHAE gene in schizophrenia, major depressive disorder and bipolar disorder in Han Chinese samples involving 1140 schizophrenia patients, 1140 major depressive disorder cases, 1140 bipolar disorder patients and 1140 normal controls. We genotyped 11 SNPs in YWHAE, including all the seven positive SNPs in Ikeda M et al.’s study (rs34041110, rs7224258, rs3752826, rs11655548, rs2131431, rs1873827 and rs28365859), and also selected additional four Tag SNPs (rs12452627, rs1532976, rs8064578, rs7225165) from the HapMap database to provide a good coverage of the gene. In schizophrenia group, only rs1873827, rs3752826, rs12452627 and rs7225165 are marginally associated with schizophrenia, but after 10,000 permutations, there was no association. In major depressive disorder group, only rs34041110, rs12452627 and rs7225165 show some marginal relationship with major depressive disorder, but no association after 10,000 permutations. In bipolar disorder group, rs1873827, rs1532976 and rs7225165 show some marginal association with bipolar disorder, but this was not remained after 10,000 permutations. In all we found that YWHAE has no association with these major mental disorders in the Han Chinese population, although several loci have marginal association with one or all of these disorders before permutations. It is a general problem that the association of one gene with complex diseases in one population cannot be replicated in another population. It may depend on the different LD blocks between different populations or on the different frequency of alleles between populations. Another reason that leads to the inconsistency may be the different endophenotypes that exist. In summary, this first case-control study based on samples in the Chinese population failed to prove the 11 SNPs of YWHAE with susceptibility to schizophrenia, major depressive disorder or bipolar disorder in general. Though we found no association, considering the current sample size and the effect size (OR=0.67) from the previous study, the power > 90% (we used the tool of GPower 3.1), our results can provide a reference for further studies of YWHAE gene in other populations. Further work with large sample size or from different populations and corresponding functional analysis is still required to fully elucidate the exact role of YWHAE in the pathogenesis of schizophrenia and other psychiatric disorders.Previous studies have reported that the cytotoxic T lymphocyte antigen-4 (CTLA-4) gene, which is related to immunological function such as T-cell regulation, is associated with psychiatric disorders. It is involved in establishing and maintaining peripheral T-cell tolerance, which controls T-cell activation and reactivity. The CTLA-4 gene is located on the long arm of chromosome 2q33. It consists of 4 exons and encodes a costimulatory molecule that is expressed on the surface of activated T cells. Based on function and experimental data, it has been suggested as a candidate gene for conferring susceptibility to autoimmune disease. Moreover, several researchers have reported the association of common SNPs in CTLA-4 with schizophrenia or major depressive disorder. 3 SNPs (rs231775, rs3087243, rs5742909) have been analyzed in previous studies. Jun et al. reported significant association of rs231775 (A49G) in exon 1 with schizophrenia but not with major depressive disorder in the Korean population. However, Jones et al. found that in males, both the promoter region rs5742909 (-318C/T) and the 3’UTR SNP rs3087243 demonstrated nominally significant association with schizophrenia in Caucasian populations. In this study, we studied the relationship between CTLA-4 and three major psychiatric disorders, schizophrenia, major depressive disorder and bipolar disorder in the Chinese Han population. We recruited 1140 schizophrenia patients, 1140 major depressive disorder patients, 1140 bipolar disorder patients, and 1140 normal controls to examine the risk conferred by 6 tag SNPs (rs231777, rs231775, rs231779, rs3087243, rs5742909, rs16840252) in the CTLA-4 gene. For schizophrenia, rs231777 (Pallele = 0.0201), rs231779 (Pallele = 0.0003, Pgenotype = 0.0016) and rs16840252 (Pallele = 0.0081, Pgenotype = 0.0117) showed nominal significance in the tests and after 10,000 permutations, rs231779 remained significant (Pallele = 0.0010, Pgenotype = 0.0145). For major depressive disorder, only rs231779 (P allele= 0.0006, Pgenotype = 0.0026) showed nominal significance in the tests and after 10,000 permutations it remained significant (Pallele = 0.0010, Pgenotype = 0.0201). For bipolar disorder, rs231777 (Pallele=0.0199), rs231779 (Pallele = 0.0004, Pgenotype = 0.0018) showed nominal significance in the tests. After 10,000 permutations, only rs231779 remained significant (Pallele = 0.0008, Pgenotype = 0.0125). This result is very interesting, as it has been hypothesized that some genes will influence the risk for relatively specific domains of psychopathology, whereas others will have a more general influence on the risk. In conclusion, our case-control study provides the evidence that CTLA-4 may play a role in the susceptibility to major psychiatric disorders in Han chinese population. Although the significant associated SNP rs231779 in this study located in the intron, we can’t exclude its possible role in the expression of CTLA-4. Our result is a clear demonstration that there is an overlap in the biological underpinnings of susceptibility to mental illness across the clinical spectrum of mood and psychotic disorders. At least some psychiatric patients with immune alterations may partially share the aetiology, development and pathogenic mechanism of the disease. However, furthermore studies for the functional role that CTLA-4 may play in the psychiatric disorders are needed, and it will give some new information which is useful for the diagnosis and therapy of these diseases. And all of this will be useful in influencing psychiatric research to move from reliance on a diagnostic and classification system that is based only on clinical description and towards a scheme that better reflects the underlying biology of the psychiatric entities encountered in our clinics. This will be of great benefit to patients. Our data, based on Han Chinese population in this study, have given some cues about this.Another part of our work is 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. CHARGE syndrome follows an autosomal dominant mode of inheritance. Its incidence of 1:8500 was evaluated in a Canadian study and is suggested to range between 0.1and1.2/10,000 live births. Mutations in the chromodomain helicase DNA-binding protein 7 (CHD7) gene in 8q12.1 were identified as causative for CHARGE syndrome. The gene has 37 coding exons and encodes a 2997-amino acid protein that belongs to the family of chromodomain helicase DNA-binding (CHD) proteins. In humans at least nine CHD genes are known. Functional domains such as chromo (chromatin organization modifier), SNF2- related helicase/ATPase and BRK were identified in the CHD7 protein. The majority of CHD7 gene mutations is unique and occurs de novo. 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