【学位级别】医学博士

【论文完成日期】2004-5-20

【论文中文题名】孤独症功能候选基因的关联研究

【论文外文题名】The association studies of candidate genes with autism in the Chinese trios

【论文著者】姓名：邬素萍 wu su ping

【论文著者】学号：B10199767

【论文著者】系别：精神卫生研究所

【论文著者】专业：精神病与精神卫生学

【论文著者】研究方向：分子遗传学

【指导教师】姓名：张岱 zhang dai

【指导教师】学校：北京大学医学部

【指导教师】系别：精神卫生研究所

【指导教师】专业：精神病与精神卫生学

【中文文摘】孤独症是一种严重的发育障碍，临床表现以社会交往障碍、兴趣与活动内容局限、刻板和重复行为为特征。一般发病于婴幼儿期，少数患儿5-6岁时症状才明显。该病在儿童群体中的患病率为1.1-2.5‰。 迄今为止，孤独症的病因及发病机制尚不清楚。近年来，孤独症双生子及家系研究结果证实遗传因素在其发病中起着重要的作用。目前，基因组扫描结果发现了一些阳性连锁区域，如染色体1p、2q、3p、6q16-21、7q31-33、13q、15q和17等，这些区域都成为孤独症易感基因的候选区域。因此，本研究根据基因功能和动物模型的研究结果，在上述热点区域选取功能候选基因，如神经递质受体基因，神经肽受体基因以及与神经发育有关的基因，用聚合酶链式反应-限制性片段长度多态性(PCR-based RFLP)分析方法，在中国汉族孤独症核心家系中进行功能候选基因的关联研究。 本论文的主要内容和研究结果如下： 第一部分 5-HT转运体基因(SLC6A4) 目的　验证5-HT转运体基因(SLC6A4)在中国汉族孤独症易感性中的作用。 方法　在175例孤独症核心家系(每例含有血缘关系的父母和一个患病子或女)中，用聚合酶链式反应-限制性片段长度多态性(PCR-based RFLP)分析方法对SLC6A4基因的八个单核苷酸多态性位点进行检测。其中三个多态性位点为Kim等发现与孤独症存在显著关联的位点。 结果　在研究的八个单核苷酸多态性位点中，有五个位点在本研究样本中不是多态性位点，其中一个位点为Kim等报道的阳性位点。其它三个多态性位点在传递不平衡检验(TDT)中，显示等位基因传递没有统计学显著性。多态性位点间的连锁不平衡检验显示这三个多态性位点明显连锁，在一个单体型块 (haplotype block)中，可以构建三个多态性位点组成的单体型模式。单体型分析(haplotype analysis)显示三个多态性位点的单体型传递也没有统计学显著性。 结论　SLC6A4基因的这些多态性位点并不增加中国汉族孤独症的患病风险，可初步排除这些位点与孤独症的关联。 第二部分 oxytocin 受体基因(OXTR) 目的　功能分析和动物模型显示OXTR基因可能在孤独症的发生与发展起一定的作用，本研究将检测OXTR基因的多态性位点与孤独症的关系。 方法　根据生物信息学的检索，分别在OXTR基因5’端非翻译区、编码区和内含子上选取八个有酶切位点的单核苷酸多态性位点进行研究。这些位点分别是：rs2254298、rs53576、rs237893和 rs237894 (内含子)，rs237911 (非翻译区)，rs2228485 (同义改变)，rs237901 和 rs810568 (错义改变)。在195例孤独症核心家系(每例含有血缘关系的父母和一个患病子或女)中，用聚合酶链式反应-限制性片段长度多态性(PCR-based RFLP)分析方法对这些多态性位点进行检测。 结果　在本研究样本中未能证实rs237901、rs810568、rs237893和rs237894是多态性位点。其它四个多态性位点在以家系为基础的关联检验(FBAT)中，有两个多态性位点显示等位基因传递具有统计学显著性(rs2254298的A：Z=2.287, P=0.022168；rs53576的A：Z= 2.573, P=0.010075)。多态性位点间的连锁不平衡检验显示三个多态性位点rs53576、rs2228485和 rs237911紧密连锁，在一个单体型块 (haplotype block)中, 可以构建三个多态性位点组成的单体型模式。单体型传递的总体检验显示OXTR基因与孤独症存在关联(χ2 = 15.237，自由度= 5，P = 0.009396)，单个单体型的分析也显示单体型ATA 在患者中过度传递，差异有显著性(Z = 2.496，P = 0.012576)。 结论　OXTR基因与孤独症存在关联，可能在孤独症的发病中起重要作用。这是首次对OXTR基因与孤独症关系的报道，尚需在不同地域，不同种族的研究中加以验证。 第三部分 neuropilin 2基因(NRP2) 目的　neuropilin 2具有轴突定向和控制神经元在中枢神经系统迁移的作用，而且，neuropilin 2基因(NRP2)定位于孤独症的易感区域染色体2q，本研究假设NRP2基因是孤独症的易感基因，检测该基因多态性位点与孤独症的关系，探讨其在孤独症的发病中的作用。 方法　根据生物信息学的检索，分别在NRP2基因编码区和内含子上选取两个有酶切位点的单核苷酸多态性位点进行研究。在166例孤独症核心家系(每例含有血缘关系的父母和一个患病子或女)中，用聚合酶链式反应-限制性片段长度多态性(PCR-based RFLP)分析方法对这两个多态性位点进行检测。 结果　以家系为基础的关联检验(FBAT)显示等位基因传递具有统计学显著性(rs849578的C：Z=2.286，P=0.022243；rs849563的A：Z=2.186，P=0.028784)。多态性位点间的连锁不平衡检验显示两个多态性位点(rs849578和 rs849563)紧密连锁，在一个单体型块 (haplotype block)中，可以构建两个多态性位点组成的单体型模式。单体型传递的总体检验显示NRP2基因与孤独症存在关联(χ2 =12.595，自由度=3，P= 0.0056)。单个单体型的分析也显示单体型 CA 在患者中过度传递，差异有显著性(Z = 2.907，P = 0.003651)。 结论　本研究首次对NRP2基因与孤独症关系进行研究，提示NRP2基因与孤独症存在关联，可能是孤独症的易感基因，但尚需进一步研究该基因其它位点并在其它种族中验证。 第四部分 γ-氨基丁酸A型受体β3亚单位基因(GABRB3) 目的 细胞遗传学研究显示染色体15q11-q13的异常与孤独症有关，有证据表明该区域内基因GABRB3的多态性与孤独症存在连锁不平衡。目前的研究很多，但是结果多不一致。由于疾病的易感位点受地域、种族和人群的影响，因此本研究将探讨GABRB3基因在中国汉族孤独症易感性中的作用。 方法 根据生物信息学的检索，分别在GABRB3基因上选取六个有酶切位点的单核苷酸多态性位点进行研究。在174例孤独症核心家系(每例含有血缘关系的父母和一个患病子或女)中, 用聚合酶链式反应-限制性片段长度多态性(PCR-based RFLP)分析方法对这六个多态性位点进行检测。 结果 在本研究样本中未能证实rs1426218, rs3212333和 rs4906886是多态性位点。其它三个多态性位点在以家系为基础的关联检验(FBAT)中，显示等位基因的传递没有统计学显著性。多态性位点间的连锁不平衡检验显示两个多态性位点(rs1432007 和rs1426208)连锁，在一个单体型块 (haplotype block)中, 可以构建两个多态性位点组成的单体型模式。单体型传递的总体检验显示GABRB3基因与孤独症不存在关联，单个单体型的分析也未显示存在关联。 结论 GABRB3基因的这些多态性位点不是孤独症的易感位点，不影响中国汉族孤独症的易感性。

【外文文摘】Autism is a neurodevelopmental disorder characterized by significant disturbances in social, communicative, and behavioral functioning. Developmental abnormalities are apparent in the first 3 years of life with a prevalence of 1.1-2.5‰. The etiology of autism is largely unknown. However, there is strong evidence for genetic components in the development of idiopathic autism. Family and twin studies indicate that autism is one of the most strongly genetic neuropsychiatric disorders. Several genome screens for autism susceptibility loci have found a mumber of chromosomal regions that show some evidence of linkage, for example, chromosome 1p, 2q, 3p, 6q16-21, 7q31-33, 13q, 15q and 17. The regions may harbor candidate genes responsible for the development of autism. Therefore, based on the interesting regions, we conducted association studies between the candidate genes and autism in the Chinese Han family trios by using the polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) analysis. The main contents and results of the study are as below: 1. Association studies of the serotonin transporter gene (SLC6A4) polymorphisms with autism in the Chinese Population Serotonin regulates several aspects of brain development, is involved in a range of behaviors frequently disturbed in autistic disorder. The serotonin transporter is a critical component of the serotonergic system. The serotonin transporter gene (SLC6A4) is of special interest given the nature of the biological findings and the reported effects of selective serotonin reuptake inhibitors of autistic symptoms. Kim et al. analyzed twenty single nucleotide polymorphisms (SNPs) and seven simple sequence repeat (SSR) polymorphisms of SLC6A4 in 115 autism trios. Seven SNP and four SSR markers showed nominally significant evidence of transmission disequilibrium by the transmission disequilibrium test (TDT). To further investigate the role of SLC6A4 in autism susceptibility we conducted an association analysis to test the relationship between SLC6A4 and autism in the Chinese population. In the present study, we chose two SNPs described by Kim et al. and the second intron SNP to conduct association analysis. Application of TDT to genotype data from these three polymorphisms, as well as the haplotype analysis, revealed no evidence for transmission disequilibrium with autism. Based on these results, it appears unlikely that SLC6A4 contributes significantly to the predisposition to develop autism in our sample. Therefore, our conclusion regarding the SLC6A4 gene does not agree with that in the report by Kim and colleagues. Thus, our analysis did not suggest an association between the SLC6A4 gene and susceptibility to autism in the Chinese population. 2. Family-based association studies of the oxytocin receptor gene (OXTR) polymorphisms with autism in the Chinese trios Autism is a neurodevelopmental disorder of unknown etiology. Impairment in social reciprocity is a central component of autism. Oxytocin (OT) plays an important role in social-affiliative behaviors. Previous study has suggested that the social impairments found in autistic disorder are associated with changes in plasma OT levels. OT’s effect appear to be mediated by a receptor. The oxytocin receptor gene (OXTR) is excellent candidate for exploration in certain psychiatric disorders, particularly those involving social impairments. For these reasons, we hypothesized that the OXTR gene may be a good candidate for genetic studies of autism. We completed an association analysis with four single nucleotide polymorphisms (SNPS) located within the OXTR region using the family-based association tests (FBAT) in 195 Chinese Han trios. We found nominally significant transmission desequilibrium between autism and two of the SNP markers (rs2254298: Z=2.287, P=0.022168; rs53576: Z= 2.573, P=0.010075). Of three SNPS (rs53576, rs2228485 and rs237911) were in strong disequilibrium by linkage disequilibrium analysis. They were in the same haplotype block. The global chi-square test for the haplotype transmission also revealed a strong association (χ2 = 15.237，df= 5，P = 0.009396). Haplotype ATA showed the excess transmission to affected offspring. The results suggest that the OXTR gene may play a role in predisposing an individual to autism. Our preliminary results provide additional evidence that the OXTR gene may be important risk factors for the development of autism. 3. Family-based association studies between the neuropilin-2 gene (NRP2) polymorphisms and autism in the Chinese trios The complex wiring of the adult nervous system depends on the occurrence during neurodevelopment of an ordered series of axon guidance decisions that ultimately lead to the establishment of precise connections between neurons and their appropriate targets. Neuropilins, which were identified neuropilin-1 and -2, are receptors for class 3 secreted semaphorins. Semaphorins 3A and 3F are axon guidance proteins during nervous system development. Neuropilin 1 and 2 are natural ligands for semaphorins 3A and 3F, respectively. Marin et al. (2001) concluded that their observations reveal a mechanism by which neuropilins mediate sorting of distinct neuronal populations into different brain structures, and provide evidence that, in addition to guiding axons, these receptors also control neuronal migration in the central nervous system. Therefore, we hypothesize that abnormalities in the NRP2 gene are involved in the etiology of some forms of autism. In the present study, we chose two SNPs located within the NRP2 region to conduct association analyses using the family-based association tests (FBAT) in 166 Chinese Han trios. The FBAT analysis for each variant showed a significant difference between two transmitted alleles (rs849578 C: Z=2.286, P=0.022243; rs849563 A: Z=2.186, P=0.028784). They were in strong disequilibrium and in the same haplotype block. The global chi-square test for the haplotype transmission also revealed a strong association(χ2 =12.595，df=3，P= 0.005600). Haplotype CA showed the excess transmission to affected offspring. The strong association between the NRP2 locus and autism suggests that the gene itself may play a role in predisposing an individual to autism. Our preliminary results provide evidence that the NRP2 gene may be important risk factors for the development of autism. 4. Association studies of GABRB3 polymorphisms with autism in the Chinese population Cytogenetic abnormalities in the Prader-Willi syndrome and Angelman syndrome critical region (15q11-q13) have been described in several individuals with autism. Previous studies have suggested that the γ-aminobutyric acidA receptor subunit β3 (GABRB3) gene in the 15q11-q13 region are possibly involved in susceptibility to autism. Cook et al. reported significant linkage disequilibrium between autistic disorder and a marker, GABRB3 155CA-2, in the GABRB3 gene on chromosome 15q11-q13. We completed an association analysis with three single nucleotide polymorphisms (SNPS) located within the GABRB3 gene using the family-based association tests (FBAT) in 174 Chinese Han trios. We found no evidence for association with the SNP markers including one SNP 2kb away from 155CA-2. The haplotype analysis also revealed no evidence for transmission disequilibrium with autism. Thus, it appears that GABRB3 do not contribute significantly to the predisposition to develop autism in our sample. Our results do not support the major role for these polymorphisms in contributing to autism susceptibility in the population.