【作者】 谢冰；

【导师】 于雅琴；

【作者基本信息】 吉林大学 ， 医学基因组学， 2011， 博士

【摘要】 精神分裂症是最常见、最严重的精神疾患,疾病负担沉重,其病因及发病机制目前尚不清楚。大量的研究发现遗传因素在精神分裂症的发病中具有重要的作用,但至今尚未发现引起精神分裂症发生的特定易感基因。目的利用分子生物学技术、遗传流行病学和生物统计学等方法,探讨MBD家族基因多态性与精神分裂症的关联,分析基因-基因交互作用对精神分裂症的影响。本研究所获得的结果将为阐明精神分裂症的遗传规律提供技术支持,为更进一步了解表观遗传机制在精神分裂症发病过程的作用提供证据支持。方法本研究以200个中国北方汉族精神分裂症患者及其健康父母组成的核心家系为研究对象,利用生物信息学方法在MBD基因家族上选择10个tag SNPs位点,包括MBD1基因上的4个tag SNPs分别为rs125555、rs140689、rs140687和rs140686,MBD2基因上的3个tag SNPs分别为rs3786254、rs7614和rs1145317,MBD3基因上的3个tag SNPs分别为rs7252741、rs4807934和rs4807122。利用聚合酶链式反应一连接酶检测反应(PCR-LDR)方法检测个体基因型。检验每个位点的基因型频数分布是否符合Hardy-Weinberg平衡；应用基于家系的关联分析方法HHRR和TDT分析单个位点与精神分裂症的关系；应用PBAT软件分析进行多位点联合分析和两个位点之间连锁不平衡程度(LD)分析；应用,检验分析MBD家族基因上的tagSNPs标记位点与精神分裂症临床症状和临床亚组的关联性,证实临床异质性与遗传异质性的存在；应用PGMDR软件进行基因-基因交互作用与精神分裂症的关联分析。结果(1)Hardy-Weinberg平衡检验MIBD1-3基因上的每个标签SNP位点的基因型在健康父母组和患者组中的分布均符合Hardy-Weinberg平衡,说明所选择的位点均可以作为遗传标记进行MBD家族基因多态性与精神分裂症的关联性研究。(2)HHRR分析在总体样本中,MBD2基因上rs1145317位点等位基因在父母组和患者组中的频数分布差异有统计学意义(χ2=5.348,P=0.021),表明rs1145317位点与精神分裂症相关联。在男性患者组中,MBD2基因上的三个位点rs1145317、rs7614和rs3786254位点等位基因在父母组和患者组中的频数分布差异均有统计学意义(χ2=7.400,P=0.007；χ2=4.723,P=0.030；χ2=4.247,P=0.039),表明MBD2基因上的三个位点与男性精神分裂症相关联。在女性患者组中,MBD3基因上rs4807934位点等位基因在父母组和患者组中的频数分布差异有统计学意义(χ2=4.832,P=0.028),表明rs4807934位点与女性精神分裂症相关联。(3)TDT分析在总体样本中,MBD2基因上rs1145317位点传递给患病子女的两个不同等位基因概率偏离了50% (χ2=4.928,P=0.026),G等位基因过多传递给了患者,表明rs1145317位点与精神分裂症相关联。在男性患者组中,MBD2基因上rs1145317、rs7614和rs3786254位点传递给患病子女的两个不同等位基因概率均偏离了50%(χ2=6.420,P=0.011；χ2=4.853,P=0.028；χ2=4.033,P=0.045),rs1145317位点G等位基因过多传递,rs7614位点A等位基因过多传递,rs3786254位点G等位基因过多传递。表明MBD2基因上的三个位点rs1145317、rs7614和rs3786254位点与男性精神分裂症相关联。在女性患者组中,MBD3基因上rs4807934位点传递给患病子女的两个不同等位基因概率也偏离了50%(χ2=4.378,P=0.036),杂合子父母双亲传递给患病子女的C等位基因过多,表明rs4807934位点与女性精神分裂症相关联。(4)多位点联合分析结果MBD1基因上的4个tag SNPs组成的6种单倍体型在患者组和父母组中的分布差异均无统计学意义(P>0.05),提示这些单倍体型与精神分裂症无关联；MBD2基因上的3个tag SNPs组成的3种单倍体型中,rs3786254-rs7614和rs3786254-rs7614-rs1145317单倍体型在患者组和父母组中的分布差异均无统计学意义(P>0.05),提示这些单倍体型与精神分裂症无关联。rs7614-rs1145317单倍体型在患者组和父母组中的分布差异存有统计学意义(P=0.029),提示rs7614-rs1145317组成的单倍体型与精神分裂症相关联；MBD3基因上的3个tagSNPs组成的3种单倍体型在患者组和父母组中的分布差异均无统计学意义(P>0.05),提示这些单倍体型与精神分裂症无关联。(5)基因-基因交互作用分析结果PGMDR方法获得rs4087122/rs4807934为多因子基因-基因交互的最佳模型。同时携带rs4807122CC和rs4807934AA基因型的个体以及同时携带rs4807122CT和rs4807934AG基因型的个体罹患精神分裂症的风险增加,相对于rs4807122TT和rs4807934GG基因型的个体风险分别增加了2.114和2.553倍(95%CI：1.125-3.971,P<0.05；95%CI：1.071-6.085,P<0.05)。(6)临床症状关联分析结果在总体样本中,MBD1基因上rs140687和rs140689位点与精神分裂症的阳性症状相关联；MBD2基因上rs1145317和rs3786254位点与精神分裂症的阳性症状相关联；MBD3基因上rs7252741、rs4807934和rs4807122位点与精神分裂症的阳性症状相关联。在男性患者组中,MBD1基因上rs125555、rs140686和rs140687位点与精神分裂症的阳性症状相关联；MBD2基因上rs7614、rs1145317和rs3786254位点与精神分裂症的阳性症状相关联；MBD3基因上rs7252741和rs4807934位点与精神分裂症的阳性症状相关联；MBD2基因上rs1145317位点与精神分裂症的病前人格相关联。在女性患者组中, MBD1基因上rs140686、rs140689和rs125555位点与精神分裂症的阳性症状相关联；MBD2基因上rs7614、rs3786254和rs1145317位点与精神分裂症的阳性症状相关联；MBD3基因上rs4807122和rs7252741位点与精神分裂症的阳性症状相关联。(7)临床亚型关联分析结果在总体样本中,MBD1和MBD2基因上各位点与偏执型和未分型精神分裂症无关联；MBD3基因rs7252741位点与偏执型精神分裂症相关联；MBD3基因各位点与未分型精神分裂症无关联。在男性患者组中,MBD2基因上rs1145317位点和rs3786254位点与男性偏执型精神分裂症相关联；MBD3基因上rs7252741位点与男性偏执型精神分裂症相关联。在女性患者组中,MBD2基因上rs7614与女性未分型精神分裂症相关联；MBD3基因上rs4807122位点与女性未分型精神分裂症相关联。结论由上述分析结果,可以得到如下结论：①MBD2(rs1145317)基因位点与精神分裂症相关联；②MBD2(rs1145317、rs7614、rs3786254)基因位点与男性精神分裂症相关联；MBD3(rs4807934)基因位点与女性精神分裂症相关联；③MBD2基因rs7614位点和rs1145317位点组成的单倍型(rs7614-rs1145317)与精神分裂症相关联；④基因-基因交互作用研究发现,同时携带rs4807122CC和rs4807934AA基因型的个体以及同时携带4807122CT和rs4807934AG基因型的个体罹患精神分裂症的风险增加；⑤MBD1、MBD2、MBD3基因中存在多个与精神分裂症临床症状相关联的位点,女性精神分裂症患者的钟情妄想、怪异行为、情感淡漠和其他妄想症状表现多于男性；⑥MBD2、MBD3基因中存在多个与精神分裂症临床亚型相关联的位点；⑦精神分裂症MBD基因的遗传存在性别差异,证实精神分裂症存在临床异质性和遗传异质性。综上所述,本研究结果证明了MBD基因家族可能与精神分裂症发病相关。不同位点间的相互作用与精神分裂症相关联,有力地支持了复杂疾病的多基因学说,同时证实了精神分裂症的临床异质性和遗传异质性。更多还原

【Abstract】 Association study between schizophrenia and gene polymorphisms of methyl-CpG-binding protein familySchizophrenia is the most common and serious mental disease, the burden of disease is very heavy. Until now, the pathogenesis of schizophrenia remains unknown. Most studies found that genetic factor may be played a major role in schizophrenia. The specific susceptibility genes associated with schizophrenia remains unidentified.ObjectiveThe purpose of this study is to identify the genetic association between the polymorphisms of methyl-CpG-binding protein (MBD) family genes and schizophrenia by using the protocols in molecular biology, genetic epidemiology and biostatistics, and to analyze the effect of gene-gene interaction. The results obtained in this study will provide technical support to clarify the inheritance of schizophrenia, and provide supporting evidence to understand the role of epigenetic mechanisms in the pathogenesis of schizophrenia.Methods200 family trios consisted of father, mother, and affected offspring with schizophrenia were collected for MBD genetic analysis.10 tag SNPs were chosen on MBD family genes including rs125555、rs140689, rs140687 and rs140686 on MBD1 gene, rs3786254、rs7614 and rs1145317 on MBD2 gene, rs7252741, rs4807934 and rs4807122 on MBD3 gene. Ten tag SNPs were genotyped using PCR-based ligase detection reaction analysis. The genotype frequency of each tag SNPs distribution was tested for Hardy-Weinberg equilibrium. The HHRR test and TDT test were used to detect the allelic association between tag SNPs of MBD gene and schizophrenia. Haplotype analysis and Linkage disequilibrium analysis was using Pedigree based association test (PBAT) software. The association between tag SNPs of MBD genes and clinical symptoms and type of schizophrenia was used byχ2 test. To elucidate the clinical heterogeneity and genetic heterogeneity based on clinical subgroups analyzed. The relationship between gene-gene interaction and schizophrenia was analyzed by Pedigree-based generalized multifactor dimensionality reduction (PGMDR) software.Results(1)H-W testThe genotype frequency distributions of each tag SNPs in patient group and parent group were not deviated from the H-W equilibrium, thus each SNP could used as genetic markers to test the relationship between MBD genes and schizophrenia.(2) HHRR testIn total samples, HHRR test showed that MBD2 gene rs1145317 allele frequency distribution was statistically significant difference in case and control group (χ2= 5.348, P=0.021). This result indicates that the rs1145317 locus associated with schizophrenia.In the male patient group, MBD2 gene of rs1145317, rs7614 and rs3786254 allele frequency distribution were statistically significant difference in case and control group (χ2=7.400, P=0.007;χ2=4.723, P=0.030;χ2= 4.247, P=0.039), show that the MBD2 gene of rs1145317. rs7614 and rs3786254 associated with schizophrenia in the male patients group.In the female patient group. MBD3 gene rs4807934 allele was statistically significant difference in case and control group (χ2= 4.832. P= 0.028). show that the rs4807934 locus associated with schizophrenia in the female patients group.(3) TDT testIn total samples, TDT analysis showed that the probability of the two different alleles in MBD2 gene rs1145317 deviated from 50% from heterozygous parents (P<0.05). G-allele was passed too much to the patient. This result indicates that the rs1145317 locus associated with schizophrenia.In the male patient group, the probability of the two different alleles in MBD2 gene rs1145317, rs7614 and rs3786254 deviated from 50% from heterozygous parents (χ2=6.420,P=0.011;χ2=4.853,P=0.028:χ2=4.033,P=0.045). G-allele on rs1145317 was passed too much to the patient. A-allele on rs7614 was passed too much to the patient. G-allele on rs3786254 was passed too much to the patient. This result indicates that the rs1145317. rs7614 and rs3786254 locus associated with schizophrenia in the male patients group.In the female patient group, the probability of the two different alleles in MBD3 gene rs4807934 deviated from 50% from heterozygous parents (χ2=4.378,P=0.036). C-allele on rs4807934 was passed too much to the patient. This result indicates that the rs4807934 locus associated with schizophrenia in the female patients group.(4) Haplotype testThere was no haplotypic association for the six haplotype system in the MBD1 gene. There was no haplotypic association for the haplotype system of rs3786254-rs7614 and rs3786254-rs7614-rs1145317 in the MBD2 gene. There was no haplotypic association for the three haplotype system in the MBD3 gene. The result indicates that these haplotype system were not associated with schizophrenia. The haplotype system of rs7614-rs 1145317 frequency distribution were statistically significant difference in case and control group (P=0.029). This result indicates that the haplotype system of rs7614-rs1145317 associated with schizophrenia.(5) Analysis for gene-gene interactionThe PGMDR method identified that the best model of rs4087122/rs4807934 to analyze the gene-gene interaction of schizophrenia. Person who carries rs4807122CC or rs4807934AA genotypes and rs4807122CT or rs4807934AG genotypes of MBD3 gene at the same time will have more risk to affect the disease. The risks were 2.114 and 2.553 times respectively compared to the person who carries rs4807122TT genotype and rs4807934GG genotype (95%CI:1.125-3.971, P<0.05 95%CI:1.071～6.085, P<0.05).(6) Analysis for clinical symptomsIn total samples, rs140687 and rs140689 on MBD1 gene; rs1145317 and on MBD2 gene; rs7252741, rs4807934 and rs4807122 on MBD3 gene were associated with some positive symptoms of schizophrenia.In the male patient group, rs125555、rs140686 and rs140687 on MBD1 gene; rs7614, rs1145317 and rs3786254 on MBD2 gene; rs7252741 and rs4807934 on MBD3 gene were associated with some positive symptoms of schizophrenia. The rs1145317 on MBD2 gene was associated with premorbid personality of schizophrenia.In the female patient group, rs140686、rs140689 and rsl25555 on MBD1 gene; rs7614、rs3786254 and rs1145317 on MBD2 gene; rs4807122 and rs7252741 on MBD3 gene were associated with some positive symptoms of schizophrenia.(7) Analysis for clinical typeIn total samples, the tag SNPs of MBD1 and MBD2 gene were not associated with paranoid and undifferentiated schizophrenia. The rs7252741 of MBD3 gene was associated with paranoid schizophrenia. The tag SNPs of MBD3 gene were not associated with undifferentiated schizophrenia.In the male patient group, the rs1145317 and rs3786254 of MBD2 gene were associated with male paranoid schizophrenia. The rs7252741 of MBD2 gene was associated with male paranoid schizophrenia.In the female patient group, the rs4807122 of MBD3 gene was associated with female undifferentiated schizophrenia.ConclusionsAccording to the results, we have obtain the following conclusions:①MBD2 gene rs1145317 locus was associated with schizophrenia;②rs1145317、rs7614、rs3786254 loci of MBD2 gene were associated with male schizophrenia; MBD3 gene rs48079334 locus was associated with female schizophrenia;③The haplotype system of rs7614-rsl 145317 associated with schizophrenia;④The best model to analyze the gene-gene interaction of schizophrenia was rs4087122/rs4807934. Person who carries rs4807122CC or rs4807934AA genotypes and rs4807122CT or rs4807934AG genotypes of MBD3 gene at the same time will have more risk to affect the disease;⑤There are many SNPs in the MBD genes, which associate with the schizophrenic clinical symptoms. The female schizophrenia has more symptoms of delusion of being loved, bizarre behavior, affective flattening and other delusions;⑥There are many SNPs in the MBD2 and MBD3 gene, which associate with the schizophrenic clinical types;⑦We found the genetic of MBD gene in schizophrenia has gender difference, and the inheritance of schizophrenia has clinical heterogeneity and genetic heterogeneity. In sum, we found the MBD genes were associated with schizophrenia. The results support the polygenic theory of complex disease. At the same time, these results prove that schizophrenia has the clinical heterogeneity and genetic heterogeneity.更多还原