Correlations of single nucleotide polymorphisms in GRM3 and DTNBP1 genes among Taiwanese schizophrenia / 黃清德 撰 .- 彰
黃清德、蔡明勳
E-mail: 354804@mail.dyu.edu.tw
ABSTRACT
Schizophrenia is one of the most serious mental illnesses. Recent studies have shown that 64~81% of schizophrenic patients with genetic heredity, and indicated that glutamate neurotransmission is pathway associated with schizophrenia. We chose GRM3 and DTNBP1 genes to analyze the corelations of genetic polymorphisms and schizophrenia. GRM3 gene product is one of the synaptic G-protein receptors of the neurotransmitter, glutamate. DTNBP1 is responsible for encoding dysbindin protein. Dysbindin is linked to brain’s commands and is one of the key proteins in both glutamate and dopamine nerve transmissions. This study included three experimental groups: the drug control well (type I), ineffective drug control (type II) of schizophrenial patients and normal persons without any known mental illness inheritance for three generations. We analyzed six single nucleotide polymorphisms (SNPs) of GRM3 and DTNBP1 genes including rs2299225, rs1468412, rs7758659, rs760666, rs875462 and rs3213207. Primers were designed for polymerase chain reaction (PCR) amplifications of sequences around SNPs, respectively. We try to know first if there are
insertions or deletions around these SNPs. Each PCR product was then sequenced and result was analyzed to understand whether there are polymorphisms. The correlations of schizophrenia and mutations were statistically analyzed. According to results of statistic analyses, there are no significant difference between type I and type II patients in the age of schizophrenial morbidity and sex. We found that the age of type I patients is between 21 to 30 years old and type II is between 21 to 30 years old. The age of onset of schizophrenia is about 15 to 45 years old. Results of individual allele frequency associations show that rs1468412 of GRM3 gene (p=0.004) and rs3213207 of DTNBP1 gene (p=0.043) is significantly associated with schizophrenia. The other four SNPs are not related with schizophrenia. Results also showed that there is a sex difference between type I and type II patients in rs1468412.
Furthermore we found that there is a nucleotide difference close to rs875462 of DTNBP1 gene associated with schizophrenia. These results show that the gene polymorphisms of GRM3 and DTNBP1 are associated with schizophrenia in Taiwan. Variations of SNPs in rs1468412 and rs3213207 may be factors causing schizophrenia in Taiwan.
Keywords : Schizophrenia、GRM3 gene、DTNBP1 gene、Single nucleotide polymorphisms Table of Contents
目錄 封面內頁 簽名頁 中文摘要 iii 英文摘要 v 誌謝 vii 目錄 viii 圖目錄 xi 表目錄 xii 1. 緒論 1 1.1 研究背景 1 1.2 研究動機 2 1.3 研究目的 3 2. 文獻回顧 4 2.1 精神分裂症介紹 4 2.2 精神分裂症分類 4 2.3 精神分裂症治療 5 2.4 精神分裂症可能致病因 素 6 2.5 基因突變與精神分裂症之關聯性 7 2.6 多巴胺傳導路徑之功能以及受器介紹 11 2.7 麩胺酸傳導路徑之功能及其受器 介紹 12 2.8 GRM3和DTNBP1基因與精神分裂症之關聯 14 2.9 如何選定GRM3與DTNBP1基因之SNPS進行精神分裂 症關聯 性分析 16 3. 材料與方法 17 3.1 實驗材料與設備 17 3.2 實驗流程 18 3.3 樣本來源 19 3.4 採血與血球分離並萃取純化染色 體DNA 21 3.5 以分光光度計分析DNA濃度再以膠體電泳分析DNA 品質 22 3.6 聚合?鏈鎖反應擴增特定的DNA片段 23 3.7 目標DNA片段大小確認與序列分析 24 3.8 統計分析 25 4. 結果與討論 26 4.1 精神分裂症病患與正常人發病年齡統計 26 4.2 精神分裂症病患之發病年齡比較分析 27 4.3 GRM3基因之對偶基因頻率統計結果 29 4.3.1 GRM3基因之SNP-rs1468412的統 計結果 29 4.3.2 GRM3基因之SNP-rs2299225的統計結果 34 4.4 DTNBP1基因之對偶基因頻率統計結果 39 4.4.1 DTNBP1基 因之SNP-rs7758659的統計結果 39 4.4.2 DTNBP1基因之SNP-rs760666的統計結果 40 4.4.3 DTNBP1基因之SNP-rs875462的 統計結果 42 4.4.4 DTNBP1基因之SNP-rs3213207的統計結果 43 4.5 SNPS實驗結果與NCBI提供序列比較 48 4.6 SNPS鄰近位 置可能和精神分裂症相關的突變 49 4.7 討論 54 5. 結論 59 參考文獻 60 附錄 68 圖目錄 圖1. 三種麩胺酸受器種類及其訊息傳 遞 14 圖2. 實驗流程圖 18 圖3. GRM3基因之rs1468412的膠體電泳圖 30 圖4. GRM3基因之rs1468412的序列統計結果 30 圖5.
GRM3基因之rs1468412的序列之男、女分別比例結果 32 圖6. GRM3基因之rs2299225的膠體電泳圖 35 圖7. GRM3基因 之rs2299225的序列統計結果 35 圖8. GRM3基因之rs2299225的序列之男、女分別比例結果 37 圖9. DTNBP1基因
之rs7758659的膠體電泳圖 39 圖10. DTNBP1基因之rs7758659的序列統計結果 40 圖11. DTNBP1基因之rs760666的膠體電泳 圖 41 圖12. DTNBP1基因之rs760666的序列統計結果 41 圖13. DTNBP1基因之rs875462的膠體電泳圖 42 圖14. DTNBP1基因 之rs875462的序列統計結果 43 圖15. DTNBP1基因之rs3213207的膠體電泳圖 44 圖16. DTNBP1基因之rs3213207的序列統計 結果 45 圖17. DTNBP1基因之rs3213207的序列之男、女分別比例結果 46 圖18. DTNBP1基因之g.15478430的序列統計結果 50 圖19. DTNBP1基因之g.15478685的序列統計結果 51 圖20. DTNBP1基因之g.15478430的序列之男、女分別比例結果 52
表目錄 表1. 樣本類型和性別、平均年齡統計表 20 表2. 有抽取到DNA的樣本類型和性別統計 21 表3. 精神分裂症病患發病 年齡分層表 27 表4. 第一型與第二型精神分裂症病患發病年齡比較 28 表5. 第一型與第二型精神分裂症男性與女性病患發病 年齡比較 28 表6. 精神分裂症與rs1468412的關聯性分析 31 表7. 精神分裂症與rs1468412的對偶基因頻率在性別上的關聯 性 分析 33 表8. 精神分裂症與rs2299225的關聯性分析 36 表9. 精神分裂症與rs2299225的對偶基因頻率在性別上的關聯 性分析 38 表10. 精神分裂症與rs3213207的關聯性分析 45 表11. 精神分裂症與rs3213207的對偶基因頻率在性別上的關聯 性分析 47 表12. SNPs序列實驗結果與NCBI資料庫比較表 48 表13. 精神分裂症與g.15478430的關聯性分析 50 表14. 精神分裂症 與g.15478685的關聯性分析 51 表15. 精神分裂症與g.15478430的對偶基因頻率在性別上的關 聯性分析 53
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