臺灣博碩士論文加值系統

癲癇是一種常見的多因子神經性疾病，其全球的盛行率大約3%。在先前的研究中認為，染色體或是基因的異常是造成癲癇疾病的重要原因，但詳細的基因與影響癲癇發生的作用機制到目前都還不清楚。本論文的研究對象為六個來自同一家族的癲癇患者，並且在臨床診斷上已排除受到後天環境影響或感染 等因素。主要的研究對象 (proband) 在臨床的診斷為複雜型局部性癲癇，而我們認為此癲癇家族的癲癇症狀主要受到遺傳因子所影響。本論文的研究目的是利用染色體核型分析與分子細胞遺傳學方法來研究是否有染色體或基因等遺傳因子的異常造成此家族性癲癇。我們收集六位曾有癲癇發作的家族成員周邊血液進行血液細胞培養，以標準流程製作成樣本玻片後以G-banding的染色方式進行染色體核型分析。此外，我們也從周邊血液中萃取基因體DNA，利用微陣列比較基因體雜交技術與即時定量聚合��鏈鎖反應技術進行染色體微差異片段的分析。
　　在染色體核型分析的研究結果中，在顯微鏡觀察下並沒有發現染色體數目或結構的異常。而微陣列比較基因體雜交技術的結果，則是發現四位癲癇成員在14號染色體長臂11.1~11.2有共同的duplication現象。我們進一步以即時定量聚合��鏈鎖反應來確認微陣列比較基因體雜交技術的結果，發現此家族六位曾有癲癇發作的成員在14號染色體長臂11.1~11.2上確實有DNA增幅的現象。此外，即時定量聚合��鏈鎖反應實驗的結果也證明此微差異片段來自於母系遺傳。
綜合以上分子細胞遺傳學分析實驗的結果， 14號染色體長臂11.1~11.2片段可能為影響此家族性癲癇的重要DNA區域，而未來可利用此段DNA增幅區域進一步研究造成癲癇的相關DNA致病序列或基因。

Epilepsy is a common and genetically complex neurological disorder that affects 3% of world population. Previous studies showed the chromosomal or genetic abnormalities might confer susceptibility to epilepsy, but the genes causing epilepsy have not been well known or characterized to date. In the present study, we reported 6 individuals with epilepsy in the same family, and the clinical work up did not reveal any environmental or infectious factors to cause the disease in this family. The proband in this epileptic family was diagnosed as complex partial seizure and we hypothesize genetic factors are major contributing factors to the epileptic seizure in this family.
To investigate the chromosomal aberrations for the familiar epilepsy, chromosome karyotyping and molecular cytogenetic techniques were used for this study. Blood samples were collected from 6 affected individuals for chromosome karyotyping. Genomic DNA was isolated from whole blood cells for array-based comparative genomic hybridization (array-CGH) analysis and DNA quantitative real-time PCR.
The karyotyping results showed that no numerical or structural abnormalities found under microscopy in these epileptic individuals. The array-CGH data demonstrated that duplication was identified in chromosome 14q11.1-11.2 from 4 epileptic individuals of this family. The identified genomic gain regions were verified in all 6 affected individuals by real-time quantitative PCR and the results were consistent with those from array-CGH. Furthermore, the real-time PCR data also revealed that the duplicated region may be gained via maternal inheritance in this family.
In the present study, the results of molecular cytogenetic analysis demonstrated that chromosome 14q11.1-11.2 might be a candidate region for causing epilepsy in this family and could be used for further investigation to determine the pathogenic DNA regions or genes involved in the familial epilepsy.

目錄
中文摘要………………………………………………………………….III
英文摘要………………………………………………………………….V
緒論………………………………………………………………………1
1. 癲癇簡介…………………………………………………………1
2. 癲癇的分類與病因………………………………………………1
3. 影響癲癇的遺傳因子……………………………………………3
4. 研究家族性癲癇之傳統細胞遺傳學與分子細胞遺傳學方法……5
5. 研究動機…………………………………………………………7
材料與方法……………………………………………………………… 9
1. 試劑與藥品………………………………………………………9
2. 研究對象與檢體來源……………………………………………10
3. 染色體核型分析……………………………………………………10
4. 基因體DNA的製備……………………………………………… 12
5. 微陣列比較基因體雜交技術………………………………………12
6. 核酸引子之設計……………………………………………………14
7. 聚合��鏈鎖反應……………………………………………………15
8. 膠片電泳分析………………………………………………………15
9. 即時定量聚合��鏈鎖反應…………………………………………16
10. 即時定量聚合��鏈鎖反應數據分析………………………………16
11. 統計分析……………………………………………………………16
結果……………………………………………………………………… 17
1. 癲癇家族分析………………………………………………………17
2. 家族性癲癇成員染色體核型分析…………………………………17
3. 以微陣列比較基因體技術分析家族性癲癇成員的基因體DNA………18
4. 以即時定量聚合��鏈鎖反應技術分析14q11.1-11.2 片段的微差異……20
5. 研究對象的家族性癲癇是否由其母系家族遺傳而來……………22
討論………………………………………………………………………23
表格………………………………………………………………………28
圖型………………………………………………………………………31
參考文獻……………………………………………………………………48

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