脊髓小腦萎縮症 (Spinocerebellar ataxia, SCA) 是一種體染色體顯性的遺傳性神經元退化性疾病。此病病人會由於小腦與神經細胞的萎縮、退化產生運動失衡的情形。目前為止SCA已被歸類為十六型，分別由SCA1到SCA16，在這十六型中，有八種屬於基因已被選殖出的類型，六種屬於已定出基因座位置，但基因未被選殖出的類型，另外兩種則僅有分類名稱而其餘資訊一無所知。在基因已選殖出之類型中，SCA的致病機制都是因為核苷酸重複序列出現了異常擴張的情形，其中多數為CAG / CTG的三核苷酸重複序列的擴增。經由遺傳，可令子代異常擴張的情形更為嚴重，造成發病的年齡更為提早。目前已發展出分子檢測的方式，可對基因已選殖出之SCA類型做出診斷，並了解三核苷酸重複序列異常擴張的長度如何。
本論文中針對了SCA4、SCA5、SCA11、SCA13、SCA14這五種僅知基因座位置的SCA類型，先以生物資訊的工具所預測之致病候選基因排定優先順序，再於致病候選基因中三核苷酸重複序列的位置附近設計引子，利用聚合酶連鎖反應以及定序凝膠電泳的方法，在病人的DNA上檢測重複序列的長度。其中SCA4計有20個致病候選基因，在9對優先順序高的引子中發現3個DNA多型性的現象；SCA5計有16個致病候選基因，於7對優先順序高的引子中並無發現任何多型性；SCA11有34個致病候選基因，在20對優先順序高的引子中發現4個DNA多型性的情形；SCA13則有52個致病候選基因，在28對優先順序高的引子中出現了5個DNA多型性的情形；而在SCA14，則是6個致病候選基因以及5對優先順序高的引子中，沒有發現到任何多型性。這結果顯示在現有的73個無法定出類型的SCA病人中，並未發現SCA4, 5, 11, 13, 14等五種SCA的致病基因。經由與Unigene資料庫比對發現這些有重複序列之多型性的候選基因，有75%分別在小腦組織的EST中也有程度不同的表現量，顯示仍值得尋找更多的病患進行測試。此外，其他的三核苷酸重複序列異常擴張之相關疾病，經以BLAST比對人類和其他生物間的疾病基因與蛋白質的序列之後，發現到在其他生物之中並未含有如同人類一般長串的三核苷酸重複序列的現象。這些觀察將可評估已有之各候選基因之優先順序。

Spinocerebellar ataxia (SCA) is an autosomal dominant hereditary neuron disorder disease. Patients with this disease show incoordination of movement because of atrophy of the cerebellum. The SCA has been classified into 16 types so far─ from SCA1 to SCA16. Out of the 16 types, 8 diseases genes have been cloned; however, another 6 have merely been mapped. We know nothing about the SCA9 and SCA15 but the names. The trinucleotide repeat (CAG / CTG) in most of the SCA disease genes expand. When the repeats expand in successive generations, the syndrome become proleptic. Molecular methods have been developed to diagnose the cloned SCA types and to measure the length of trinucleotide repeat expansion.
My thesis has studied 5 SCA types that do not have a disease gene─ SCA4, SCA5, SCA11, SCA13, and SCA14. After setting up the priority of predicted candidate genes, I designed primers near the repeat site of high priority candidate genes. Polymerase chain reaction (PCR) and electrophoresis were used to measure the length of repeats in DNA samples from patients. There were 20 candidate genes in SCA4; 3 out of the 9 high priority set of primers are polymorphic. There are 16 candidate genes in SCA5 and non- polymorphic in 7 set of primers. There are 34 candidate genes in SCA11; 4 out of the 20 set of primers are polymorphic. There are 52 candidate genes in SCA13 and 5 out of the 28 set of primers are polymorphic. There are 6 candidate genes in SCA14 and none of the 5 primers is polymorphic.
These results reveal that no disease genes were found in SCA4, 5, 11, 13, 14 types among the 73 unclassified SCA patients. After searching UniGene database, 75 percent of the polymorphic repeat-containing genes are expressed in different extent in cerebellum. Thus, it is still worth to test these primers set on more patients to hunt for the disease gene. Besides, the trinucleotide repeat length in animals were obviously shorter than that in human beings. This observation will be useful to evaluate the priority of testing candidate genes in the future.

致謝..........................................................I
中文摘要......................................................Ⅱ
英文摘要......................................................Ⅲ
縮寫表........................................................Ⅳ
目錄與圖表目錄.................................................V
一、緒論......................................................1
脊髓小腦萎縮症之簡介...........................................1
SCA的病理、病徵與致病原因......................................2
脊髓小腦萎縮症之分類...........................................3
關於三核苷酸重複序列...........................................4
以生物資訊學的方法對未知的SCA類型疾病蒐集致病候選基因............7
以實驗方式發現已知基因座位置，但基因未選殖出的SCA類型
之疾病基因....................................................7
利用生物資訊學方法分析不同生物中三核苷酸重複序列的相關
疾病基因......................................................8
研究策略總整理................................................9
二、實驗材料與方法............................................10
實驗材料.....................................................10
人類基因體DNA (Human Genomic DNA)............................10
引子.........................................................10
聚合酶.......................................................10
聚合酶連鎖反應試劑............................................10
套裝系統 (Kit)................................................10
其他化學藥品..................................................11
放射性同位素..................................................11
PCR熱循環器...................................................11
實驗方法......................................................11
引子的設計....................................................11
使用Primer3工具網站輔助設計....................................11
聚合酶連鎖反應 (Polymerase chain reaction, PCR)...............14
熱循環參數1...................................................15
熱循環參數2...................................................16
熱循環參數3...................................................16
DNA凝膠電泳...................................................16
Agarose凝膠電泳...............................................16
Polyacrylamide凝膠電泳........................................17
定序凝膠電泳 (sequencing gel).................................17
製作定序用標記 (Sequencing marker)............................18
真空凝膠乾燥..................................................19
自動放射顯影及磷光顯像儀操作...................................19
實驗上利用到的資料庫說明.......................................20
三、結果......................................................22
蒐集致病候選基因的程序及結果...................................22
對致病候選基因設定優先順序的原因................................24
對各致病候選基因測試最佳化PCR條件...............................25
利用PCR方法來篩選病人樣本......................................28
對已知類型的SCA疾病基因用相同的PCR篩選方法當作對照組.............29
篩選後所觀察到的結果...........................................31
出現DNA多型性的致病候選基因介紹.................................34
具有DNA多型性的致病候選基因可以核對到某些基因與蛋白質............34
以生物資訊的方法分析三核苷酸重複序列相關疾病的結果...............37
四、討論......................................................40
實驗結果與對照組的比較.........................................40
實驗設計及實驗方法優缺點的討論..................................40
致病候選基因可能性的探討.......................................42
推測DNA多型性的意義............................................44
對三核苷酸重複序列相關疾病的分析結果討論其意義...................45
後續的展望....................................................46
五、參考文獻..................................................47
附錄..........................................................52

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