第八型脊髓小腦運動失調症(spinocerebellar ataxia type 8；簡稱SCA8)為一種遺傳之神經退化性疾病，通常伴隨著小腦功能障礙，但外顯率不完全。和SCA8相關的基因包括CTG三核苷重複擴增的ATXN8OS基因與反向表現CAG三核苷重複擴增的ATXN8基因。為探究SCA8的低外顯率，本論文檢視了ATXN8OS選擇性裁接的exon B及exon A附近序列，結果發現了兩個新穎的多型性點：SNP1 T/C、SNP2 C/T。此兩個多型性點組成的四種單套型中，T-T單套型在15個SCA8 CTG/CAG重複擴增病人族群中，出現的頻率較87個正常人族群來的低(4.7% vs. 22.8%，P = 0.041)。為探討ATXN8、ATXN8OS基因的轉錄調控，本論文將此兩基因的近端啟動子片段構築於pGL3-basic質體上。啟動子刪除分析實驗顯示，相較於ATXN8 -1035~-6、-518~-6片段，-99~-6片段的轉錄活性最強。位於ATXN8啟動子片段-62位置的SNP2 C/T雖可能影響轉錄因子CEBP的結合，但CEBPcD共轉移的實驗卻看不出SNP2 C/T對ATXN8啟動子活性的影響。在ATXN8OS的啟動子方向，反轉錄-聚合酶鏈式反應(RT-PCR)實驗則推測出ATXN8OS的啟動子位置位於表現子D5的上游。在ATXN8OS的細胞模式研究上，短暫大量表現CTG重複擴增的ATXN8OS在細胞內會形成ribonuclear foci。進一步的利用ATXN8OS誘導細胞株，發現CTG三核苷重複擴增數目為88或157時，annexin染到的細胞會顯著增加多於不含ATXN8OS CTG三核苷重複的細胞，而誘導ATXN8OS表現後，含23或157個ATXN8OS CTG三核苷重複擴增的細胞，其細胞週期停在G1/G0期也顯著多於不含ATXN8OS cDNA的細胞。本論文的研究結果或有助於釐清SCA8低外顯率及分子致病機轉。

Spinocerebellar ataxia type 8 (SCA8) is a hereditary neurodegenerative disorder, manifesting itself as a slowly progressive cerebellar ataxia. The penetrance of SCA8 is incomplete. SCA8 involves the expression of a CTG/CAG expansion mutation from opposite strands producing CUG expansion transcripts (ATXN8OS) and a polyglutamine expansion protein (ATXN8) known to be pathogenic on other disorders. To investigate incomplete penetrance of the disease, the alternative splicing donor and acceptor sites flanking exons B and A were amplified and sequenced. Two novel SNPs, SNP1 T/C and SNP2 C/T, were identified. Among the four haplotypes constructed by SNP1 and SNP2, the T-T haplotype frequency is notably low in 15 cases with SCA8 expansion mutations compared to the 87 controls (4.7% vs. 22.8%, P = 0.041). To investigate the transcriptional pathways regulating ATXN8OS and ATXN8 expression, the ATXN8OS and ATXN8 promoter fragments were cloned into pGL3-basic vector. Promoter deletion analysis suggested that the ATXN8 -99~-6 proximal promoter fragment displayed highest promoter activity in comparison to -1035~-6 and -518~-6 fragments. Although SNP2 C/T at -62 position may affect transcription factor CEBPbinding, CEBPcD co-transfection did not demonstrate the effect of SNP2 C>T on ATXN8 expression. For ATXN8OS promoter, RT-PCR revealed that the ATXN8OS promoter region was located upstream of exon D5. In ATXN8OS cell model study, transiently expressed ATXN8OS cDNA carrying expanded CTG repeats formed ribonuclear foci. Furthermore, using inducible ATXN8OS cell lines, a repeat-length related increase in annexin-V-positive cells were observed. Upon induction of ATXN8OS expression, cell cycle analysis revealed a significant increase in the cell population at G1/G0 phase for cells carrying 23 and 157 CTG repeats compared to the vector cells. The study results may shed insights into the reduced penetrance and the pathogenesis of SCA8.

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