帕金森氏症(Parkinson’s disease，簡稱PD)為中腦黑質細胞退化導致多巴胺分泌不足所引發的漸進性神經性退化疾病。現今研究尚無法明確指出PD 的病理機制，只知PD 與環境及多種遺傳因子相關。先前本實驗室對台灣部分發病年齡低於50 歲的PD 病患進行Parkin 基因突變篩檢，發現一新穎的內含子9 插入突變c.1084intron+，此插入突變與內含子9 的-6g>a 的多型性點相關。進一步PD 病例-對照組分析結果顯示-6a 等位基因顯著和高PD 感受性相關(Wu et al., 2010)。本研究選殖Parkin 全長cDNA，並於末端in-frame 接上綠螢光蛋白後，再於表現子9、10 間依序插入包含內含子9 裁接donor 序列、-6g>a 多型性序列及內含子9 裁接acceptor 序列。將此Parkin cDNA 裁接質體轉染入人類腦癌細胞SK-N-SH 後，利用西方轉漬及活細胞影像儀，分析Parkin-綠螢光融合蛋白的表現量。結果顯示帶有-6a 多型性的內含子9 裁接質體，裁接效率低於帶有-6g 的內含子9 裁接質體。故推論內含子9 的-6g>a 多型性可能因提高c.1084intron+插入突變發生的機率，而與PD的感受性相關。本研究結果或能提供臨床診斷諮詢的參考。

Parkinson’s disease (PD) is a common neurodegenerative disorder caused by loss of dopaminergic neurons in the brain’s nigrostriatal pathway. The etiology of PD has not been fully elucidated. An interaction between environmental factors and genetic predisposition are thought to cause PD. Previously we screened Parkin mutations in early-onset PD patients and identified a novel 86-bp IVS9 insertion (c.1084intron+). The c.1084intron+ was due to a g>a polymorphism at position -6 of a cryptic splice acceptor site within IVS9. A case-control study in a cohort of PD and ethnically matched controls revealed a trend toward increase in risk of developing PD (Wu et al., 2010). To investigate the effect of the -6g>a polymorphism on intron 9 splicing, we cloned the in-frame Parkin-EGFP gene and inserted DNA fragments containing 5’ donor splice site, -6g>a polymorphism and 3’ acceptor splice site of intron 9 between exon 9 and exon 10. The effect of -6g>a polymorphism on intron 9 splicing was examined by transfection into neuroblastoma SK-N-SH cells of the Parkin-EGFP splicing construct. Both western blot using GFP antibody and fluorescence microscopy examination revealed that the Parkin-EGFP cDNA
containing -6a showed low efficiency of splicing. The studies may provide a reference for clinical diagnosis and counseling.