Parkin基因內含子9 g>a多型性的功能性分析

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(superscript +)). The c.1084intron(superscript +) 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.