DNM1L表現變異與巴金森病：啟動子多型性,轉錄調控及治療開發研究

Background: Parkinson’s disease (PD) is an age-related neurodegenerative disorder that is diagnosed in about 1% of individuals over the age of 65 and ranks only second behind Alzheimer’s disease in prevalence. PD is characterized by gradually lost motor control due to a selective loss of dopaminergic neurons in the substantia nigra (SN). Mitochondrial dysfunction has been suggested to contribute to the pathogenesis of PD. Dynamin 1 like (DNM1L, also known as DRP1), a key protein controlling mitochondrial fission, undergoes posttranslational modifications and translocation to the mitochondrial outer membrane to regulate mitochondrial fission. Purpose: Single nucleotide polymorphisms (SNPs) were found within 1 kb promoter region (-960 ~ -311) of DRP1 gene in East Asia populations (NCBI database of SNP). In a study of genes involved in the biogenesis and function of mitochondria, significantly increased DRP1 mRNA level was found in peripheral blood mononuclear cells of PD patients as compared with normal controls. This study aimed to analyze DRP1 gene promoter variation in a cohort of PD patients and normal controls (NC) and explore promoter variation affecting DRP1 expression. Methods: A cohort of unrelated Taiwanese PD subjects and normal controls were recruited. DRP1 promoter fragments (-714 ~ -125) were PCR amplified and sequenced (n=63 for PD and n=63 for NC). A large-scale case-control association study (n=555 for PD and n=552 for NC) was conducted with -556 G/A SNP. In addition, in silico search and cloning of potential transcription factors binding to these polymorphic sites, and construction of polymorphic DRP1 promoter reporter plasmids and functional assays were performed. Results: Through directing sequencing, polymorphisms -556 G/A (rs565216693), -318 -/AAT, -315 A/T (rs201231372), and -311 A/T (rs11423175) were found in the DRP1 promoter region. Polymorphisms -318 -/AAT and -315 A/T were completely linked in the sequenced 126 samples. A significant difference was found in both genotype and allele distribution (P = 0.011). Individuals carrying GA genotype exhibited an increased risk of developing PD (odds ratio: 5.71; 95% CI: 1.26–25.89, P = 0.020). Individuals carrying polymorphic A genotype also exhibited an increased risk of developing PD (odds ratio: 5.66; 95% CI: 1.25–25.60, P = 0.020). Upon transfection of putative TFAP2A, CEBPB, or FOXA1 transcription factors into human SH-SY5Y cells, A allele-binding CEBPB and FOXA1 significantly raised endogenous DRP1 expression. Five promoter reporter plasmids containing polymorphic changes have been prepared. Through cDNA trans-activation assay, the effects of these promoter variations will be assayed on SH-SY5Y cells. Conclusion: Our data suggest that DRP1 -556 G/A polymorphism plays a role in Taiwanese PD susceptibility.