Pyrosequencing analysis of methylation levels of clock genes in leukocytes from Parkinson's disease patients

DNA methylation of neuronal PAS domain protein 2 (NPAS2) and cryptochrome circadian clock 1 (CRY1) promoters may be associated with Parkinson's disease (PD). However, there is no simple and cost-effective method to quantify DNA methylation in these regions. Additionally, it is not clear whether DNA methylation of NPAS2 and CRY1 promoters is altered in peripheral blood of PD patients, especially newly diagnosed drug-naïve PD patients, and thus can be used as a PD biomarker. In the present study, we utilized bisulfite pyrosequencing assays to examine DNA methylation levels of six CpG sites in the NPAS2 promoter and five CpG sites in the CRY1 promoter. We compared DNA methylation levels at these sites in leukocytes from 80 medicated PD patients, 30 drug-naïve PD patients, and 80 healthy controls. Our results indicate that NPAS2 hypomethylation occurs at the early stage of PD and is a moderate biomarker for distinguishing PD patients from healthy subjects.