Bisulfite Specific PCR-based Sequencing method was used for semi-quantitative detection of methylation status of CpG islands in SNCA and LRRK2 promoter regions. Bisulfite Specific Cloning-based Sequencing method was used for further quantitative examination of CpG-2 methylation of SNCA. mRNA level was also detected in leukocytes.
Semi-quantitative detection showed that the methylation status of SNCA CpG-2 differed between PD patients and normal controls, while there was no difference in CpG-1 of SNCA or in LRRK2 promoter. Further quantitative analysis by clonal assay showed that the CpG-2 of SNCA was hypomethylated in PD patients compared with the normal control (5.90% versus 7.69%, P = 0.034). Moreover, among the 14 CpG sites of CpG-2, the 2nd, 4th and 9th CpG sites were significantly hypomethylated in PD patients. In subgroups of PD, the methylation level decreased in the early-onset PD patients (P = 0.001). RT-PCR examination showed that SNCA mRNA was increased in PD patients compared with normal control (P = 0.003).
Our results indicated that the methylation level of SNCA CpG-2, especially that of the 2nd, 4th and 9th CpG sites in leukocytes might have great potential to be a useful and informative biomarker in PD diagnosis and treatment.