Study Of Methylation Levels Of The Parkin Gene Promoter And Mutation Analysis Of DJ-1 In Parkinson's Disease Patients

Background:Parkinson's disease (PD) is the second most frequent neurodegenerative movement disorder after dementia, with a prevalence of 1%-2% among people over the age of 65 years. Genome-wide linkage analysis and positional cloning methods have meant that a number of genes associated with this disease have been identified in recent decades. Four mutations (PARKIN/PARK2, PINK1/PARK6, DJ-1/PARK7, PARK9/ATP13A2) have been significantly linked to autosomal recessive early-onset Parkinson's disease (EOPD). Loss of function of parkin is the predominant genetic cause of autosomal recessive EOPD.Up to the day, over 100 different point mutations, as well as exon deletions, duplication, and triplications, have been found in parkin. Many recent studies have identified the parkin gene as a target for loss of heterozygosity (LOH) in cell lines and tumor tissues from ovarian, breast, and hepatocellular carcinomas. Overall, these results suggest that parkin maps to the common fragile site (CFS) FRA6E, and could be a potential tumor suppressor gene (TSG) candidate, with an important role in tumorigenesis and cancer development. Epigenetic research in human leukemia has demonstrated that abnormal methylation of the PARK2 promoter may play a role in the pathogenesis and development of this hematologic neoplasm. It is well known that DNA methylation can silence gene expression. Over expression of the parkin gene can protect against several different insults that may promote the development of PD. Based on these facts, we hypothesized that abnormal methylation of the parkin promoter might reduce the expression of parkin, which would in turn contribute to the pathogenesis of PD. In this study, we compared the frequencies of methylation of CpG sites in the parkin promoter region among PD patients with and without parkin mutations, and normal controls, to determine the role of this epigenetic mechanism in the pathogenesis of PD.During the known genes to hereditary autosomal recessive EOPD, DJ-1 mutations are the second more common genetically factor after parkin mutation. However, DJ-1 mutation screening for different ethnic groups showed that the mutation rate just accounted for 1%-2% or lower. The exact frequency of DJ-1 mutation in Chinese Han population with EOPD was still unclear, and if some certain polymorphisms increase the prevalence of PD should be studied.Objective:We detect the levels of methylation of parkin gene promoter in Parkinson's disease patients using bisulfite-specific polymerase chain reaction (BSP). Furthermore, we compared the frequencies of methylation of CpG sites in the parkin promoter region among PD patients with and without parkin mutation, and normal controls, to determine the role of this epigenetic mechanism in the pathogenesis of PD. By screening DJ-1 mutations, we detect the prevalence of the DJ-1 mutation in EOPD patients of China and analyze the association of the certain polymorphic marker g.168₁85del in intronl and PD.Methods:A total of 44 subjects were included in the study. They were divided into 17 PD patients with parkin mutations,17 PD patients without parkin mutations, and 10 control subjects. PD patients with parkin mutations were all confirmed to have exon rearrangement mutations or heterozygous point mutations in the parkin gene using quantitative polymerase chain reaction (PCR) and direct sequencing. At first, genomic DNA of all subjects was extracted from peripheral blood. After treated with bisulfite, promoter region of parkin gene was specifically amplified. The PCR products were directly sequenced after T-A clone. At last, we observe and compare the status of methylation of parkin promoter region through the analysis of the online computer program. Aiming to confirm the presence of pathogenic DJ-1 mutations, we screened all seven exons and exon-intron boundary regions of DJ-1 by PCR and direct nucleotide sequencing in 90 Chinese patients with EOPD. We also investigated whether the 18 bp deletion polymorphism in intron 1 of DJ-1 (g.168₁85del) might be a risk factor for PD in the 90 patients and 105 controls from the Chinese population.Results:First, we identified CpG islands between -801 bp upstream and +350 bp downstream from the transcription initiation site of parkin (designated as 0) using the online methylation analysis programme. One CpG island from -335 to +437 bp was found in this region. We selected a region from -341 to +9 bp in the CpG island, which contained 34 CpG sites, for the bisulfite primer amplifications.Second, the results showed hypomethylation of parkin gene promoter region in PD patients with and without parkin mutations.Third, there were no significant differences in the total methylation percentages of CpG sites between PD patients and normal controls, as between PD patients with and without parkin mutstions.Fourth, we did not found any pathogenic mutations in DJ-1 gene of the 90 EOPD patients. We did identify four known polymorphic variants including the 18 bp deletion g.168₁85del in intron 1, as well as g.5027G>A (rs17523802). g.5065T>C (rs226249), and g.5094C>T (rs11121064) within exon 1.Fifth, We observed no significant difference in the allele frequencies(χ2=0.21, df =1. P=0.64) or the genotype frequencies (χ2=0.22, df=1, P=0.64) of the g.168₁85del polymorphism between EOPD patients and controls. Conclusion:First, we first reported that promoter region of parkin gene in PD patients revealed the hypomethylation status. There were no significant differences in the total methylation percentages of CpG sites between PD patients and normal controls. The methylation state may be related to the pathogeneses and development of PD.Second, we have not found any types of pathogenic DJ-1 mutation contributing to EOPD among 90 Chinese patients. We did identify four known polymorphic variants, among of which study of association between g.168₁85del polymorphic and PD risk is the hot point.Third, we also observed no association between the DJ-1 intron 1 g.168₁85del polymorphism and PD risks, which may be association with the ethic, age, sex and so on.