Analysis Of ATXN3Promoter Methylation In Peripheral Blood Of SCA3/MJD Patients

Objective:To study the methylation levels of ATXN3promoter, the ATXN3mRNA levels in PBMCs of SCA3/MJD patients and controls; to evaluate the CAG repeats size of ATXN3in SCA3/MJD patients; to explore the relationship between DNA methylation levels and mRNA expression, as well as CAG repeats size; and to analyze whether DNA methylation plays roles in the pathogenesis of SCA3/MJD.Methods:141SCA3/MJD patients from71families and112health controls were enrolled in our study. Density gradient centrifugation was used to isolate PBMCs of all subjects. The DNA methylation levels of ATXN3promoter were detected by BSP. The ATXN3mRNA levels were measured by qRT-PCR. Recombinant DNA technology with T-vector cloning followed by direct DNA sequencing was applied to evaluate the CAG repeats size of ATXN3.Results:1. BSP results showed the methylation levels of ATNX3promoter. Hypermethylation status was observed in the first CpG island. Predominantly non-methylated status was observed in the second CpG island.2. Statistically significant difference was found when comparing the first CpG island methylation levels between the SCA3/MJD patients and controls (82.61±3.97vs78.97±2.22, p=0.00). In6out of7kindreds, higher DNA methylation levels were observed in the patients with earlier age at onset than the patients with later age at onset who shared the same CAG repeats size in the same family.3. In the SCA3/MJD group, higher levels of DNA methylation were observed in the parents from SCA3/MJD kindreds with an intergenerational CAG repeats instability than those without intergenerational CAG repeats instability (86.04±2.68vs80.04±2.8, p=0.00)4. No statistically significant difference of ATNX3mRNA levels was observed between SCA3/MJD patients and controls(0.51±0.25vs0.46±0.19, p=0.27), and no correlation was observed between ATNX3mRNA levels and the DNA methylation levels of ATXN3promoter.Conclusions:1. This is the first study of DNA methylation levels of ATXN3promoter using BSP technology. Hypermethylation status was observed in the first CpG island in contrast to hopomethylation status in the second CpG island.2. The levels of DNA methylation in the promoter of the ATXN3gene may be correlated with anticipation of SCA3/MJD patients.3. The levels of DNA methylation in the promoter of the ATXN3gene may affect the stability of CAG repeats in the ATXN3gene.