Parkinson’s disease-associated, sex-specific changes in DNA methylation at PARK7 (DJ-1), ATXN1, SLC17A6, NR4A2, and PTPRN2 in cortical neurons

Evidence for epigenetic regulation playing a role in Parkinson’s disease (PD) is growing, particularly for DNA methylation. Approximately 90% of PD cases are due to a complex interaction between age, genes, and environmental factors, and epigenetic marks are thought to mediate the relationship between aging, genetics, the environment, and disease risk. To date, there are a small number of published genome-wide studies of DNA methylation in PD, but none accounted for cell-type or sex in their analyses. Given the heterogeneity of bulk brain tissue samples and known sex differences in PD risk, progression, and severity, these are critical variables to account for. In this first genome-wide analysis of DNA methylation in an enriched neuronal population from PD post-mortem parietal cortex, we report sex-specific PD-associated methylation changes in PARK7 (DJ-1), SLC17A6 (VGLUT2), PTPRN2 (IA-2β), NR4A2 (NURR1), and other genes involved in developmental pathways, neurotransmitter packaging and release, and axon and neuron projection guidance.

Methods
DNA extraction, bisulfite treatment, and EPIC arrays

DNA was isolated from enriched parietal cortex NeuN+ nuclei using the Qiagen QIAamp DNA Micro Kit (Cat. # 56304), with some modifications to maximize yield. Given that samples were already homogenized during nuclei isolation, the sample lysis and incubation steps of the QIAamp DNA Micro Kit protocol were removed. Instead, 20 uL of proteinase K were added directly to each MACS eluate. Samples were then vortexed for 15 seconds and incubated at room temperature for 15 minutes. In addition, the optional carrier RNA was added to Buffer AL, the incubation time after the addition of 100% ethanol was increased to 10 minutes, the incubation time for the elution buffer was increased to 5 minutes, and the final elution step was repeated using 10 mM Tris-HCl pH 8.0.

Intact genomic DNA yield was quantified by Qubit fluorometry (Life Technologies). Bisulfite conversion was performed on 500 ng genomic DNA using the TrueMethyl Array kit (Cambridge Epigenetix). All conversion reactions were cleaned using SPRI-bead purification and eluted in Tris buffer. Following elution, BS-converted DNA was denatured and processed through the EPIC array protocol. The EPIC array contains ~850,000 probes that query DNA methylation at CpG sites across a variety of genomic features, including CpG islands, RefSeq genic regions, ENCODE open chromatin, ENCODE transcription factor binding sites, and FANTOM5 enhancer regions. To perform the assay, converted DNA was denatured with 0.4 N sodium hydroxide. Denatured DNA was then amplified, hybridized to the EPIC bead chip, and an extension reaction was performed using fluorophore-labeled nucleotides per the manufacturer's protocol. Array BeadChips were scanned on the Illumina iScan platform.