DOI: 10.5061/dryad.7d7wm37w0
title: Parkinson’s disease-associated, sex-specific changes in DNA methylation at PARK7 (DJ-1), ATXN1, SLC17A6, NR4A2, and PTPRN2 in cortical neurons

description: 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.
description: <em>DNA extraction, bisulfite treatment, and EPIC
 arrays</em> DNA was isolated from enriched
 parietal cortex NeuN<sup>+</sup> 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.
description: Data is included for N=100 samples in the form of IDAT
 files. In our modeling, one sample was removed during
 analysis; details are as follows: After estimation of neuronal vs. glial
 cell proportion in each sample with the <em>CETS</em> R
 package, samples with estimated glial cell to neuronal cell proportion
 &gt; 0.90 were removed from analysis. This removed one sample --
 sample ID = 03_51 -- from the male data set, leaving n=62 males and a
 total N=99 sample size in our modeling.