Epigenetic DNA Methylation Profiling with MSRE: A Quantitative NGS Approach Using a Parkinson's Disease Test Case

Abstract

Epigenetics is a rapidly developing field focused on deciphering chemical fingerprints that accumulate on human genomes over time. As the nascent idea of precision medicine expands to encompass epigenetic signatures of diagnostic and prognostic relevance, there is a need for methodologies that provide high-throughput DNA methylation profiling measurements. Here we report a novel quantification methodology for computationally reconstructing site-specific CpG methylation status from next generation sequencing (NGS) data using methyl-sensitive restriction endonucleases (MSRE). An integrated pipeline efficiently incorporates raw NGS metrics into a statistical discrimination platform to identify functional linkages between shifts in epigenetic DNA methylation and disease phenotypes in samples being analyzed. In this pilot proof-of-concept study we quantify and compare DNA methylation in blood serum of individuals with Parkinson's Disease relative to matched healthy blood profiles. Even with a small study of only six samples, a high degree of statistical discrimination was achieved based on CpG methylation profiles between groups, with 1008 statistically different CpG sites (p < 0.0025, after false discovery rate correction). A methylation load calculation was used to assess higher order impacts of methylation shifts on genes and pathways and most notably identified FGF3, FGF8, HTT, KMTA5, MIR8073, and YWHAG as differentially methylated genes with high relevance to Parkinson's Disease and neurodegeneration (based on PubMed literature citations). Of these, KMTA5 is a histone methyl-transferase gene and HTT is Huntington Disease Protein or Huntingtin, for which there are well established neurodegenerative impacts. The future need for precision diagnostics now requires more tools for exploring epigenetic processes that may be linked to cellular dysfunction and subsequent disease progression.

Genome-wide brain DNA methylation analysis suggests epigenetic reprogramming in Parkinson disease

“…6,7 Several studies have found differential methylation in SNCA and MAPT . 8–11 Furthermore, significant changes in DNA methylation were identified in multiple genes in both blood 10,12,13 and brain. 10 Relevant to the current study, dysregulation of Wnt signaling due to methylation was observed in the frontal cortex and midbrain sections of PD brains.…”

Epigenetic machine learning: utilizing DNA methylation patterns to predict spastic cerebral palsy

“…DNA libraries were prepared from methyl-sensitive restriction endonuclease (MSRE) [ 30 , 31 ] fragmented genomic DNA (gDNA) using HpaII, which recognizes C (CpG) G sites. A standard sequencing protocol was then performed including randomized shearing (Covaris, Woburn, MA) and synthesis of a gDNA fragment library using Illumina TruSeq Nano library synthesis kits (San Diego, CA).…”

Cytosine Methylation Within Marine Sediment Microbial Communities: Potential Epigenetic Adaptation to the Environment

“…CpG methylation was calculated using a commercial bioinformatic pipeline to reconstruct probabilities of methylation at CpG sites (as used in Marsh and Pasqualone, 2014; Marsh et al., 2016; Crowgey et al., 2018; Genome Profiling LLC, Newark, DE). An overview of the pipeline workflow is available in Supplementary Figure S1.…”

“…Computational reconstruction of CpG methylation is based on a null selection model, where the distribution of m5C modifications at any single CpG site is expected to be 50% in a large population of cells (genome copies) for CpG sites that are non-functional or silent. Where CpG methylation status is important for cellular fitness and thus there is a selection force pushing the m5C distributions away from a 50:50 equilibrium, methylation of those CpG sites among all the cellular genome copies sampled can be measured (Marsh and Pasqualone, 2014; Marsh et al., 2016; Crowgey et al., 2018).…”