Nanopore sequencing reveals methylation changes associated with obesity in circulating cell-free DNA from Göttingen Minipigs

Epigenetics. 2023 Dec;18(1):2199374. doi: 10.1080/15592294.2023.2199374.

Abstract

Profiling of circulating cell-free DNA (cfDNA) by tissue-specific base modifications, such as 5-methylcytosines (5mC), may enable the monitoring of ongoing pathophysiological processes. Nanopore sequencing allows genome-wide 5mC detection in cfDNA without bisulphite conversion. The aims of this study were: i) to find differentially methylated regions (DMRs) of cfDNA associated with obesity in Göttingen minipigs using Nanopore sequencing, ii) to validate a subset of the DMRs using methylation-specific PCR (MSP-PCR), and iii) to compare the cfDNA DMRs with those from whole blood genomic DNA (gDNA). Serum cfDNA and gDNA were obtained from 10 lean and 7 obese Göttingen Minipigs both with experimentally induced myocardial infarction and sequenced using Oxford Nanopore MinION. A total of 1,236 cfDNA DMRs (FDR<0.01) were associated with obesity. In silico analysis showed enrichment of the adipocytokine signalling, glucagon signalling, and cellular glucose homoeostasis pathways. A strong cfDNA DMR was discovered in PPARGC1B, a gene linked to obesity and type 2 diabetes. The DMR was validated using MSP-PCR and correlated significantly with body weight (P < 0.05). No DMRs intersected between cfDNA and gDNA, suggesting that cfDNA originates from body-wide shedding of DNA. In conclusion, nanopore sequencing detected differential methylation in minute quantities (0.1-1 ng/µl) of cfDNA. Future work should focus on translation into human and comparing 5mC from somatic tissues to pinpoint the exact location of pathology.

Keywords: Cell-free DNA; Nanopore sequencing; diagnostics; epigenetics; methylation; obesity.

Plain language summary

Oxford nanopore sequencing can reveal changes in methylation patterns associated with obesity in minute quantities of cell-free DNA from serum.Bisulphite conversion and methylation-specific PCR can be used to validate differentially methylated regions in cell-free DNA.A differentially methylated region in an intronic region of the PPARGC1B gene was found associated with obesity.Differentially methylated regions in cell-free DNA could be useful as early risk markers of certain diseases and pathologies.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell-Free Nucleic Acids* / genetics
  • DNA
  • DNA Methylation
  • Diabetes Mellitus, Type 2* / genetics
  • Humans
  • Nanopore Sequencing*
  • Obesity / genetics
  • RNA-Binding Proteins / genetics
  • Swine
  • Swine, Miniature / genetics

Substances

  • DNA
  • Cell-Free Nucleic Acids
  • PPARGC1B protein, human
  • RNA-Binding Proteins

Grants and funding

This work was supported by the Novo Nordisk Foundation Center for Basic Metabolic Research, an independent research center at the University of Copenhagen partially funded by an unrestricted donation from the Novo Nordisk Foundation (NNF18CC0034900). The animal study was funded by Novo Nordisk A/S, the Innovation Fund Denmark, and the LifePharm Centre at University of Copenhagen, Faculty of Health and Medical Sciences, Department of Veterinary and Animal Sciences.