TY - JOUR

T1 - Nanopore sequencing reveals methylation changes associated with obesity in circulating cell-free DNA from Göttingen Minipigs

AU - Drag, Markus Hodal

AU - Debes, Karina Poulsdóttir

AU - Franck, Clara Sandkamm

AU - Flethøj, Mette

AU - Lyhne, Mille Kronborg

AU - Møller, Jacob Eifer

AU - Ludvigsen, Trine Pagh

AU - Jespersen, Thomas

AU - Olsen, Lisbeth Høier

AU - Kilpeläinen, Tuomas O.

N1 - Publisher Copyright: © 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.

PY - 2023

Y1 - 2023

N2 - 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.

AB - 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.

KW - Cell-free DNA

KW - diagnostics

KW - epigenetics

KW - methylation

KW - Nanopore sequencing

KW - obesity

U2 - 10.1080/15592294.2023.2199374

DO - 10.1080/15592294.2023.2199374

M3 - Journal article

C2 - 37032646

VL - 18

JO - Epigenetics

JF - Epigenetics

SN - 1559-2294

IS - 1

M1 - 2199374

ER -