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

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

[68Ga]Ga-NODAGA-E[(cRGDyK)]2 Angiogenesis PET/MR in a Porcine Model of Chronic Myocardial Infarction

Angiogenesis is crucial in tissue repair and prevents scar tissue formation following an ischemic event such as myocardial infarction. The ischemia induces formation of new capillaries, which have high expression of integrin αvβ3. [68Ga]Ga-NODAGA-E[(cRGDyK)]2 ([68Ga]Ga-RGD) is a promising PET-radiotracer reflecting angiogenesis by binding to integrin αvβ3. A Göttingen mini-pig underwent transient catheter-induced left anterior descending artery (LAD) occlusion for 120 min, and after 8 weeks was imaged on a Siemens mMR 3T PET/MR system. A large antero-septal infarction was evident by late gadolinium enhancement (LGE) on the short axis and 2–4 chamber views. The infarcted area corresponded to the area with high [68Ga]Ga-RGD uptake on the fused PET/MR images, with no uptake in the healthy myocardium. To support the hypothesis that [68Ga]Ga-RGD uptake reflects angiogenesis, biopsies were sampled from the infarct border and healthy myocardium. Expression of αvβ3 was evaluated using immunohistochemistry. The staining showed higher αvβ3 expression in the capillaries of the infarct border compared to those in the healthy myocardium. These initial data confirm in vivo detection of angiogenesis using [68Ga]Ga-RGD PET in a translational model, which overall support the method applicability when evaluating novel cardio-protective therapies