1University of Heidelberg Department of Internal Medicine III, Precision Digital Health Heidelberg, Deutschland; 2Universitätsklinikum Heidelberg Klinik für Innere Med. III, Kardiologie, Angiologie u. Pneumologie Heidelberg, Deutschland
Background: The development of dilated cardiomyopathy (DCM) is heavily determined by genetic factors. Genome instability and genetic variations have been demonstrated to play a crucial role in developing DCM and to have a significant correlation with poor outcome. One of the features of genome instability is represented by formation of extrachromosomal circular DNA particles (eccDNA) which have recently attracted the attention of the scientistic community. Presence and number of certain eccDNAs are linked to worse prognosis in multiple cancer types and associated with aging and physical inactivity in skeletal muscles of H.sapiens and C.livia domestica. The role of this type of structural variation (SV) in the development of DCM or related heart diseases has not yet been studied. Considering a negative role of circular DNA particles in other pathologies, we hypothesised that eccDNAs may also play a role in DCM development and progression.
Objectives: The aim of this study was to identify and describe eccDNA elements in heart tissues of patients with DCM and investigate its specificity in the disease.
Methods and Results: We isolated eccDNA from cardiac left ventricular explants of 21 DCM patients (20 mg each) applying four steps that included tissue lysis, column purification, removal of linear DNA, and rolling-circle amplification. Enriched eccDNA were sequenced on Illumina NovaSeq 6000 using TrueSeqNano protocol for library preparation. In total 150-500 million 150 bp pair-end reads have been generated. Bioinformatical detection of eccDNA from NGS data was based on split and discordant reads.
We detected more than 700,000 eccDNA, with a median of 25,000 eccDNA per individual. Detected eccDNA were sized between 30 bp and 51.5 kb, with a median size of 3 kb. The vast majority of eccDNA were unique for each individual, and less than 1% of eccDNA had a similar circular DNA in another sample. eccDNAs are formed from across the genome with a tendency to regions with repetitive elements and more transcriptionally active chromosomes like chr1 and chr11.
About 5% of all detected eccDNA contained a full gene sequence including the promoter region, potentially enabling transcription. Among those, 3.000 eccDNA carried protein coding genes. Pathway enrichment analysis of all genes showed a broad activation of important pathways like “Cytosolic DNA-sensing” (3.1-fold, FDR=0,0012) or “Lipid and atherosclerosis” (1.8-fold, FDR=0,013). We did not see an enrichment in pathways related to heart failure, however, in several patients, we detected eccDNA to carry full length genes important for heart failure development.
Bulk RNA-seq from the same samples did not show statistically significant enrichment in expression of eccDNA-carried genes. However, for particular eccDNA we could confirm eccDNA-based transcription, by comparing single nucleotide variants in linear DNA to newly formed variants in the detected eccDNA and transcripts deriving from it.
Conclusion: We showed that eccDNA elements are formed in human DCM heart tissue. We can further prove transcriptional activity of those elements, potentially contributing to a DCM induced cellular reaction. Further studies may lead to new diagnostic or therapeutic avenues.