NEW YORK (GenomeWeb) – Extrachromosomal circular DNA (eccDNA) elements are common in healthy human tissue, and some are even large enough to house one or more complete genes that could be expressed, a new analysis has found.
While the human genome is typically organized into stable chromosomes, tumor cells have been known to accumulate eccDNA. But researchers led by the University of Copenhagen's Birgitte Regenberg suspected that such circular DNA might also accumulate in normal cells, especially as deleted and damaged DNA are marked for destruction.
They isolated and sequenced about 100,000 eccDNA elements from muscle and blood samples obtained from 16 healthy men. As they reported today in Nature Communications, the researchers found that about half the eccDNA they uncovered harbored a gene or a gene fragment. While they found that eccDNAs could be generated from any part of the genome, some spots — such as the region harboring the titin muscle gene — were more likely to form eccDNA. Additionally, they reported that some eccDNAs appeared to be transcribed.
"Thus, somatic genomes are rich in chromosome-derived eccDNAs that may influence phenotypes through altered gene copy numbers and transcription of full-length or truncated genes," Regenberg and her colleagues wrote in their paper.
The researchers collected skeletal muscle biopsy and blood leukocyte samples from the 16 men, half of whom exercised regularly and half of whom were sedentary. They noted that the two groups have different life expectancies and different levels of oxidative stress, which could affect DNA damage levels.
Using an adapted Circle-Seq method, they first isolated DNA from the samples, then removed linear DNA, performed rolling-circle amplification, and then sequenced and mapped the pair-end reads to the human genome.
From that approach, Regenberg and her colleagues identified about 100,000 unique eccDNAs, which they then ranked based upon their coverage support. Of the eccDNAs detected within muscle, 43,960 were high confidence. In leukocytes, 6,253 eccDNAs were high confidence. The team found no difference in eccDNA found in physically active versus sedentary men.
The eccDNAs the researchers uncovered ranged in size from 0.05 kilobases to 57.8 kb, though 99 percent were smaller than 25 kb. Still, more than 1,000 eccDNAs were derived from chromosomal breakpoints that were more than 25 kb apart and, as the average length of a human gene is 27 kb, the researchers noted that eccDNAs could include full genes or parts of genes.
Though eccDNA was generated from all chromosomes, the researchers found that more than half came from genic or pseudogenic regions. For instance, 130 eccDNAs mapped to the 0.3-Mb titin gene (TTN), which the researchers confirmed using outward PCR and Sanger sequencing. TTN, they noted, is one of the most highly transcribed genes, particularly in muscle where it contributes to resting-state plasticity.
As some eccDNAs are recurrent, the researchers said this indicates there are hotspots for circularization.
Regenberg and her colleagues also analyzed mRNA samples from muscle tissue and found that some eccDNAs are transcribed. In particular, they reported split-transcript reads from TTN that matched circularized TTN as well as the reads from the Huntington disease-linked HIP1 gene. This suggests that some eccDNAs are present in the nucleus and are not sequestered in parts of the cell where they wouldn't be transcribed.
The researchers further noted that eccDNAs could affect phenotypic variation through their expression. "We find that eccDNAs are transcriptionally active and we suggest that eccDNA contributes to phenotypic variation through expression of full-length and/or truncated genes," the authors wrote.