Amplicon rearrangements during the extrachromosomal and intrachromosomal amplification process in a glioma

Abstract

The mechanisms of gene amplification in tumour cells are poorly understood and the relationship between extrachromosomal DNA molecules, named double minutes (dmins), and intrachromosomal homogeneously staining regions (hsr) is not documented at nucleotide resolution. Using fluorescent in situ hybridization and whole genome sequencing, we studied a xenografted human oligodendroglioma where the co-amplification of the EGFR and MYC loci was present in the form of dmins at early passages and of an hsr at later passages. The amplified regions underwent multiple rearrangements and deletions during the formation of the dmins and their transformation into hsr. In both forms of amplification, non-homologous end-joining and microhomology-mediated end-joining rather than replication repair mechanisms prevailed in fusions. Small fragments, some of a few tens of base pairs, were associated in contigs. They came from clusters of breakpoints localized hundreds of kilobases apart in the amplified regions. The characteristics of some pairs of junctions suggest that at least some fragments were not fused randomly but could result from the concomitant repair of neighbouring breakpoints during the interaction of remote DNA sequences. This characterization at nucleotide resolution of the transition between extra- and intrachromosome amplifications highlights a hitherto uncharacterized organization of the amplified regions suggesting the involvement of new mechanisms in their formation.

MYC amplification in multiple marker chromosomes and EZH2 microdeletion in a man with acute myeloid leukemia

“…These studies also report similar deletion of MYC from chromosome 8, suggesting that the region was excised from the normal locus and amplified episomally. A recent study also showed that dmins with a 4.5 Mb amplicon similar in size to our case may integrate into homogeneously staining regions without changing the size of the amplicon (12). It is possible, therefore, that the marker chromosomes in this report may represent linear acentric aggregates of an approximately 4.4 Mb region of chromosome 8q24.…”

“…The mechanism of MYC amplification in dmins or HSRs in AML has been proposed to occur by excision from an intact chromosome 8q24, circularization, and amplification by mutual recombination (12). A few reports have suggested that MYC amplicons originate from a similar approximately 4.5 Mb region of chromosome 8q24 (12,13). The pathogenic role of amplified MYC in AML is poorly understood.…”

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

“…Studies of eccDNAs in germline cells could be used to measure germline mutation rates and assess sperm quality, for example in livestock. Thus, Circle-Seq has the potential to yield insights into the rate at which genetic variation arises in the form of copy number variation, and lead to a novel understanding of diseases that involve gene copynumber variation [38][39][40] .…”

Circularization of genes and chromosome by CRISPR in human cells

“…Next, we tested induction of DNA circularization by CRISPR-pairs at two different genic loci in both normal and cancer cells as endogenous eccDNAs also are reported to derive from genic 40 sequences (8,10,13,18,19 ] and [TRIM28 circle exon 1-2 ], gave evidence of end-joining events. This supports the previous notion that the major DNA-repair pathway that appears to mediate eccDNA formation in 5 mammalian cells is non-homologous end joining (NHEJ) (20,21).…”

“…Only few studies have previously looked at eccDNA stability over time (11,19,27). We established, from high-throughput FACS measurements, that eccDNA expression quickly 20 becomes silenced in cells and eccDNAs are gradually lost over time.…”