Fusion of short telomeres in human cells is characterized by extensive deletion and microhomology, and can result in complex rearrangements

Abstract

Telomere fusion is an important mutational event that has the potential to lead to large-scale genomic rearrangements of the types frequently observed in cancer. We have developed single-molecule approaches to detect, isolate and characterize the DNA sequence of telomere fusion events in human cells. Using these assays, we have detected complex fusion events that include fusion with interstitial loci adjacent to fragile sites, intra-molecular rearrangements, and fusion events involving the telomeres of both arms of the same chromosome consistent with ring chromosome formation. All fusion events were characterized by the deletion of at least one of the telomeres extending into the sub-telomeric DNA up to 5.6 kb; close to the limit of our assays. The deletion profile indicates that deletion may extend further into the chromosome. Short patches of DNA sequence homology with a G:C bias were observed at the fusion point in 60% of events. The distinct profile that accompanies telomere fusion may be a characteristic of the end-joining processes involved in the fusion event.

Figure 1.

Illustrating the single molecule telomere fusion assay. (A) Diagrammatic representation of oligonucleotide primers used for the PCR reaction; the XpYp and 17p primers are specific to those chromosome end; the 21q group primers are capable of amplifying the telomeres of 1q, 2q, 5q, 6q, 6p, 8p, 10q, 13q, 19p, 19q, 21q, 22q and 2q13; the 16p group amplifies the telomeres of 1p, 9p, 12p, 15q 16p and XqYq as well as the 2q14. (B) Examples of telomere fusion gels, showing the improvement in the sensitivity of the PCR reaction when the additional telomeres are included in the reaction, as indicated on the left. HEK293, and MRC5 E6E7 with population doublings (PD), are indicated above. Each reaction contains 100 ng of DNA, and the fusion products were detected with the XpYp telomere adjacent probe. The frequency at which fusion was detected is indicated below each analysis, molecular weight markers are indicated on the right.

Figure 2.

Examples of the DNA sequences telomere fusion events. (A) XpYp:XqYq, showing a deletion of 5601 bp in the XqYq telomere-adjacent DNA and fusion between two arms of the same chromosome. (B) 17p:10q, showing telomere TTAGGG repeats on either side of the fusion point. (C) 17p:2q14, example of fusion with an interstitial telomeric locus. (D) XpYp:10q, an example of a large insertion of non-telomeric DNA derived from 8q24. (E) 1p:XpYp, an example of an insertion from the same telomere (XpYp) in reverse orientation, SNPs that identify the XpYp telomeric-allele involved in the fusion event are identified with arrows above the sequence. (F) XpYp:XqYq, an example of a small insertion as well as fusion between two arms of the same chromosome. Telomere-adjacent DNA is shown in the multicolour, insertions in brown, TTAGGG repeats in black and telomere repeat variants in red. The number of contiguous blocks of TTAGGG (T) and variants (V) are detailed below each sequence.

Figure 3.

Histograms summarizing the data from the telomere fusion analysis in telomerase-positive epithelial HEK293 (open bars) and telomerase-negative MRC5 cells expressing HPV oncoproteins E6E7 from all three PD points sampled (solid bars). (A–D) The deletion size from the start of the telomere repeat arrays for16p group, 21q group, XpYp and 17p, respectively. (E) Size of 100% sequence homology at the fusion point. (F) Number of contiguous TTAGGG repeats at the fusion point. Means ± SD for each data set are indicated.

Figure 4.

Examples of the telomere variant repeat structure derived from sequence analysis of the telomeres adjacent to single telomere fusion events. The telomere codes run left to right (proximal to distal), the fusion point is at the right hand of each code, the numbering above indicates the number of repeat units from the start of the telomere repeat array. The key indicates the lettering system for each type of telomere repeat variant identified. Gaps (−) in the codes were introduced to improve the alignment, these gaps therefore indicate putative expansions or contractions of repeat blocks. An example of duplication is indicated with an arrow, deletions and insertions are indicated with parenthesis.

Figure 5.

Illustrating a model, for the sequence of events, that may account for intra-allelic inverted insertion that accompanies a subset of telomere fusion events. This model is using the fusion sequence depicted in Figure 2F as an example, the deletion points are indicated as distances from the start of the telomere repeat array. (A–B) Short telomeres become de-protected and are subjected to nucleolytic resection, yielding a 3′-overhang within the telomere-adjacent DNA. (C) The 3′-end of the overhang (−111) folds back upon itself and anneals adjacent to the −596 position, this is ligated and endonucleolytic cleavage creates a 5′-overhang structure which can be rendered double-stranded by strand extension (D). Further resection (E) to the −356 position creates a substrate (F) for annealing and ligation of the 1p sub-telomere at position −1260 (G). Endonucleolytic cleavage removes the unpaired 3′-overhang from XpYp (G), strand extension and ligation complete the fusion (H–I).