Complex reorganization and predominant non-homologous repair following chromosomal breakage in karyotypically balanced germline rearrangements and transgenic integration

Abstract

We defined the genetic landscape of balanced chromosomal rearrangements at nucleotide resolution by sequencing 141 breakpoints from cytogenetically interpreted translocations and inversions. We confirm that the recently described phenomenon of 'chromothripsis' (massive chromosomal shattering and reorganization) is not unique to cancer cells but also occurs in the germline, where it can resolve to a relatively balanced state with frequent inversions. We detected a high incidence of complex rearrangements (19.2%) and substantially less reliance on microhomology (31%) than previously observed in benign copy-number variants (CNVs). We compared these results to experimentally generated DNA breakage-repair by sequencing seven transgenic animals, revealing extensive rearrangement of the transgene and host genome with similar complexity to human germline alterations. Inversion was the most common rearrangement, suggesting that a combined mechanism involving template switching and non-homologous repair mediates the formation of balanced complex rearrangements that are viable, stably replicated and transmitted unaltered to subsequent generations.

Figure 1. Circos plots of chromothripsis in human germline balanced rearrangements and a transgenic animal

Three plots are shown for the two most intricately rearranged balanced SV subjects (BSID42 and BSID43) and the multiple fragmented integrations associated a transgenic animal (G₀/6). In each plot, lines connect each of the inter and intra-chromosomal junction fragments sequenced and the chromosomes are labeled outside of the circle. 1A. BSID42: Fourteen junctions were confirmed between chromosomes 5 and X. 1B. BSID43: Eleven junction fragments were confirmed between chromosomes 3, 5, and 7. 1C. G₀/6: Sequencing in a transgenic sheep revealed that the transgene was apparently ‘shattered’ and integrated into six different chromosomes, including one interchromosomal excision of sheep chromosome 8 (OAR8) and insertion into the junction fragment between OAR7 and the transgene, mimicking a translocation junction. All junction fragments were confirmed by capillary sequencing. Additional validation was performed using FISH to further confirm these results (Supplementary Material). All positions are hg19.

Figure 2. Delineation of two subjects with germline chromothripsis

Complete sequence resolution of all 25 chromosomal breakpoints in two highly complex chromosomal rearrangements. On the top are diagrams detailing the fully reconciled exchanges of material between chromosomes with lines and arrows connecting the junctions and below are reconstructions of the resultant chromosomal organization. Coordinate distances are not to scale. 2A. Sequencing revealed a similar pattern of the ‘shattering’ and aberrant reorganization of localized genomic regions from karyotypically balanced germline structural variations to those recently reported in cancer cells (i.e., chromothripsis). There were 14 junction fragments from two shattered regions of 5q14.3 and resultant re-organization of both 5q14.3 and the integration sites of Xq26.3 in both derivative chromosomes of the reciprocal translocation. These exchanges were generally balanced (6,357 bp lost from all exchanges combined), with frequent oscillations of strand orientation at junction fragments, inverted insertions of many fragments, and apparent induction of intrachromosomal inverted excision/insertion events in both derivative chromosomes. 2B. Two independent karyotypic analyses indicated a balanced reciprocal translocation between chromosomes 3q and 5q; however, sequencing revealed the shattering of chromatin from 7q and re-integration of 7q DNA shards into the junction fragments of both derivative chromosomes, resulting in no direct 3q–5q junctions. There were four different inverted excision/insertion events, including intrachromosomal excision, inversion, and re-insertion in 7q with a co-occurring inverted insertion of an intact 3.5 Mb segment of 3q, all of which involved only 1,551 bases of total DNA imbalance. All positions are hg19. See also Supplementary Movies 1 and 2.

Figure 3. Complex rearrangements in transgenic animals

Results from sequencing of two transgenic mice (A) and five transgenic sheep (B). Each transgene prior to pronuclear injection is provided in the first line and resultant transgene integration site and internal structure is shown below for each animal. Genomic integration sites are given by the gray lines flanking the transgene and the most parsimonious transgene structure is shown (R6/1,2 mice and G₀/1,2,4,5,6 sheep). The fragment injected into the R6 lines (1,905 bp) comprised the human HTT promotor, exon 1 (~130 CAG repeats), and 264 bp of human intron 1. The fragment injected into the sheep (11,625 bp) included the human HTT promoter and cDNA (69 CAG repeats), followed by exon 4, intron 4, and exon 5 of the bovine growth hormone (BGH) gene. Arrows represent strand orientation. *This transgene was fragmented and inserted into multiple chromosomal locations; one of the small fragments and resultant complex rearrangements of the host genome involving three independent chromosomes is shown here (see also Fig. 1, Supplementary Fig. 1). **Head-to-tail junctions and read depth analyses indicate multiple copies of the segment, however the precise number of duplications could not be determined. Mouse positions are mm9 and sheep positions are OAR2.

Figure 4. Breakpoint sequence signatures from balanced structural variations and copy number variation from independent population-based studies

Histograms of nucleotide distribution for each sample. Breakpoints with no homology or inserted sequences are in orange (blunt ligation), green bars represent sequence microhomology between breakpoints, and blue bars represent the number of inserted nucleotides at the breakpoints. 4A. Breakpoint sequence distribution from karyotypically and presumably pathogenic balanced rearrangements sequenced in this study. 4B. Sequence distribution of 545 breakpoints from six tumors (two CLLs, one colorectal cancer, one thyroid cancer, one renal cancer, and one small cell lung cancer) localized by paired-end sequencing and confirmed with PCR by Stephens et al. (2011). 4C. Sequence distribution of 315 deletions captured and sequenced in normal individuals by Conrad et al. (2010). 4D. Breakpoint signatures of 16,783 deletion breakpoints from the 1,000 Genomes Pilot Project 1 analyzed with the same pipeline as the karyotypically balanced rearrangements in this study (4A) showing an identical distribution of breakpoint sequences to those seen in Mills et al. (2011).

Figure 5. Comparison of observed chromosomal rearrangements to random simulations

5A. Histogram density chart showing homology length of samples with 0 bp or more at the breakpoint. Green bars are observed breakpoints, blue bars are a distribution of 1,000 chimeras of two random genomic sequences. 5B–C. Histogram distribution of breakpoints falling in LINE and SINE elements from 10,000 simulated sets of breakpoints equal in size to the observed set. The arrow indicates the observed dataset (91^st and 52^nd percentile for SINEs (5B) and LINEs (5C), respectively).