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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.

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Figure 1: Circos plots of chromothripsis in human germline balanced rearrangements and a transgenic animal.
Figure 2: Delineation of two subjects with germline chromothripsis.
Figure 3: Complex rearrangements in transgenic animals.
Figure 4: Breakpoint sequence signatures from balanced structural variations and copy-number variation from independent population-based studies.
Figure 5: Comparison of observed chromosomal rearrangements to random simulations.

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Acknowledgements

We thank all study participants, their families and the referring clinicians for their invaluable contributions. We thank T. Gillis, M.A. Anderson, W. Varney, J. Ruliera, T. Henderson, C. Zhang, L. Griffin, The Genomics Core of the Massachusetts General Hospital Center for Human Genetic Research, the Genome Sequencing Platform at the Broad Institute and the Partners Center for Personalized Genomic Medicine for technical assistance. We thank M. Sun for performing the multiplex ligation-dependent probe amplification (MLPA) experiments. We also thank the Partners Research Computing at Massachusetts General Hospital. This work was funded by grants from the US National Institutes of Health (GM061354, HD065286, MH087123, NS32765 and NS16367), the Simons Foundation Autism Research Initiative, Autism Speaks and the CHDI Foundation. J.C.J. was the recipient of the Philip Wrightson Fellowship of the Neurological Foundation of New Zealand, C.E. received a Bisby Fellowship from the Canadian Institute for Health Research (CIHR).

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Authors and Affiliations

Authors

Contributions

M.E.T. and J.F.G. wrote the manuscript, which was edited by all coauthors. C.C., J.C.J., C.E., M.J.D., M.E.M., J.F.G. and M.E.T. designed the experiments. J.C.J., C.E., C.H. and A.M.L. performed the molecular studies. C.C., R.E.M., M.L.B., T.K.O., M.J.D., I.M.H. and M.E.T. designed the bioinformatic and statistical analyses. C.C., A.H., A.K., I.B., T.K.O., I.M.H., R.E.M. and M.E.T. performed the analyses. A.M.L. and C.C.M. performed the FISH analyses. J.C.J., C.C.M., A.M.L., S.R.R., C.J.M., C.S.B., S.J.R., R.L.M.F., R.G.S., Y.S., M.J.D., M.E.M. and J.F.G. provided the subjects and transgenic animals, and C.L. provided the clinical microarray data for the human chromothripsis samples.

Corresponding author

Correspondence to Michael E Talkowski.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Note, Supplementary Figures 1–9 and Supplementary Tables 1–3 (PDF 7241 kb)

Supplementary Table 4

Delineation of all breakpoint sequences, microhomology, and inserted sequences (Excel file) (XLSX 63 kb)

Supplementary Movie S1

Animation of chromosomal breakage and reconstitution of BSID42. Karyotype analysis indicated a 5q to Xq balanced translocation. However sequencing revealed a 'shattering' and aberrant reorganization of localized genomic regions similar to those recently reported in cancer cells (i.e., chromothripsis). (MOV 1152 kb)

Supplementary Movie S2

Animation of chromosomal breakage and reconstitution of BSID43. Two independent karyotype 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. (MOV 1547 kb)

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Chiang, C., Jacobsen, J., Ernst, C. et al. Complex reorganization and predominant non-homologous repair following chromosomal breakage in karyotypically balanced germline rearrangements and transgenic integration. Nat Genet 44, 390–397 (2012). https://doi.org/10.1038/ng.2202

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