Article

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

Center for Human Genetic Research, Massachusetts General Hospital, Boston, Massachusetts, USA.
Nature Genetics (Impact Factor: 29.65). 03/2012; 44(4):390-7, S1. DOI: 10.1038/ng.2202
Source: PubMed

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.

Download full-text

Full-text

Available from: Toshiro K. Ohsumi, Jul 07, 2015
0 Followers
 · 
126 Views
  • Source
    • "Replication fork stalling and template switching has been hypothesized to underpin some of the complex genomic rearrangements found in constitutional genomic disorders (Liu et al. 2011b). One study, for example, showed that 19% of individuals with chromosomal abnormalities had evidence for complexity of genomic rearrangements at the breakpoints (Chiang et al. 2012). In many cases, these complexes of structural variation are driven by clustered rearrangements with a striking resemblance to those described here, and cause severe developmental disorders (Liu et al. 2011b). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Mutation is associated with developmental and hereditary disorders, aging and cancer. While we understand some mutational processes operative in human disease, most remain mysterious. We used C. elegans whole genome sequencing to model mutational signatures, analyzing 183 worm populations across 17 DNA repair-deficient backgrounds, propagated for 20 generations or exposed to carcinogens. The baseline mutation rate in C. elegans was ~1/genome/generation, not overtly altered across several DNA repair deficiencies over 20 generations. Telomere erosion led to complex chromosomal rearrangements initiated by breakage-fusion-bridge cycles and completed by simultaneously acquired, localized clusters of breakpoints. Aflatoxin-B1 induced substitutions of guanines in GpC context, as observed in aflatoxin-induced liver cancers. Mutational burden increased with impaired nucleotide excision repair. Cisplatin and mechlorethamine, DNA crosslinking agents, caused dose- and genotype-dependent signatures among indels, substitutions and rearrangements. Strikingly, both agents induced clustered rearrangements resembling 'chromoanasynthesis,' a replication-based mutational signature seen in constitutional genomic disorders, suggesting interstrand crosslinks may play a pathogenic role in such events. Cisplatin mutagenicity was most pronounced in xpf-1 mutants, suggesting this gene critically protects cells against platinum chemotherapy. Thus, experimental model systems combined with genome sequencing can recapture and mechanistically explain mutational signatures associated with human disease.
    Genome Research 07/2014; 24(10). DOI:10.1101/gr.175547.114 · 13.85 Impact Factor
  • Source
    • "This mechanism generates complex rearrangements formed by the incorrect ligation of ends with little or no sequence of homology and the insertion of some nucleotides, with the further loss of some fragments to generate deletions (Kloosterman et al. 2011; Holland and Cleveland 2012). The other mechanism that can be involved in the formation of complex rearrangements is chromoanasynthesis, which is generally involved in constitutional rearrangements that show complexity, microhomology at breakpoints , and occasionally the fusions of distant sequences and is indicative of a DNA replication-based mechanism as the causative agent, including fork stalling and template switching (FoSTeS) and/or microhomology-mediated break-induced replication (MMBIR) (Zhang et al. 2009; Liu et al. 2011; Chiang et al. 2012; Holland and Cleveland 2012). FoSTeS occurs when the DNA replication forks stall due to a DNA lesion or an error, allowing the replication fork to invade another fork through an area of microhomology. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Genome rearrangements are caused by the erroneous repair of DNA double-strand breaks, leading to several alterations that result in loss or gain of the structural genomic of a dosage-sensitive genes. However, the mechanisms that promote the complexity of rearrangements of congenital or developmental defects in human disease are unclear. The investigation of complex genomic abnormalities could help to elucidate the mechanisms and causes for the formation and facilitate the understanding of congenital or developmental defects in human disease. We here report one case of a patient with atypical clinical features of the 1p36 syndrome and the use of cytogenomic techniques to characterize the genomic alterations. Analysis by multiplex ligation-dependent probe amplification and array revealed a complex rearrangement in the 1p36.3 region with deletions and duplication interspaced by normal sequences. We also suggest that chromoanagenesis could be a possible mechanism involved in the repair and stabilization of this rearrangement.
    MGG Molecular & General Genetics 07/2014; DOI:10.1007/s00438-014-0876-7 · 2.83 Impact Factor
  • Source
    • "Although new methods such as fluorescence in situ hybridization (FISH) and array-based comparative genomic hybridization have been used to detect BCA-associated breakpoints in conjunction with karyotyping and other cytogenetic techniques (i.e., chromosome sorting), they are laborious and also unable to achieve single base pair resolution [Veltman et al., 2003; Chen et al., 2010]. Massive parallel sequencing has been demonstrated to accurately detect BCA-associated breakpoints, but this technique is highly dependent on prior knowledge of the affected G-band region [Chen et al., 2008; Chen et al., 2010; Sobreira et al., 2011; Talkowski et al., 2011; Chiang et al., 2012; Talkowski et al., 2012; Schluth-Bolard et al., 2013]. Therefore, for the routine clinical detection of BCA events, and to facilitate mapping of BCA-associated breakpoints, a highly accurate, cost-effective, and robust detection approach is desirable. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Balanced chromosomal rearrangement (or balanced chromosome abnormality, BCA) is a common chromosomal structural variation. Next-generation sequencing has been reported to detect BCA-associated breakpoints with the aid of karyotyping. However, the complications associated with this approach and the requirement for cytogenetics information has limited its application. Here, we provide a whole-genome low-coverage sequencing approach to detect BCA events independent of knowing the affected regions and with low false positives. First, six samples containing BCAs were used to establish a detection protocol and assess the efficacy of different library construction approaches. By clustering anomalous read pairs and filtering out the false-positive results with a control cohort and the concomitant mapping information, we could directly detect BCA events for each sample. Through optimizing the read depth, BCAs in all samples could be blindly detected with only 120 million read pairs per sample for data from a small-insert library and 30 million per sample for data from non-size-selected mate-pair library. This approach was further validated using another 13 samples that contained BCAs. Our approach advances the application of high-throughput whole-genome low-coverage analysis for robust BCA detection—especially for clinical samples—without the need for karyotyping. This article is protected by copyright. All rights reserved
    Human Mutation 05/2014; 35(5). DOI:10.1002/humu.22541 · 5.05 Impact Factor