Replication stress induces genome-wide copy number changes in human cells that resemble polymorphic and pathogenic variants - PubMed (original) (raw)

Replication stress induces genome-wide copy number changes in human cells that resemble polymorphic and pathogenic variants

Martin F Arlt et al. Am J Hum Genet. 2009 Mar.

Abstract

Copy number variants (CNVs) are an important component of genomic variation in humans and other mammals. Similar de novo deletions and duplications, or copy number changes (CNCs), are now known to be a major cause of genetic and developmental disorders and to arise somatically in many cancers. A major mechanism leading to both CNVs and disease-associated CNCs is meiotic unequal crossing over, or nonallelic homologous recombination (NAHR), mediated by flanking repeated sequences or segmental duplications. Others appear to involve nonhomologous end joining (NHEJ) or aberrant replication suggesting a mitotic cell origin. Here we show that aphidicolin-induced replication stress in normal human cells leads to a high frequency of CNCs of tens to thousands of kilobases across the human genome that closely resemble CNVs and disease-associated CNCs. Most deletion and duplication breakpoint junctions were characterized by short (<6 bp) microhomologies, consistent with the hypothesis that these rearrangements were formed by NHEJ or a replication-coupled process, such as template switching. This is a previously unrecognized consequence of replication stress and suggests that replication fork stalling and subsequent error-prone repair are important mechanisms in the formation of CNVs and pathogenic CNCs in humans.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Examples of APH-Induced CNCs Detected by aCGH Representative data illustrating (A) a deletion at 15q22, (B) a duplication at 10q25, and (C) a tandem deletion and duplication at Xq21.

Figure 2

Figure 2

APH-Induced Replication Stress Creates Submicroscopic CNCs in Normal Human Fibroblasts (A) Percent of untreated (n = 11) and APH-treated (n = 14) clones containing one or more CNCs. (B) Average number of CNCs per clone in untreated and APH-treated clones. (C) Mean number of deletions (gray bars) and duplications (white bars) in untreated and APH-treated clones. Error bars indicate standard error.

Figure 3

Figure 3

CNCs Identified in APH-Treated Human Fibroblast Clones CNCs are mapped onto chromosome ideograms. Red bars to the left of a chromosome indicate deletions. Blue bars to the right of a chromosome indicate duplications.

Figure 4

Figure 4

Mapping of CNCs that Colocalize within the Same Chromosomal Bands or with Bands Containing Common Fragile Sites (A) Deletions (red bars) and duplications (blue bars) from chr. 3 hybrid clones and human fibroblast clones mapping to the same chromosomal bands. Asterisks (∗) indicate CNCs whose breakpoints were sequenced. (B) Examples of deletions (red bars) from human fibroblast clones mapping to chromosomal bands containing common fragile sites (black bars). Asterisks indicate CNCs whose breakpoints were sequenced.

Figure 5

Figure 5

PCR Strategy for Amplifying and Sequencing Deletion and Duplication Breakpoints Arrows represent PCR primers, designated P1 and P2, designed to flank deletion (A) and duplication (B) breakpoint junctions. Primers are designed to amplify breakpoints of duplications in any orientation.

Figure 6

Figure 6

Example of a Complex Deletion at 3q26.31 Detected in Chr. 3 Hybrid Clone 6-30 The red line indicates a deletion predicted by SegMNT at a 5000 bp interval average. Arrows indicate the positions of PCR assays used to detect the presence (+) or absence (−) of human chromosome 3 sequences in the clone. Blue areas represent undeleted regions, and pink areas represent deleted regions.

Similar articles

Cited by

References

    1. Iafrate A.J., Feuk L., Rivera M.N., Listewnik M.L., Donahoe P.K., Qi Y., Scherer S.W., Lee C. Detection of large-scale variation in the human genome. Nat. Genet. 2004;36:949–951. - PubMed
    1. Redon R., Ishikawa S., Fitch K.R., Feuk L., Perry G.H., Andrews D., Fiegler H., Shapero M.H., Carson A.R., Chen W. Global variation in copy number in the human genome. Nature. 2006;444:428–429. - PMC - PubMed
    1. Sebat J., Lakshmi B., Troge J., Alexander J., Young J., Lundin P., Måner S., Massa H., Walker M., Chi M. Large-scale copy number polymorphism in the human genome. Science. 2004;305:525–528. - PubMed
    1. Sharp A.J., Locke D.P., McGrath S.D., Cheng Z., Bailey J.A., Vallente R.U., Pertz L.M., Clark R.A., Schwartz S., Segraves R. Segmental duplications and copy-number variation in the human genome. Am. J. Hum. Genet. 2005;77:78–88. - PMC - PubMed
    1. Freeman J.L., Perry G.H., Feuk L., Redon R., McCarroll S.A., Altshuler D.M., Aburatani H., Jones K.W., Tyler-Smith C., Hurles M.E. Copy number variation: new insights in genome diversity. Genome Res. 2006;16:949–961. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources