Hydroxyurea induces de novo copy number variants in human cells

Abstract

Copy number variants (CNVs) are widely distributed throughout the human genome, where they contribute to genetic variation and phenotypic diversity. Spontaneous CNVs are also a major cause of genetic and developmental disorders and arise frequently in cancer cells. As with all mutation classes, genetic and environmental factors almost certainly increase the risk for new and deleterious CNVs. However, despite the importance of CNVs, there is limited understanding of these precipitating risk factors and the mechanisms responsible for a large percentage of CNVs. Here we report that low doses of hydroxyurea, an inhibitor of ribonucleotide reductase and an important drug in the treatment of sickle cell disease and other diseases induces a high frequency of de novo CNVs in cultured human cells that resemble pathogenic and aphidicolin-induced CNVs in size and breakpoint structure. These CNVs are distributed throughout the genome, with some hotspots of de novo CNV formation. Sequencing revealed that CNV breakpoint junctions are characterized by short microhomologies, blunt ends, and short insertions. These data provide direct experimental support for models of replication-error origins of CNVs and suggest that any agent or condition that leads to replication stress has the potential to induce deleterious CNVs. In addition, they point to a need for further study of the genomic consequences of the therapeutic use of hydroxyurea.

Fig. 1.

HU induces de novo CNVs in normal human fibroblasts. (A) Incidence of de novo CNVs in normal, hTERT-immortalized human fibroblasts treated with 100–300 μM HU or 0.4 μM APH for 72 h. Fifteen independent clones each of untreated, 100 μM, 200 μM, and 300 μM HU-treated cells and 14 clones of APH-treated cells were analyzed. Error bars indicate SE. (B) Incidence of de novo CNVs in cells cultured in the presence or absence of HU or APH for varying lengths of time corresponding to two population doublings under each treatment (NT, 48 h; APH, 67 h; HU, 120 h). De novo CNVs from 10 independent clones each of untreated, 100 μM HU-treated, and 0.4 μM APH-treated cells and nine clones of 200 μM and 300 μM HU-treated cells were analyzed. Error bars indicate SE. (C) Colony-forming ability of HU-treated cells was reduced compared with untreated cells. Error bars indicate SD. (D) Poisson distributions illustrating the differences in de novo CNV incidence between HU-treated (red) and untreated (blue) cells. Graph includes all doses of HU from both experiments summarized in A and B. (E) Size distribution of CNVs. Graph showing the fraction of CNVs by size for two treatment groups, 0.4 μM APH (blue circles), 100–300 μM HU (red squares), and untreated (green triangles).

Fig. 2.

Locations of replication stress-induced CNVs. Red circles indicate HU-induced CNVs, blue squares indicate APH-induced CNVs, and green triangles indicate spontaneously arising CNVs in untreated cells. Markers above and below chromosomes represent duplications and deletions, respectively. Three large terminal deletions were also observed and are shown, although such events were considered to be a different class of alteration than smaller CNVs. Ideograms were adapted from the University of California, Santa Cruz genome browser (http://genome.ucsc.edu) (58). Precise coordinates for all de novo CNVs are listed in Dataset S1.

Fig. 3.

Clustering of HU- and APH-induced CNVs at 3q13.31 and 7q11.22. Clustering was defined as a region of the genome containing four or more overlapping or closely adjacent CNVs within a 2.5-Mb window (Materials and Methods). Although overlapping CNVs were found in these regions, all had distinct breakpoints.

Fig. 4.

Example of a complex de novo CNV induced by HU treatment. (A) Breakpoint junction sequencing revealed that this CNV at 3q26.31 in clone H2C21, which was called as a deletion on the basis of array data, was in fact a complex rearrangement involving an inversion of 5013 bp (red arrow), flanked by 48,309-bp and 14-bp deletions (blue bars). One of the two resulting breakpoint junctions was characterized by an insertion of 7 bp. This insertion was identified as an inverted duplication of 7 bp inserted 9 bp downstream of its location in the reference genome (gray arrows). (B) Diagram of the putative replication path taken to generate this complex CNV. (C) CNV breakpoint junction sequences from this complex CNV. The strand of DNA is indicated as (+) or (-). The inverted duplication is highlighted in gray, whereas the original position of the duplicated sequence in the reference genome is underlined. Regions of homology at the junction are underlined and highlighted in yellow. The two breakpoint junctions from this duplication had only a single base of homology. The other junction had 2 bp of microhomology at the junction. Mate-pair sequence analysis of the parental hTRT-090 (43) revealed several constitutional CNVs with a similar structure, suggesting that the replication stress-induced CNVs observed in vitro closely model the processes that occur in vivo.