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.

Large transcription units unify copy number variants and common fragile sites arising under replication stress

“…Table S1). Forty-eight human and 41 mouse breakpoint junctions in these CNVs have been sequenced and all showed microhomologies, blunt ends, or small insertions (Arlt et al 2009(Arlt et al , 2011(Arlt et al , 2012(Arlt et al , 2014. These large sizes and junction properties identify the experimental CNVs examined here as being most similar to the nonrecurrent class of human pathogenic CNVs.…”

“…We previously noted a partial correspondence of replication-stressinduced CNVs and known CFSs (Arlt et al 2011) but were uncertain of the extent of this connection given that fibroblasts show cell-type-specific CFS patterns (Murano et al 1989;Le Tallec et al 2011). We therefore scored APH-induced metaphase chromosome breakage in our human 090 fibroblast cell line (Supplemental Fig.…”

“…CFSs are thought to be cytogenetic markers of loci prone to structural alterations, an idea supported by CFS rearrangements in multiple tumor types (Arlt et al 2006;Bignell et al 2010). In addition, many of the spontaneous and replication stress-induced CNVs seen in experimental cell systems overlap in specific genomic regions called hotspots, some of which correspond to CFSs (Durkin et al 2008;Arlt et al 2011). However, other CFSs were devoid of CNVs, making the nature of the CNV-CFS relationship uncertain.…”

“…Both simple and complex CNVs with nonhomologous breakpoint junctions arise sporadically and are induced in normal human cells by partial inhibition of replication with low-doses of aphidicolin (APH), hydroxyurea (HU), or ionizing radiation (IR) ( Fig. 1A; Arlt et al 2009Arlt et al , 2011Arlt et al , 2014. Similar CNVs were also induced in mouse embryonic stem (mES) cells in the presence or absence of XRCC4, a key NHEJ protein, reinforcing the idea that nonrecurrent CNVs are mainly created by replicative repair mechanisms when fork progression is impaired (Arlt et al 2012).…”

Oncogenes create a unique landscape of fragile sites

“…To date, most mapped FSs have been induced by low concentrations of aphidicolin, an inhibitor of the DNA polymerases a and d 15 . An additional chemical inducer of FSs is hydroxyurea, an inhibitor of a key nucleotide biosynthesis gene, ribonucleotide reductase, which leads to low nucleotide pools and slow replication fork progression 16 . In the past, most FSs were mapped in lymphocytes.…”