Replication Inhibitors Modulate Instability of an Expanded Trinucleotide Repeat at the Myotonic Dystrophy Type 1 Disease Locus in Human Cells

Abstract

Gene-specific CTG/CAG repeat expansion is associated with at least 14 human diseases, including myotonic dystrophy type 1 (DM1). Most of our understanding of trinucleotide instability is from nonhuman models, which have presented mixed results, supporting replication errors or processes independent of cell division as causes. Nevertheless, the mechanism occurring at the disease loci in patient cells is poorly understood. Using primary fibroblasts derived from a fetus with DM1, we have shown that spontaneous expansion of the diseased (CTG)(216) allele occurred in proliferating cells but not in quiescent cells. Expansions were "synchronous," with mutation frequencies approaching 100%. Furthermore, cells were treated with agents known to alter DNA synthesis but not to directly damage DNA. Inhibiting replication initiation with mimosine had no effect upon instability. Inhibiting both leading- and lagging-strand synthesis with aphidicolin or blocking only lagging strand synthesis with emetine significantly enhanced CTG expansions. It was striking that only the expanded DM1 allele was altered, leaving the normal allele, (CTG)(12), and other repeat loci unaffected. Standard and small-pool polymerase chain reaction revealed that inhibitors enhanced the magnitude of short expansions in most cells threefold, whereas 11%-25% of cells experienced gains of 122-170 repeats, to sizes of (CTG)(338)-(CTG)(386). Similar results were observed for an adult DM1 cell line. Our results support a role for the perturbation of replication fork dynamics in DM1 CTG expansions within patient fibroblasts. This is the first report that repeat-length alterations specific to a disease allele can be modulated by exogenously added compounds.

Hijacking of the mismatch repair system to cause CAG expansion and cell death in neurodegenerative disease

“…Thus, their role in causing expansion is difficult to test. Further, tests of expansion among cell types have not always painted a consistent picture due to the fact that the effects of strand breaks cannot be separated from the effects of replication in proliferating cells [67][68][69][70][71][72][78][79][80]. However, the use of transgenic animals has been particularly informative [70,[80][81][82][83][84][85][86].…”

Altered Replication in Human Cells Promotes DMPK (CTG)n · (CAG)n Repeat Instability

“…Correlated with (CTG) n · (CAG) n expansion from normalsize (n ϭ 5 to 37 repeats) to disease-associated (n Ͼ 80 repeats) lengths are changes in the local chromatin structure, including DNA methylation, induction of heterochromatin, alteration of nucleosome positioning, decreased nuclease hypersensitivity, and decreased gene expression of the DMPK gene and the neighboring SIX5 gene (13). Suggesting a role for the perturbation of replication fork dynamics in DMPK (CTG) n · (CAG) n instability, it has been reported that DNA replication inhibitors and DNA-damaging chemotherapeutic drugs can modulate instability at the DMPK locus in DM1 patient-derived cells (41,94) and transgenic-mouse cells (36).…”

Patients with primary cataract as a genetic pool of DMPK protomutation

“…The molecular mechanisms that determine the instability of triplet repeats during meiosis are unclear, implicating not only meiotic recombination but also DNA replication and repair (Pearson 2003;Malter et al 1997;De Temmerman et al 2004), CpG islands methylation, or its absence in the flanking region of the DMPK repeats and other epigenetic germline-specific modifications (Lemmers et al 2004), the involvement of nucleosomes (Wang et al 1994), and the defect in the DNA mismatch repair trans-acting mechanism (Kramer et al 1996). Also, especially in DM1, a role for the perturbation of replication in instability is strongly supported (Yang et al 2003).…”