Why Cockayne syndrome patients do not get cancer despite their DNA repair deficiency

Abstract

Cockayne syndrome (CS) and xeroderma pigmentosum (XP) are human photosensitive diseases with mutations in the nucleotide excision repair (NER) pathway, which repairs DNA damage from UV exposure. CS is mutated in the transcription-coupled repair (TCR) branch of the NER pathway and exhibits developmental and neurological pathologies. The XP-C group of XP patients have mutations in the global genome repair (GGR) branch of the NER pathway and have a very high incidence of UV-induced skin cancer. Cultured cells from both diseases have similar sensitivity to UV-induced cytotoxicity, but CS patients have never been reported to develop cancer, although they often exhibit photosensitivity. Because cancers are associated with increased mutations, especially when initiated by DNA damage, we examined UV-induced mutagenesis in both XP-C and CS cells, using duplex sequencing for high-sensitivity mutation detection. Duplex sequencing detects rare mutagenic events, independent of selection and in multiple loci, enabling examination of all mutations rather than just those that confer major changes to a specific protein. We found telomerase-positive normal and CS-B cells had increased background mutation frequencies that decreased upon irradiation, purging the population of subclonal variants. Primary XP-C cells had increased UV-induced mutation frequencies compared with normal cells, consistent with their GGR deficiency. CS cells, in contrast, had normal levels of mutagenesis despite their TCR deficiency. The lack of elevated UV-induced mutagenesis in CS cells reveals that their TCR deficiency, although increasing cytotoxicity, is not mutagenic. Therefore the absence of cancer in CS patients results from the absence of UV-induced mutagenesis rather than from enhanced lethality.

Keywords: RNA pol II; apoptosis; dipyrimidines; mutagenesis; transcription arrest.

Fig. 1.

Survival of normal adult (WT), normal neonatal (NHF-D), XP-C [XPC(1) and (2)], CS-A [CSA(2)], CS-B [CSB(1) and (2)], and GM17536 (originally designated “CS-A,) [CSA(1)] fibroblasts. Error bars represent SD of two survival determinations.

Fig. S1.

UV-induced cytotoxicity. Survival of normal fibroblasts (GM05659) and keratinocytes treated with UVB (A) and UVC (B).

Fig. S2.

GM17536 shows no difference from normal primary fibroblasts when exposed to UV or illudin S. (A) Survival of GM17536 (blue squares) and normal (red squares) primary fibroblasts exposed to increasing UV doses. (B) Survival of GM17536, XP-C (XP226BA), CS-A (GM1856), CS-B (GM01428), and normal (GM03440) primary fibroblasts exposed to increasing concentrations of illudin S. (C) Subclonal UV-specific mutations (C:G→T:A at Py–Py sites) in GM17536 and normal (GM05659) primary fibroblasts.

Fig. 2.

UV induces unselected subclonal (<20% clonal) mutations in normal primary fibroblasts and keratinocytes. (A) Spectrum of subclonal mutations in adult (WT) and neonatal (NHF-D) fibroblasts treated with UVC and in keratinocytes (kerat) treated with UVB. (B) Subclonal frequencies of UV-specific mutations in adult and neonatal fibroblasts treated with UVC and in keratinocytes treated with UVB. Solid bars represent UV-specific mutations (C:G→T:A mutation at Py–Py sites); hashed bars represent C:G→T:A mutations at non-Py–Py sites. Frequencies were calculated by dividing the number of mutations of each type by the number of times the wild-type base of each mutation type was sequenced.

Fig. 3.

UVC induces increased UV-specific mutations in primary XP-C cells, relative to primary normal cells, but not in primary CS cells. (A and B) Frequency of all subclonal (<20% clonal) mutations (A) and UV-specific mutations (B) in normal adult (WT) and neonatal (NHF-D) primary fibroblasts and in XP-C [XPC(1) and (2)] primary fibroblasts. (C and D) Frequency of all subclonal mutations (C) and UV-specific mutations (D) in normal adult (WT) and neonatal (NHF-D) primary fibroblasts and in CS-A [CSA(2)] and CS-B [CS-B(1) and (2)] primary fibroblasts. (E) UV-specific mutations in XP-C [XPC(1) and (2)] and CS-A [CSA (2)] and CS-B [CS-B(1) and (2)] primary fibroblasts. (F) Subclonal frequencies of UV-specific mutations, oxidative-signature mutations, and all other mutations in primary neonatal (NHF-D), CS-A [CSA (2)], and CS-B [CS-B(1) and (2)] fibroblasts. Open bars represent UV-specific mutations (C:G→T:A mutation at Py–Py sites); solid bars represent oxidative-signature mutations (G:C→T:A); hashed bars represent all other mutations. Frequencies were calculated by dividing the number of mutations of each type by the number of times the wild-type base of each mutation type was sequenced. Error bars represent 95% confidence intervals calculated from Wilson scores of the mutation frequency for each sample.

Fig. S3.

UVC-induced mutation frequencies versus survival in normal and CS-A and CS-B primary fibroblasts. (A and B) C:G→T:A (A) and C:G→T:A (B) mutations at CC:GG dinucleotides in normal adult (WT) and neonatal (NHF-D) fibroblasts and in CS-A [CSA(2)] and CS-B [CSB(1) and (2)] fibroblasts. Percent survivals were derived from cell survival rates in Fig. 1C. Frequencies were calculated by dividing the number of mutations of each type by the number of times the wild-type base of each mutation type was sequenced.

Fig. S4.

UVC-induced mutations accumulate preferentially in the inactive genes of XP cells and in the coding strand of the active genes of CS cells. (A–C) Mutation spectrum in active (black bars) versus inactive (gray bars) genes in primary normal neonatal (A), CS-B (B), and in XP-C (C) fibroblasts. Hashed bars indicate mutation frequencies in control groups; solid bars indicate mutation frequencies in pooled UV-treated groups. (D–F) Mutation spectrum in the coding (nontranscribed) strand versus the template (transcribed) strand primary normal neonatal (D), CS-B (E), and XP-C (F) fibroblasts. Frequencies were calculated by dividing the number of mutations of each type by the number of times the wild-type base of each mutation type was sequenced. Hashed bars indicate mutation frequencies in active genes of control groups; solid bars indicate mutation frequencies in active genes of pooled UV-treated groups.

Fig. S5.

UV-induced mutation frequency changes in hTERT-immortalized cells. (A) High mutation frequencies in untreated hTERT-immortalized normal (navy) and CS-B (green) cells and reduction by UV exposure. (B) Reduction in subclonal variant frequencies with UV dose representing UV-induced population bottle-necking in hTERT-immortalized normal (navy) and CS-B (green) cells.

Fig. S6.

Gene amplification in hTERT⁺ cells. (A) Copy number for selected genes in hTERT-transfected fibroblasts that had been grown continuously for at least 2 y. Probes used are indicated in the x axis. GM05659T, blue bars; GM01428T, red bars. Error bars indicate SD. (B) Large GM05659T cell labeled with probes for 17q24 (red) and chromosome 6 (green); the outline of the cell is marked by the dashed white line. (C) GM01429T cell labeled with probes for 6q14 (red) and chromosome 5 (green); the outlines of three cell nuclei are marked by white dashed lines. (D) GM05659T chromosome spread labeled with probes for 17q24 (red), chromosome 2 (green), and chromosome 9 (blue). (E) GM01428T chromosome spread labeled with probes for 17q24 (red), chromosome 2 (green), and chromosome 9 (blue). (Scale bars: 20 μ.)