Candidate mutator genes in mismatch repair-deficient thymic lymphomas: no evidence of mutations in the DNA polymerase {delta} gene

Marcia R. Campbell1, Thy Y. Thang1, Frank R. Jirik2 and Susan E. Andrew1,3

1 Department of Medical Genetics, University of Alberta, Edmonton,Alberta, Canada T6G 2H7 and
2 Centre for Molecular Medicine and Therapeutics and the Department of Medicine, University of British Columbia, Vancover, British Columbia, Canada V5Z 4H4


    Abstract
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 Abstract
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DNA mismatch repair (MMR) proteins recognize nucleotides that are incorrectly paired. Deficiencies in MMR lead to increased genomic instability reflected in an increased mutation frequency and predisposition to tumorigenesis. Mice lacking the MMR gene, Msh2, develop thymic lymphomas that exhibit much higher mutational frequencies than other Msh2–/– tumours and Msh2–/– normal thymic tissue, suggesting that an additional mutator may have been acquired in a tissue-specific manner. Clustered mutations observed exclusively in the thymic lymphomas suggests that a gene(s) associated with the replication machinery might have become altered during tumorigenesis. Based on mutation studies in Saccharomyces cerevisiae lacking Msh2 and DNA polymerase {delta} (DNA pol {delta}), we hypothesized that the acquisition of mutations in DNA pol {delta} could contribute to the hypermutator phenotype and tumorigenesis in Msh2–/– thymic tissue. Furthermore, previous reports have suggested that genes containing mononucleotide repeats are non-random mutational targets in the absence of MMR. Therefore, we sequenced all 26 exons of the DNA pol {delta} catalytic subunit, including the six exons containing mononucleotide repeats of >5 bp, from nine Msh2–/– thymic lymphomas and two wild-type controls. No DNA pol {delta} pathogenic mutations were found in the thymic lymphomas, although several DNA base differences compared with published DNA pol {delta} sequences were observed. We conclude, therefore, that inactivating mutations in DNA pol {delta} are not a contributing factor in the development of the hypermutator phenotype in MMR-deficient murine thymic lymphomas.

Abbreviations: DNA pol {delta}; DNA polymerase {delta}; MMR, mismatch repair; PCR, polymerase chain reaction.


    Introduction
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 Abstract
 Introduction
 References
 
Tumour development is hypothesized to require the accumulation of multiple genetic alterations within a particular cell. A mutator phenotype, characterized by an increased mutation rate, allows acquisition of sufficient mutations within key tumour suppressors or oncogenes necessary for tumorigenesis. Thus, a mutator phenotype, leading to the disruption of cell cycle regulation mechanisms, is hypothesized to be a common feature in the development of many neoplasms (1).

A lack of DNA mismatch repair (MMR) has been shown to result in a mutator phenotype (2,3). MMR is a post-replicative repair system that acts on mispaired nucleotides as well as small insertion/deletion loops generated by misincorporation during DNA synthesis. This repair system is therefore instrumental in maintaining the fidelity of DNA. In the mouse, Msh2 is responsible for the initial recognition of a mispair and subsequent recruitment of additional proteins necessary for MMR. To study mammalian MMR, Reitmair et al. (4) generated homozygous Msh2–/– mice by gene targeting technology. These mice were viable, but rapidly developed tumours, primarily lymphoid, starting at about 2 months of age. To study mutation frequency in the absence of MMR, Msh2–/– mice were bred with transgenic mice carrying the lacI reporter gene (5,6). Using the lacI mutation detection assay, Msh2–/– mice demonstrate a 10- to 15-fold increase in spontaneous mutation frequency as compared with Msh–/+ or wild-type mice (6). DNA isolated from the Msh2–/– thymic lymphomas revealed an even greater increase in mutational frequency, with values 3- to 17-fold higher than in non-tumour Msh2–/– thymic tissue (7). Other Msh2–/– tumours did not demonstrate such elevated mutation frequencies, but were similar to values seen in Msh2–/– non-tumour tissues, suggesting that the increase in mutation frequency was specific to the thymic lymphomas. Interestingly, the lacI assay revealed closely clustered mutations that were not observed in non-tumour tissue or other Msh2–/– tumours (7).

The greatly elevated mutation frequency in Msh2–/– thymic lymphomas, the latency period before tumours appear and the closely clustered multiple mutations specific to the lymphomas all suggest subsequent mutations in additional `mutator' genes with an additive or synergistic mutator effect, have been acquired in MMR-deficient thymi. Aberrant replication is one such mechanism that has been shown to induce such a mutator phenotype (8). DNA replicative slippages as a result of a mutated DNA polymerase may account for the observed hypermutator effect and is therefore a likely hypothesis to explain the increase in mutation frequency. Thus, mutations in DNA polymerase genes that alter the enzyme fidelity could be responsible for the above characteristics of thymic lymphomas. For example, DNA polymerase ß mutations have been shown to result in a mutator phenotype associated with several forms of cancer (911). DNA polymerase {delta} (DNA pol {delta}), is another potential polymerase that has an intrinsic 3'–5' exonuclease activity and has been implicated in DNA repair (12,13). When DNA pol {delta} is mutated, its catalytic properties are abnormal suggesting a potential mutator activity possibly resulting in a mutator phenotype (14). These results are consistent with the findings that Saccharomyces cerevisiae strains with mutations in the 3'–5' exonuclease domain of DNA pol {delta} are hypermutable (15). Supporting this result, Tran et al. have shown that an error-prone (or abnormal) DNA pol {delta} results in a mutator phenotype in S.cerevisiae and that double Msh2–/–/Pol{delta} mutations have hypermutation frequencies above either mutation alone, suggesting that Msh2 and DNA pol d act synergistically (16). Da Costa et al. examined the 3'–5' exonuclease domain of DNA pol {delta} and found that three out of seven colon cancer cell lines contained a DNA alteration causing an amino acid change, suggesting that a mutated DNA pol {delta} leads to tumorigenesis (17). Further implicating DNA pol {delta} in tumorigenesis, Flohr et al. identified point mutations in the catalytic subunit of DNA pol {delta} in human colon cancer cell lines and in primary human colon cancers (18). Additional support for the involvement of DNA pol {delta} in Msh2–/– tumorigenesis arises from the observation that, in the absence of MMR, some genes are non-random targets for mutations due to the presence of coding mononucleotide repeats of >=5 bp. Several genes containing coding mononucleotide repeats have been identified as downstream target genes in MMR-deficient tumours. Coding mutations in such mononucleotide runs have been identified in BAX, caspase-5, IGFRII, transforming growth factor ß receptor II, MSH6 and MSH3 in microsatellite unstable (MSI+) colorectal and endometrial cancers (1924). DNA pol {delta} contains six exons with mononucleotide runs of >5 bp and therefore justified a similar candidate gene screening approach assaying for a mutated DNA polymerase in murine MMR-deficient thymic lymphomas. Other DNA polymerases, such as DNA polymerase ß, display mutator phenotypes but are less likely candidates because they lack coding mononucleotide tracts. From the above data and in particular, the existence of six coding mononucleotide runs, we hypothesized that a mutated DNA pol {delta} is a likely candidate mutator gene in Msh2–/– murine thymic lymphomas. It is likely that a mutation in the catalytic subunit of DNA pol {delta} would cause decreased DNA copying fidelity and so lead to increased genomic instability and progression to malignancy.

Mice lacking the DNA MMR gene, Msh2, have been developed as a model for studying the relationship between a mutator phenotype and development of cancer. Msh2–/– mice develop thymic lymphomas with a hypermutator phenotype. We hypothesized that, in Msh2–/– thymic lymphomas, an additional mutator gene (in conjunction with MMR loss) was responsible for this increased hypermutator effect.

To identify the possible role of the DNA pol {delta} catalytic subunit in the development of Msh2–/– thymic lymphomas, all 26 exons of the gene were sequenced from genomic DNA in nine Msh2–/– thymic lymphomas and two control (non-tumorous) liver and brain samples from wild-type mice. DNA was extracted from tissues as previously described (25). Intronic primers (26) (GenBank accession number AF024570) (Table IGo) were used to amplify thymic lymphoma genomic DNA and control genomic DNA by polymerase chain reaction (PCR) (Table IIGo) and products were examined for mutations by 33P sequencing (Thermo Sequenase Cycle Sequencing, Amersham Pharmacia). Comparison of the two published DNA pol {delta} catalytic subunit sequences revealed sequence differences at seven locations (Table IIIGo). These differences in published sequence probably demonstrate sequencing errors of the cDNA or genomic DNA or differences in genetic backgrounds of the mice. The published genomic DNA sequence of DNA pol {delta} (26) is from the 129SVJ mouse strain and the published cDNA sequence (12) is from the BalbC/9 strain. The Msh2 mice are on a mixed genetic background of BalbC and 129 mouse strains.


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Table I. Intronic primer sets for amplification of exons 1–26 of the DNA pol {delta} gene
 

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Table II. DNA pol {delta} PCR conditions
 

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Table III. Nucleotide sequence variations found in Msh2–/– thymic lymphomas and Msh2+/+ control tissues compared with published DNA pol {delta} sequences
 
Our results confirmed the polymorphisms seen in the published sequences (Table IIIGo). Sequence from the thymic lymphomas (tumours A to I) and controls was identical to the published genomic DNA sequence for exons 3, 16, 20 and 24 and identical to the published cDNA sequence for exons 18 and 23.

DNA pol {delta} sequence from the thymic lymphomas revealed three locations where the sequence was different from both published sequences (exons 12, 13 and intron 17). One change was silent (exon 12), one was intronic (intron 17) and one resulted in an amino acid alteration (exon 13) (Table IIIGo). All changes were confirmed by repeat sequencing. Within exon 3 at nucleotide position 1532, three of the nine tumours (tumours B, C and H) contain a T residue instead of a T residue as in the published sequence (Table IIIGo) and at nucleotide position 4876 in exon 12, four of the nine tumours (tumours B, C, D and G) and both control tissues contain a T residue instead of a C residue as in the published sequences. The exon 12 alteration is silent. The intronic sequence alteration in intron 17 was seen in three tumours (tumours B, D and I) and in the controls; it is believed to be non-pathogenic as it does not alter the splice site junction of intron 17–exon 17. Exon 13 is the only alteration resulting in a polymorphic amino acid change that differed from both the cDNA and genomic DNA published sequences. This alteration was seen in four of the nine tumours sequenced (tumours B, C, D and H). However, the alteration is not considered to be disease associated as two non-tumour wild-type control tissues demonstrated the same nucleotide sequence alteration (Table IIIGo). Furthermore, this DNA alteration results in a conservative substitution of a serine replacing a glycine; both of these are neutral, uncharged amino acids, so this substitution is not predicted to result in altered protein function. Although this change is not likely to be associated with tumorigenesis, it is located adjacent to a conserved polymerization II (Pol II) domain of DNA pol {delta} (12), so the close proximity of this polymorphism to the Pol II polymerization domain could subtly affect enzyme function or fidelity. Further functional analyses are required to confirm this possibility.

In the absence of MMR, mononucleotide repeats of >=5 bp are prone to mutation through expansion and contraction of these repeats. Exons 3, 8, 16, 18 and 22 each contain mononucleotide repeats of >=5 bp, which were hypothesized to be non-random locations for polymerase slippage. No mutations were seen at these locations, in normal or tumour DNA. Contrary to our hypothesis, no disease-associated mutations were found in the coding sequence of DNA pol {delta} in the nine Msh2–/– thymic lymphomas that were screened (Table IIIGo), suggesting that polymerase slippages in the absence of MMR do not result in mutations of the mononucleotide tracts of DNA pol {delta}.

Several polymorphic DNA alterations were seen in the tumours and controls (Table IIIGo). The identified changes seen in our lymphomas are unlikely to be disease associated as they were observed in both tumour and non-tumour control tissue and probably represent differences in the strain of mice used or errors in the published cDNA or genomic DNA sequences.

Evidence supporting the involvement of DNA pol {delta} in the development of Msh2–/– thymic lymphoma and the presence of six coding mononucleotide repeats led us to sequence the 26 exons of the DNA pol {delta} gene in nine Msh2–/– thymic lymphomas and two wild-type controls from Msh2–/– littermates. No lymphoma-associated mutations were found in the coding region of DNA pol {delta}. Notably, the six coding mononucleotide repeat sequences were not mutated. The absence of repeat tract mutation indicates that the DNA pol {delta} gene is not a non-random target for hypermutation-driven mutagenesis in Msh2–/– murine thymic lymphomas. Continued investigation of other possible candidate genes, including other DNA polymerases such as DNA polymerase ß, is required to understand further the molecular events underlying the development of thymic lymphoma in the Msh2-deficient mouse.


    Notes
 
3 To whom correspondence should be addressed Email: seandrew{at}gpu.srv.ualberta.ca Back


    Acknowledgments
 
We thank Dr A. Reitmair for the Msh2–/– mice and Dr Agnes Baross-Francis for her work collecting the Msh2–/– thymic lymphomas. This work was supported by the Leukemia Research Foundation of Canada. MRC is supported by a Seventy-Fifth Anniversary Faculty of Medicine and Dentistry Graduate Student Award. S.E.A. is a Medical Research Council scholar and Alberta Heritage Foundation for Medical Research scholar.


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Received February 9, 2000; revised August 18, 2000; accepted August 29, 2000.





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