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A Functional Assay for Mutations in Tumor Suppressor Genes Caused by Mismatch Repair Deficiency

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A Functional Assay for Mutations in Tumor Suppressor Genes Caused by Mismatch Repair Deficiency © 2001 Oxford University Press Human Molecular Genetics, 2001, Vol. 10, No. 24 2737–2743 A functional assay for mutations in tumor suppressor genes caused by mismatch repair deficiency Hanlee P. Ji and Mary-Claire King* Department of Medicine, Division of Medical Genetics, University of Washington, Seattle, WA 98195-7720, USA Received May 25, 2001; Revised and Accepted September 19, 2001 The coding sequences of multiple human tumor MMR-deficient strains of S.cerevisiae has increased our suppressor genes include microsatellite sequences understanding of the role of MSI in human tumors (5–7). that are prone to mutations. Saccharomyces cerevisiae In cancers from individuals with HNPCC, the elevation in strains deficient in DNA mismatch repair (MMR) can mutation rates causes inactivation of specific tumor suppressor be used to determine de novo mutation rates of these gene, producing a selective growth advantage and thus human tumor suppressor genes as well as any other contributing to stepwise neoplastic progression (8). Multiple genes have been identified with microsatellite mutations gene sequence. Microsatellites in human TGFBR2, including TGFBR2, IGFR2, MSH3, MSH6, PTEN, BAX and PTEN and APC genes were placed in yeast vectors MBD4 (8–15). The majority of these microsatellites consist of and analyzed in isogenic yeast strains that were wild- mononucleotide repeats which accumulate 1 bp deletions or type or deletion mutants for MSH2 or MLH1. In MMR- insertions. deficient strains, the vector containing the (A)10 Observational studies have identified these microsatellite microsatellite sequence of TGFBR2 had a mutation mutation hotspots; candidate genes are sequenced for micro- rate (mutations/cell division) of 1.4 × 10–4, compared satellite mutations in a series of tumors and cell lines exhib- to a mutation rate of 1.7 × 10–6 in the wild-type strain. iting MSI. Recently, criteria have been developed to In MMR-deficient strains, mutation rates in PTEN and standardize diagnostic testing of MSI in primary human APC were also elevated above background levels. tumors, permitting comparison among genes that display high PTEN mutation rates were higher in both msh2 MSI (MSI-H) versus those with mutations attributable to back- ground levels (MSS or MSI-L) (16). Whereas these tumor (4.4 × 10–5) and mlh1 strains (2.3 × 10–5). APC muta- × studies accurately identify mutations and their frequency tion rates in the msh2 strain (2.4 10–6) and the mlh1 among the tumor sample sets, they lack the ability to quantify × strain (1.7 10–6) were also significantly, but less rates of mutation caused by MMR deficiency (17). In addition, dramatically, elevated over background. Mutations there are inherent difficulties in analyzing de novo mutations in selected for in the yeast screen were identical to coding region microsatellites. Fluctuation analysis with those previously observed in human tumor samples mammalian cell lines has been developed for artificial dinucleo- with microsatellite instability (MSI). This functional tide tracts and is technically challenging (17–20). As an added assay has applicability in providing quantitative data complication of tumor studies, comparing the effects of about microsatellite mutation rates caused by MMR individual MMR genes on MSI is difficult because tumor deficiency in any human tumor suppressor gene samples and cell lines are frequently not isogenic and often- times have not been fully characterized for loss of MSH2 or sequence. It can also be applied as a genetic screen MLH1. to identify new genes that are vulnerable to such We have adapted a plasmid frameshift assay to evaluate MSI microsatellite mutations and thus may be involved in within the coding regions of human tumor suppressor genes in the neoplastic development of tumors with MSI. yeast strains lacking either msh2 or mlh1 (5). Relying upon the highly conserved function of MMR genes between humans INTRODUCTION and S.cerevisiae, this genetic screen permits the quantitative analysis and identification of de novo mutations caused by Hereditary non-polyposis colorectal cancer (HNPCC) is a MMR deficiency in any human microsatellite sequence. familial cancer syndrome caused by germline mutations in Several plasmids (pHJ series) were constructed in which a DNA mismatch repair (MMR) genes such as hMSH2 and portion of the tumor suppressor cDNA sequence was placed hMLH1 (1). The loss of these MMR genes causes micro- upstream and in-frame of URA3. These vectors were trans- satellite instability (MSI), a phenomenon where mutations formed into three isogenic haploid strains: wild-type, msh2 occur at significantly elevated rates in microsatellite tracts of deletion mutant and mlh1 deletion mutant. De novo mutations genomic DNA (2). The study of MMR genes in Saccharomyces in the tumor suppressor sequence lead to frameshifts that cerevisiae was vital in defining the role of MMR in human prevent the appropriate translation of the URA3 marker. These cancer (3,4). Similarly, the study of microsatellite tracts in events can be seen as growth on 5-fluororotic acid (FOAR) and *To whom correspondence should be addressed. Tel: +1 206 616 4294; Fax: +1 206 616 4295; Email: [email protected] 2738 Human Molecular Genetics, 2001, Vol. 10, No. 24 subsequently be scored (21). For fluctuation analysis, the Using the method of the median, fluctuation analysis was method of the median was used to determine mutation rates, repeated independently three to four times for each plasmid to the number of FOAR events per cell division (22). determine mutation rates in each strain and the results were For this initial study, we measured the mutation rates of micro- averaged (22). For all tumor suppressor genes, mutation rates satellite sequences in three tumor suppressor genes, TGFBR2, were significantly higher in MMR-deficient strains in compar- PTEN and APC. Transforming growth factor β-receptor II ison with the wild-type strain (Table 1). The plasmid pHJ9, containing the TGFBR2 (A) tract, showed dramatically inhibits cell proliferation and contains an (A)10 tract that is 10 frequently mutated in MSI-positive tumors such as colon elevated mutation rates in comparison with the other vectors. adenocarcinomas (8). The phosphatase tumor suppressor The TGFBR2 plasmid mutation rates were similar between either msh2 or mlh1 backgrounds. Mutation rates in pHJ3, PTEN contains two (A)6 tracts, one in exon 7 and the other in exon 8, the latter being the primary mutation hotspot in containing the PTEN microsatellites, were slightly higher in endometrial carcinomas with MSI (11,13). The adenomatous msh2 strains than mlh1 strains (1.84 times). For pHJ4, polyposis gene APC plays a key role in sporadic and familial containing APC microsatellites, mutation rates were surpris- colon cancers and contains a large number of microsatellites. ingly low with only 11- and 8-fold increases over wild-type mutation rates for msh2 and mlh1, respectively. We chose a region of APC with two (A)5 and one (T)5 micro- satellites previously shown to have mutations in MSI-positive colon carcinomas (9). Analysis of tumor suppressor microsatellite mutations Mutations caused by MMR deficiency were identified in the RESULTS tumor suppressor regions of the vectors (Table 2). Plasmids were recovered from FOAR colonies and the tumor suppressor Mutation rate analysis of positive control and background regions sequenced. Plasmids were isolated from independent levels in MMR-deficient strains experiments, separate samples and represent independent mutations. FOAR mutations were detected more frequently in As a positive control to test mutation rates in the MMR-deficient TGFBR2 than in PTEN and APC, the difference attributable to strains, we designed a centromeric plasmid (pHJ5) with a significantly higher mutation rates of the TGFBR2 region. dinucleotide tract, (GT)16, upstream and in-frame of URA3 Overall, the types of mutations from the msh2 and mlh1 strains (Fig. 1A). This plasmid also contained a LEU2 marker for had a similar distribution to those found in MSI-positive plasmid retention. As has been demonstrated in previous work, tumors, with 90% being 1 bp deletions. this microsatellite tract mutates at significantly elevated rates Plasmids lacking mutations in the TGFBR2, PTEN and APC in MMR-deficient yeast strains (5,6,23). In our hands, tumor suppressor microsatellites had mutations in URA3 as mutation rates (FOAR/cell division) in msh2 were 4.6 × 10–4, a was determined in a subset that was sequenced. URA3 muta- 71.4-fold elevation over wild-type background and in mlh1 tions occurred mostly in PTEN and APC plasmids, given the were 3.4 × 10–4, a 53-fold elevation over wild-type. lower mutation rates of their microsatellites. These mutations As an additional control, vector alone (pCI-HA) was occurred in three microsatellites tracts in URA3: (A)5 codons transformed into all strains to measure background mutation 58–60, (T)5 codons 67–69 and (T)6 codons 85–87. To account rates which can occur in URA3 alone, leading to FOAR. for the difference caused by mutations occurring in the tumor Vector alone had the lowest mutation rates among all the suppressor sequence versus URA3, we calculated an estimated plasmids tested. In wild-type strains, the mutation rate for tumor suppressor mutation rate using the ratio of plasmids with vector alone was 2.0 × 10–7. Mutation rates were 1.0 × 10–6 tumor suppressor mutations over the total number sequenced in msh2, a 5-fold increase over wild-type and were 0.8 × 10–6 in (Fig. 2). Given that no mutations were identified in the PTEN mlh1, a 4-fold increase over wild-type. These mutation rates and APC plasmids rescued from wild-type background, we –8 are a direct reflection of mutations occurring in URA3. used 1.0 × 10 which is an estimate of the general background mutation rate. In concordance with the plasmid mutation rate, Analysis of mutation rates in tumor suppressor the estimated tumor suppressor mutation rates of the TGFBR2 microsatellites in MMR-deficient strains sequence were significantly higher than mutation rates of the PTEN or APC sequences.
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