Human Molecular Genetics, 2000, Vol. 9, No. 3 347-352
© 2000 Oxford University Press
Microsatellite instability in tumors as a model to study the process of microsatellite mutations
Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Caixa Postal 486, 30161-970 Belo Horizonte, MG, Brazil and 1Laboratory of Cancer Genetics, Ludwig Institute for Cancer Research, Rua Professor Antônio Prudente, 109-4º andar, 01509-010 São Paulo, Brazil
Received 14 July 1999; Revised and Accepted 1 December 1999.
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ABSTRACT |
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We screened 42 sporadic gastric tumors and found that seven of them had significant microsatellite instability. These were then studied at 26 microsatellite loci, comprising di-, tri- and tetranucleotide repeats. The instability level of individual microsatellites in the tumors was found to be positively correlated with the population average heterozygosity and variance of repeat number of the microsatellite loci, as predicted by the stepwise mutation model. Moreover, as is known to occur in human populations, instability was strongly correlated with the number of repeats at each microsatellite locus and with the perfection of the reiterated sequence. These results demonstrate that microsatellite mutations in unstable tumors show similarities to germline mutations and suggest that their study may be useful in understanding the mechanisms that generate microsatellite variability in human populations. We used this model to test the claims that the microsatellite mutation process is biased towards increased size and heterozygosity with wide differences in allele sizes. These assertions were not confirmed.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Microsatellites are repeating units of 1–6 bp that are ubiquitous, abundant and highly polymorphic in eukaryotic genomes (1–3). The high level of variation observed at these loci is undoubtedly due to their high mutation rate, estimated to be of the order of 10–3–10–4 per generation (4). Several studies have attempted to ascertain whether the heterozygosity and allele frequency distributions in human populations fit more closely predictions based on the stepwise mutation model (5) or on the infinite allele model (6). Most of such studies have suggested that microsatellite mutations fit a stepwise model (7–10). Thus, studies of microsatellite mutations in in vitro human cell lines (4), human pedigrees (11) and artificial constructs in yeast (12) have shown that most microsatellite mutations involve the addition or subtraction of only one or two repeat units.
The degree of polymorphism of a microsatellite probably reflects two balancing factors: the frequency of strand slippage events during replication (13) and the efficiency of repair of resulting mismatches. Given the complexities of DNA replication and repair, the rates and the types of alteration are likely to be controlled at multiple steps, reflecting the probabilities of strand dissociation, misalignment during reassociation, correction of displaced bases by proofreading exonucleases and correction by mismatch repair (14).
Many structural aspects of microsatellites have been implicated in influencing mutation rates, including the length and base composition of the repeat motif (4,15,16), the number of repeats (17–19), the base composition of flanking sequences (20,21) and the degree of perfection of the repeats (17,20,22,23). Besides, there have been controversial claims of trends in the germline mutation process of microsatellites towards a preponderance of expansions over contractions (24,25) and also a preference for microsatellite mutations to occur in heterozygotes in which there is a large size difference between alleles (26). These issues have been difficult to resolve because of methodological limitations for the study of the microsatellite mutation process. Analyses based on pedigrees are work-intensive and generally yield only a small number of mutations (11). On the other hand, studies based on the evolution of lymphoblastoid cell lines in vitro (4) are potentially subject to distortions caused by the nature of the transformation process. Population variation studies can be especially revealing in those cases in which diversity levels can be compared directly with microsatellite sequence. However, variability at the population level depends also on population history and eventual selective forces. Then, genome-specific effects (selection for or against linked sites) will obscure the relationship between the mutation process and variability. Thus, new experimental approaches for the study of the microsatellite mutation process could make a substantial contribution to the precise understanding of this process.
Dramatic increases in the rate of somatic mutation of microsatellites [microsatellite instability (MSI)] have been observed in an autosomal dominant form of colorectal cancer known as hereditary non-polyposis colorectal cancer (HNPCC), associated with germline mutations in the DNA repair genes hMSH2, hMLH1, hPMS1 or hPMS2 (27–29). Mismatch repair defects also occur in a proportion of sporadic cancers, especially those belonging to the HNPCC spectrum (29). Although lack of expression of hMSH2 and hMLH1 genes is associated with sporadic tumors with MSI, as seen in HNPCC, the overwhelming majority of such tumors do not show mutations in these mismatch error repair genes (30–32). Rather, it appears that MSI in sporadic colorectal cancer more often results from epigenetic inactivation of hMLH1 associated with DNA methylation (33,34), in association with the large number of replications that occur in the rapidly dividing tumor tissue. Nevertheless, sporadic human malignant tumors displaying MSI may provide us with a useful system to study the mutation process of microsatellite loci, with the advantage that they may allow the dissection of the factors influencing the mutational event per se from those influencing mechanisms of mismatch repair. In the present study we report on the analysis of mutations observed at 26 different microsatellite loci in seven human sporadic gastric tumors with MSI. The results show that in general the mutation process in tumors with MSI is similar to that which generates population variation at human microsatellites, and that these malignancies thus constitute a useful model for the study of microsatellite evolution.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Tumors displaying MSI selected from a battery of 42 gastric adenocarcinomas were identified by the amplification of the BAT-26 locus, which has been shown to be almost always unstable in tumors with MSI (35). Seven of the 42 human gastric adenocarcinoma tumors showed microsatellite abnormalities at this locus (data not shown) and were studied further with 26 microsatellites, including di-, tri- and tetranucleotide repeat loci. MSI was considered to have occurred when there were extra bands in microdissected tumor tissue as compared with adjacent normal tissue. Figure 1 shows a representative example of alterations observed in one of these microsatellites. Assuming that the extra band(s) observed in the tumor tissue was mutated from the closest allele seen in normal tissue, we found that the vast majority (89%) of our mutations were single step, as has been shown for germinative mutations (11). Thus, we feel justified in adopting a stepwise mutation model for our analysis.
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The microsatellite loci analyzed, together with the instability level of each locus are shown in Table 1. The instability level was defined as the ratio of the number of tumors displaying mutations in each locus to the total number of tumors with MSI. Table 1 also lists the structure of each microsatellite according to GenBank or dbEST, map location and the corresponding heterozygosity rates and variance of the allele repetition number observed in at least 50 unrelated Brazilian Caucasian individuals. To compare the processes of microsatellite somatic mutations in tumors with those underlying population variation, non-parametric tests were used to assess the correlation between the instability level in tumors and both the population heterozygosity rate and variance of repeat number for each locus. Both associations were tested by Spearman’s rank correlation and in both cases a significant positive association was found: rs = 0.44, P < 0.02 for correlation between instability and variance of repeat number; rs = 0.53, P < 0.005 for instability and heterozygosity rate. Under the stepwise mutation model the variance of repeat numbers is linearly related to the mutation rate E(S2) = Nµ
2 (36). Linear regression analysis of the relationship between MSI in tumors and population variance also resulted in a significant linear relationship [CF (1,23) = 6.8, P < 0.016], as shown in Figure 2A. On the other hand, under the stepwise mutation model, the predicted heterozygosity is related to the mutation rate by the equation H = 1 – 1/(1 + 8Neµ). In Figure 2B this theoretical curve is superimposed on the data comparing tumor instability and population heterozygosity. In both analyses (comparison of MSI with variance and comparison of MSI with heterozygosity), the expected relationship between tumor instability and the population data is thus obtained supporting the view that somatic and germline mutations share the same fundamental characteristics of the stepwise mutation model.
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For di-, tri- and tetranucleotide repeats, the average number of repeats of microsatellite alleles had a very strong positive correlation with its instability level in tumors (rs = 0.78, P = 0.0001). Figure 3 shows the plot of the relationship between number of repeats of perfect microsatellites and mutations in tumors. When loci with imperfect repeats are included in the analysis, a much weaker but still significant correlation between number of repetitions and instability was obtained (rs = 0.64, P < 0.0005). Again, these data mirror those obtained when the population mutation rate of microsatellite loci is compared with repeat numbers and repeat perfection.
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There have been controversial assertions of trends in the germline mutation process of microsatellites towards a preponderance of expansions over contractions (24). In addition, a preference for microsatellite mutations has been claimed to occur in heterozygotes in which there is a large size difference between alleles (26). First, we tested for possible asymmetry of gains over losses in the somatic mutations of the tumors. For this, we examined all mutations observed at 21 different loci (76 mutation events). We assumed that the extra band(s) observed in the tumor tissue were mutated from the closest allele seen in normal tissue and then ascertained whether the mutation was an expansion or a contraction (Table 2). In the cases where the mutation was equidistant from the two alleles, the origin was considered unknown (indicated by a question mark in Table 2). In some tumors, there was more than one mutant band. In Table 2 we only scored the direction of clear mutant bands. Often there were minor bands of questionable significance and whenever there were doubts about the veracity of a mutation we did not include it. In total, there was a preponderance of contractions (59%) over expansions (41%), allowing us to reject the hypothesis of a trend for microsatellite growth. Next we examined whether mutations observed in tumors occurred disproportionately in loci whose alleles differed greatly in length. We tested this claim using our data at 18 different loci at all seven tumors with MSI (58 mutation events). The exact allele length differences at loci D2S123, D2S177 and D2S119 could not be determined with certainty and the data pertaining to these loci were excluded from this analysis. We calculated the total numbers and proportions of the mutations in relation to the total observations in comparison with the length differences at the microsatellites (Table 3). No significant correlation between mutations and length differences of alleles in somatic mutations occurring in this set of tumors was detectable using Fisher’s exact test.
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DISCUSSION |
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Defective mismatch repair, recognized phenotypically as somatic alterations in microsatellite repeat sequences, represents a novel mechanism of tumorigenesis. In order to verify whether such tumor-associated somatic instability could be considered a model for the germline mutational process of microsatellites, we characterized mutations at diverse microsatellite loci in sporadic gastric adenocarcinomas displaying MSI. Our principal objective was to verify whether mutation frequency was associated with microsatellite population parameters that are indicators of microsatellite variability. We found a positive correlation between heterozygosity rate and the variance of repeat number and locus instability in tumors in accordance with theoretical predictions of the stepwise mutation model. Recently, in a study also using tumor data, Di Rienzo et al. (36) detected a positive correlation between variance of repeat number scored at three populations from different ethnic backgrounds and the mean squared of mutation size estimated from somatic mutations in tumors. The authors also demonstrated a concordance between the observed and the expected relationships of the variables, assuming a general stepwise mutation model. These results indicate that the somatic mutation process is subject to the same constraints as the germline mutation process that generates microsatellite polymorphisms. Most of them apparently operate at steps preceding mutation repair by mismatch repair system, maybe increasing the probabilities of strand dissociation and/or misalignment during reassociation of DNA strands in slippage events.
We also found a positive correlation between the mean number of repeats and the instability of loci in tumors. To assess the effect of imperfections on instability, we compared the regression lines fitted using data of all microsatellites and of only the perfect ones. This analysis demonstrated that imperfect microsatellite loci have a lower instability, as expected from their length, when compared with perfect loci. These results are in agreement with surveys of population diversity that have found a positive correlation between microsatellite diversity and length (22,37,38). In this context, we have previously shown using the same tumors with MSI, that mutations in D13S308E, an asymmetric trinucleotide repeat microsatellite containing an 11 repeat imperfect portion and a 15 repeat perfect portion, occur exclusively in perfect repeats (39).
Based on our results and those obtained by Di Rienzo et al. (36) we conclude that human tumors with MSI may indeed be useful models for the study of the germline mutation process of short tandem repeat loci. As well as being of practical value for examining in more detail the microsatellite mutational process, our finding of the underlying similarities between somatic and germline mutations of these loci is also of biological significance. If it can be shown absolutely that somatic and germline mutations are controlled by similar biological variables and are in fact different manifestations of the same underlying process, then the apparently disparate phenomena of evolution- and age-associated, mutation-dependent diseases such as cancer might be mechanistically related (40). Such a relationship might lead to important conclusions such as the essential inevitability of cancer due to the evolutionary selection of a relatively elevated mutation rate in man (41–43). Others have compared the characteristics of germline and somatic mutations in a number of genes. In HPRT, for example, the same percentage of G:C to T:A transitions at CpG is thought to result from the spontaneous deamination of endogenously somatic mutations (44,45). Similar data are obtained when germline mutations in p53 in patients with Li–Fraumeni syndrome are compared with sporadic mutations in those tumors that are thought to be relatively little influenced by exogenous mutagens such as those of the intestinal tract. In Li–Fraumeni patients 53% of all p53 mutations are G:C to A:T transitions at CpG, whereas 47% of p53 mutations in colon tumors are of the same form. To these comparisons, which suggest that germline and somatic mutations arise from the same endogenous events, we can now add the evidence of microsatellite mutations that appear, following detailed analysis, to arise from and be influenced by the same factors in the somatic and germline lineages. The importance of these data is that whereas the similarity of gene mutations could be argued to be due to convergence selected for by phenotypic pressures, the neutral microsatellite mutations studied here are not subject to selective pressure.
The model of microsatellite mutations in sporadic tumors was used to test the controversial claims of trends in the microsatellite mutations towards expansions over contractions (24) and of a preference for microsatellite mutations to occur in heterozygotes in which there was a large size difference between alleles (26). Our results were not in agreement with either of these propositions. Similarly, Di Rienzo et al. (36) and Hoff-Olsen et al. (46) did not find any microsatellite direction bias in mismatch-deficient tumors, although the latter called attention to heterogeneity in the preponderant direction of mutation between different loci. We did not find evidence of such heterogeneity beyond what would be expected by chance (Table 2), and we believe that their conclusions can be explained by the small number of loci that they studied (4). In conclusion, our tumor results, as well as the others (36,46), agree perfectly well with the recent observations of Brinkmann et al. (11) and Sajantila et al. (47), who studied germline microsatellite mutations in paternity cases and did not observe any preferential tendency for gain or loss of repeat units in the mutational process.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Population and patient samples
Fifty unrelated Brazilian Caucasian individuals were randomly selected from a population that was submitted to paternity tests at GENE—Núcleo de Genética Médica (Belo Horizonte, Brazil). The DNA was extracted from peripheral blood according to standard procedures (48).
Tumor and corresponding normal tissues were sampled from 42 patients who underwent surgery for sporadic gastric adeno- carcinoma at Hospital do Câncer (São Paulo, SP, Brazil). All patients gave their express consent to this study, which was also approved by the ethics committee. DNA was extracted by a phenol–chloroform procedure as reported previously (49).
MSI and population typing
The presence of tumors displaying MSI at 42 sporadic gastric adenocarcinomas was detected by amplification of the BAT26 locus (35). Mutations were found in seven tumors, which were studied further with 10 loci containing dinucleotide repeats (D2S119, D2S123, D2S134, D2S136, D2S147, D2S177, D10S89, D12S43, D13S71, D17S250), 11 loci of trinucleotide repetitions (D13S308E, D2S196E, EST586, EST13299, EST05486, HHEA48B, D2S1353, D8S1119, D6S1031, CDH2, PPGB) and five loci of tetranucleotide repetitions (ACTBP2, D5S2501, D9S938, D12S67, TH01). PCR for dinucleotide loci was performed in a 25 µl reaction volume containing 50 ng of DNA, PCR buffer with 1.5 mM MgCl2, 4 pmol of each selected primer, each deoxynucleotide (200 µM), 1 U of Taq polymerase and 0.1 µM [
-32P]dCTP (Amersham Pharmacia Biotech, Uppsala, Sweden). PCR products were separated by denaturing electrophoresis in 6% polyacrylamide gels containing 7 M urea and visualized by autoradiography. The other loci were amplified using primer sequences and PCR conditions according to the following sources: D13S308E (50); D2S196E (51); EST586, EST1329, EST05486 and HHEA48B (52); CDH2 and PPGB (53); ACTBP2 (54); TH01 (55); D2S1353, D8S1119, D6S1031, D5S2501, D9S938 and D12S67 (Genome Data Base, http://www.gdb.org ). PCR products were denatured and analyzed on 8% polyacrylamide gels containing 7 M urea and stained with silver salts according to Santos et al. (56). The population samples were genotyped by the same protocols described above.
Statistical analysis
To measure the association between variables, we have used the Spearman rank correlation coefficient (rs), a non-parametric test.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr Álvaro José Romanha of the Centro de Pesquisas René Rachou for encouragement and technical support. We are also grateful to Eduardo Tarazona Santos for helpful discussions. This work was supported by CNPq, FAPESP and Fundação Oswaldo Cruz.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +55 31 227 3496; Fax: +55 31 227 3792; Email: spena@dcc.ufmg.br
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  O. Paun and E. Horandl Evolution of Hypervariable Microsatellites in Apomictic Polyploid Lineages of Ranunculus carpaticola: Directional Bias at Dinucleotide Loci Genetics, September 1, 2006; 174(1): 387 - 398. [Abstract] [Full Text] [PDF]
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 S. Demokan, Y. Suoglu, D. Demir, M. Gozeler, and N. Dalay Microsatellite instability and methylation of the DNA mismatch repair genes in head and neck cancer Ann. Onc., June 1, 2006; 17(6): 995 - 999. [Abstract] [Full Text] [PDF]
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 Y. Lai and F. Sun The Relationship Between Microsatellite Slippage Mutation Rate and the Number of Repeat Units Mol. Biol. Evol., December 1, 2003; 20(12): 2123 - 2131. [Abstract] [Full Text] [PDF]
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M. Colombino, A. Cossu, A. Arba, A. Manca, A. Curci, A. Avallone, G. Comella, G. Botti, F. Scintu, M. Amoruso, et al. Microsatellite instability and mutation analysis among southern Italian patients with colorectal carcinoma: detection of different alterations accounting for MLH1 and MSH2 inactivation in familial cases Ann. Onc., October 1, 2003; 14(10): 1530 - 1536. [Abstract] [Full Text] [PDF]
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M. Colombino, A. Cossu, A. Manca, M. F. Dedola, M. Giordano, F. Scintu, A. Curci, A. Avallone, G. Comella, M. Amoruso, et al. Prevalence and prognostic role of microsatellite instability in patients with rectal carcinoma Ann. Onc., September 1, 2002; 13(9): 1447 - 1453. [Abstract] [Full Text] [PDF]
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 A. L. Bacon, M. G. Dunlop, and S. M. Farrington Hypermutability at a poly(A/T) tract in the human germline Nucleic Acids Res., November 1, 2001; 29(21): 4405 - 4413. [Abstract] [Full Text] [PDF]
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 U. Holtkemper, B. Rolf, C. Hohoff, P. Forster, and B. Brinkmann Mutation rates at two human Y-chromosomal microsatellite loci using small pool PCR techniques Hum. Mol. Genet., March 1, 2001; 10(6): 629 - 633. [Abstract] [Full Text] [PDF]
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A. L. Bacon, S. M. Farrington, and M. G. Dunlop Sequence interruptions confer differential stability at microsatellite alleles in mismatch repair-deficient cells Hum. Mol. Genet., November 1, 2000; 9(18): 2707 - 2713. [Abstract] [Full Text] [PDF]
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