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Short Mononucleotide Repeat Sequence Variability in Mismatch Repair-deficient Cancers¹

Johns Hopkins Oncology Center, Baltimore, Maryland 21231 [L. Z., J. Y., K. W. K., B. V.]; Program of Human Genetics, Johns Hopkins School of Medicine, Baltimore, Maryland 21231 [J. Y., K. W. K., B. V.]; Department of Medicine and Ireland Cancer Center, University Hospitals of Cleveland and Case Western Reserve University, Cleveland, Ohio 44106 [J. K. V. W., S. D. M.]; and Howard Hughes Medical Institutes, Chevy Chase, Maryland 20815 [S. D. M., B. V.]

	ABSTRACT

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Mismatch repair-deficient cancers are characterized by widespread insertions and deletions in microsatellite sequences, including those comprised of mononucleotide repeats. Such alterations have been observed in relatively short mononucleotide tracts in several genes and often are interpreted to indicate that the affected genes normally act as tumor suppressors. To aid in the interpretation of such changes, we have systematically assessed their frequency within transcribed regions of the genome that are unlikely to play a tumorigenic role. The advent of the complete human genomic sequences of chromosome 22 allowed us to select 29 genes for this analysis, spaced at 1-Mb intervals. Each of the selected genes had an (A)₈ or a (G)₈ tract deep within intronic sequences that was not included in the processed transcript. Surprisingly, we found that there was substantial variation in the prevalence of mutations among these tracts. Some tracts were altered in <5% of the mismatch repair-deficient cancers studied, whereas other tracts were altered in nearly half of the cancers. In particular, (G)₈ tracts were considerably more prone to mutation than (A)₈ tracts, and the sequences or chromatin structures surrounding the mononucleotide tracts seemed to affect their mutability significantly.

	INTRODUCTION

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MSI³ was first observed in a subset of sporadic CRCs (1, 2, 3) and subsequently shown to be characteristic of cancers developing in HNPCC patients (4) . Studies in bacteria and yeast suggested that this form of instability might be attributable to MMR defects (5) , stimulating experiments that showed that defective MMR was responsible for MSI in human cancers. In HNPCC patients, the MMR defect is attributable to inherited mutations of one of several genes whose products participate in the process (6 , 7) . The genes most commonly defective are hMSH2 and hMLH1. In sporadic CRC, MMR defects are attributable to somatic mutations of the same genes in a subset of cases (8) and are associated with silencing of hMLH1 through epigenetic changes in the remainder (9) . Approximately 3% of the CRC in the United States are attributable to hereditary defects in MMR, leading to HNPCC, whereas approximately 10% are associated with somatically acquired defects (6 , 10) .

At the biochemical level, colorectal tumors appear to result from alterations of specific pathways that control cell birth and cell death. These pathways are similar in MMR-deficient and MMR-proficient cancers, but the mutational mechanisms that drive these tumors are distinct (11) . For example, the APC gene appears to initiate the neoplastic process and is mutated in both MMR-deficient and MMR-proficient cancers, but the nature of the mutations found in the former is different from that in the latter (12 , 13) . Moreover, the prevalence of APC mutations is somewhat less in MMR-deficient cancers than in MMR-proficient cancers. Many of the MMR-deficient tumors without APC mutations contain mutations of ß-catenin, a gene whose product binds to APC and mediates its oncogenic effects (14 , 15) . A similar situation exists with the TGFß pathway. MMR-proficient tumors often have mutations in SMAD4 or SMAD2 (16 , 17) , which function as downstream mediators of the TGFß signal. Most MMR-deficient tumors, in contrast, contain inactivating mutations of the TGFßRII gene and are thereby resistant to the effects of this growth-inhibitory cytokine (18) . Either type of defect (RII mutations in MSI cancers, SMAD2/4 mutations in chromosomal instability cancers) alters the same regulatory pathway. Similarly, MMR-deficient tumors have a lower prevalence of p53 mutations than MMR-proficient tumors, and it has been proposed that mutations of the BAX gene may substitute for p53 mutations in such tumors (19) .

Most of the mutations in TGFßRII and BAX genes, as well as those in several other genes implicated in the pathogenesis of MMR-deficient tumors, result in small deletions or insertions of SMTs. Unfortunately, mutations in such tracts in MSI tumors do not necessarily imply a causal role for the affected gene, as presciently noted in the first study describing such alterations (2) . Investigators have therefore provided evidence that control genes containing tracts of identical size were mutated less frequently in the same cancers. Whether such control genes have provided an accurate estimate of the true extent of alteration in SMTs is a question of fundamental significance to this field.

To investigate this issue in greater depth, we carried out a systematic study of the prevalence of mutations in SMTs in a set of cancers exhibiting MSI. Using information recently obtained from the human genome project (20) , we analyzed 29 genes that had mononucleotide tracts deep within their introns distributed every 1 Mb on chromosome 22. We found a surprisingly high variation in mutation rates in these sequences. These results have important implications for the interpretation of mutations of similar tracts in other genes as well as for basic features of MMR mechanisms in human cells.

	MATERIALS AND METHODS

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Cell Lines and Xenografts.
Surgically removed colorectal tumors were disaggregated and implanted into nude mice or into in vitro culture conditions as described previously (21) . DNA was purified from 17 MMR-deficient cell lines and seven xenograft tumors (22) . MMR status was assessed on the basis of the evaluation of numerous mononucleotide and dinucleotide repeat markers, as described. (23) .

PCR and Gel Electrophoresis.
One of the two PCR primers was labeled with [³²P]ATP by T4 polynucleotide kinase (Epicentre). The selected SMTs were amplified for 35 cycles using a four-stage touch-down protocol as follows: 95°C for 2 min for the initial denaturation; stage 1, four cycles of 95°C for 30 s, 64°C for 30 s, and 70°C for 2 min; stage 2, four cycles of 95°C for 30 s, 61°C for 30 s, and 70°C for 2 min; stage 3: four cycles of 95°C for 30 s, 58°C for 30 s, and 70°C for 2 min; and stage 4, 23 cycles of 95°C for 30 s, 55°C for 30 s, and 70°C for 2 min. PCR products were denatured and separated on 6% denaturing polyacrylamide gels, which were subsequently dried and subjected to autoradiography.

	RESULTS

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The choice of mononucleotide tracts for these experiments was based on the following three factors: (a) we wished to study tracts whose alterations were unlikely to play a role in the neoplastic process, thereby minimizing the possibility that mutations of such tracts could be selected for or against during tumorigenesis; (b) we wished to study tracts within genes (rather than within intergenic regions), because some repair processes, including MMR, can be affected by transcription (24) ; this choice allowed more direct comparison with previously reported alterations in transcribed genes; and (c) we wished to study a relatively large number of mononucleotide tracts in a systematic manner.

To fulfill these objectives, we chose to analyze mononucleotide tracts within the introns of genes spaced approximately every 1 Mb on chromosome 22. The availability of the complete genomic sequence of this chromosome allowed us to choose mononucleotide tracts that were far from intron/exon borders, thus ensuring that changes in such tracts would not have any functional effect on the processing of transcripts. Most of these genes were well characterized and transcribed in CRC cells, as indicated by serial analysis of gene expression databases (25) . The genes chosen for analysis and their positions along the chromosome are shown in Table 1 . The mononucleotide repeats selected for this analysis, their minimum distance from an intron/exon junction, the primers used for amplification, and the PCR product sizes are provided in Table 2 .

Examples of the results obtained with the new markers on chromosome 22 are shown in Fig. 1 and Fig. 2 . The tracts exhibited in Fig. 1 were mutated infrequently, often in none of the 24 cancers tested. In contrast, tracts such as those exhibited in Fig. 2 were mutated quite commonly, some in close to half of the cancers. Six facts were apparent from these data:

View larger version (57K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Mononucleotide repeats infrequently mutated in MMR-deficient tumors. , a deletion mutation; , an insertion. Lanes 1–17 contain PCR products derived from cell lines; Lanes 18–24 contain PCR products derived from xenografts.

View larger version (66K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Mononucleotide tracts frequently mutated in MMR-deficient tumors. , a deletion mutation; , an insertion. Double signs indicate that both alleles were mutated. The orders of Lanes are the same as in the legend to Fig. 1 .

(b) There was substantial variation in the prevalence of mutations of tracts composed of the same nucleotide, particularly for (A)₈ tracts. Thus, eight of the (A)₈ tracts were mutated in none of the lines, whereas one was mutated in more than one-third of the lines.

(c) (G)₈ tracts were defined as those which contained either (G)₈ or (C)₈ in the transcribed strand. This difference did not appear to play a role in mutability. The mutation prevalence among the three genes containing the (G)₈ tracts was 25 to 54.2%, whereas it was 12.5 to 45.8% among the five genes containing (C)₈ tracts. There was no apparent difference in mutation rates between the tracts containing (A)₈ versus (T)₈ in the transcribed strand.

(d) There was no particular subset of cancers that contained the bulk of the alterations. Thus, the average number of markers exhibiting mononucleotide alterations per tumor was 4.3 ± 1.7, and none of the 24 tumors contained alterations in more than nine markers or in fewer than two markers.

(e) Of the alterations detected, 41% were 1 bp insertions, 55% were 1 bp deletions, and 4% involved shifts of 2 bp. There was no apparent difference between the (G)₈ tracts and (A)₈ tracts in the ratio of insertions:deletions.

(f) The majority of the alterations affected only one allele, but in many cases both alleles were affected. For example, one allele of marker 29 [an (A)₈ tract] was altered in seven cancers, and both alleles were altered in two cancers. One allele of marker 14 [a (G)₈ tract] was altered in nine cancers, and both alleles were altered in two cancers (Fig. 2) .

Because some of the tracts were altered so commonly in MMR-deficient cancers, it was of interest to determine whether they were also altered in normal cells. To investigate this issue, we examined these tracts in the normal cells of patients whose tumors contained alterations. Of 33 variants encountered in the cancers, 29 represented somatic mutations not present in the corresponding normal tissues. Thus the great majority of alterations studied here represented somatic changes specifically associated with MMR-deficiency.

	DISCUSSION

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Although microsatellites containing repeats of 2–4 bp are affected by MMR-deficiencies in human cells, it has been rigorously demonstrated that homopolymeric sequences are particularly prone to mutation and are thus uniquely valuable to assess instability in such cells (26) . Mononucleotide repeat markers such as BAT40 and BAT26 have been shown to be altered in >95% of MMR-deficient cancers (23) , and it has been proposed that the evaluation of even a single such marker provides as sensitive an indicator of MMR-deficiency as a panel of several microsatellite markers (27) .

It is also well known, from the study of both mammalian and lower eukaryotic cells, that the frequency of mutation in microsatellite tracts is dependent on the number of the repeats in the tract (26 , 28) . The mutation frequency is very high in (A)_n tracts when n is >20, and even tracts as short as 10 bp are frequently mutated (23) . However, it is thought that tracts where n is <10 are less subject to mutation in MMR-deficient cells (29) . Moreover, (A)_n tracts are usually not reduced to sizes smaller than eight bases even during the many cell doublings and clonal bottlenecks through which tumors evolve (23) .

In most published studies of genes that are potential targets of MMR (i.e., potentially selected through a positive effect on growth), the prevalence of SMT mutations in control, presumably unselected genes has been noted to be low. The study of such control genes has in general indicated that A or G tracts of 9 bp or fewer are mutated in <5% of cancers with MSI. It is important to note that our results do not conflict with these previous studies. The majority of mononucleotide tracts we studied (16 of 29) were mutated in fewer than 10% of the cancers, and 8 were not altered in any of the cancers. The novel finding reported here is that this frequency varies widely, depending on the exact mononucleotide tract studied. Both the nature of the mononucleotide within the tract (A:T versus G:C bases) and the sequences surrounding the tract seem to be important. It is important to note that the tracts we examined were very distant from exonic sequences (Table 2) , and alterations in these tracts would not be expected to have functional consequences.

These results suggest that previously unrecognized features of the sequences or chromatin structure within or surrounding individual tracts contribute to mutability. They also have implications for evaluating potential targets of MMR. As explained above, "targets" comprise those genes that are mutated by virtue of MMR-deficiency and play a significant role in neoplastic growth. Such targets must be distinguished from genes that undergo passenger mutations and are present simply because they happened to occur in a cell that subsequently developed a clonal growth advantage. In light of the large number of cell divisions and clonal bottlenecks through which a malignant tumor passes before becoming clinically apparent, such passenger mutations are expected to far outnumber target mutations in MMR-deficient cancers (30 , 31) .

All of the mutations observed in our study are presumably passenger mutations. Our results demonstrate that a significant prevalence of mutation in a given gene in MSI cancers is not a reliable indicator that such genes are targets rather than passengers, even when the mutated tract is small. How, then, does one implicate a gene as a target rather than as a passenger in such tumors? This is extremely difficult, and compelling evidence in addition to the prevalence of mutations is needed, as pointed out in a recent workshop (31) . Functional evidence can help, but it is rarely definitive. Additional genetic evidence supporting the role of a gene as a target, however, can be obtained through at least two routes. First, as all mononucleotide tract mutations are presumably inactivating, it is reasonable to suggest that only when both alleles of the putative target gene are mutated is it highly likely that the gene is a target. Biallelic inactivation usually occurs through alterations of the mononucleotide tracts of both alleles, but it can also occur through mononucleotide tract alteration in one allele and a different mutation (not within the mononucleotide tract) in the second allele. Thus, evidence in favor of the role of TGFßRII was provided by the finding that both alleles are inactivated in most CRCs, through either mononucleotide tract or non-mononucleotide tract alterations (18) . Biallelic mutations of BAX have also been observed (19) . Second, it is notable that most of the genes that are mutated in MMR-proficient cancers are also mutated in MMR-deficient cancers, though the spectra and absolute prevalence of mutation sometimes varies (11) . By analogy, it is reasonable to assume that MMR gene targets will also be mutated in at least a small subset of MMR-proficient tumors. In this regard, it is notable that several MMR-proficient cancers contain mutations of TGFßRII (32 , 33) . Hopefully, attention to these issues will help to distinguish passengers from valid targets and lead to the discovery of new pathways of growth control that are relevant to both MMR-proficient and MMR-deficient cancers.

	ACKNOWLEDGMENTS

 
We thank Dr. Victor E. Velculescu for help with informatics, A. de la Chapelle, and the members of our laboratories for their critical comments.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by the Clayton Fund and NIH Grants CA 43460, CA 62924, and GM07184.

² To whom requests for reprints should be addressed, at Johns Hopkins Oncology Center, 1650 Orleans Street, Baltimore, MD 21231. E-mail: vogelbe{at}welch.jhu.edu

³ The abbreviations used are: MSI, microsatellite instability; CRC, colorectal cancer; HNPCC, hereditary nonpolyposis colorectal cancer; MMR, mismatch repair; APC, adenomatous polyposis coli; TGFß, transforming growth factor ß; TGFßRII, TGF-ß type II receptor; SMT, short mononucleotide tract.

Received 12/21/00. Accepted 2/28/01.

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