A novel sensitive method to detect frameshift mutations in exonic repeat sequences of cancer-related genes
N. Mironov1,
L.A.M. Jansen,
W.-B. Zhu,
A.-M. Aguelon,
G. Reguer and
H. Yamasaki
Unit of Multistage Carcinogenesis, International Agency for Research on Cancer, 69372 Lyon Cedex 08, France
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Abstract
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We have investigated frameshift mutations in exonic repeats in the ATR, BRCA1, BRCA2, PTCH, CTCF, Cx26, NuMa and TGFßRII genes, using human tumor samples from stomach, esophagus, breast and skin and melanoma, as well as colon cancer and endometrial cancer cell lines (125 samples in total). We developed a sensitive method to detect mutations in the repeats, using the introduction of an artificial restriction site into a repeat. The method detects a single mutant among 103 normal genes. Thus, an alteration in a repeated sequence can be detected unambiguously. The (A)8 repeat of BRCA2 was found mutated in only two of five colon cell lines with microsatellite instability (MI+). The ATR gene has an (A)10 repeat which was altered in two of three MI+ stomach cancer samples and one of three MI+ endometrial cell lines. The TGFßRII gene [with an (A)10 repeat] had the maximal frequency of mutations: 10 out of 13 MI+ samples. At least one sample from all types of cancers, except melanomas, was positive for TGFßRII gene mutations. No mutations were found in repeats in the BRCA1, PTCH, CTCF, NuMA and Cx26 genes in any types of tumors examined. In conclusion, our study indicates that repeats were altered only in MI+ cells and that the mutation frequencies in the genes studied differ among tumor types. Based on these results, we discuss meaningful and meaningless alterations in exonic repeats.
Abbreviations: HNPCC, human non-polyposis colorectal cancer; MI, microsatellite instability; RER, replication error.
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Introduction
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It is widely accepted that the accumulation of several genetic alterations, which are necessary for cancer development, is associated with a mutator phenotype (1). A mutator phenotype, revealed as microsatellite instability (MI), was first described for human colon cancer, both human non-polyposis colorectal cancer (HNPCC) and sporadic (2,3), and subsequently for many other cancers, including endometrium (4), stomach (5) and esophagus (6).
DNA in tumors of HNPCC patients frequently has microsatellite alterations and this reflects, in many cases, a defect of the mismatch repair system. Five genes participating in the DNA mismatch repair system have been found to be mutated in HNPCC families: hMSH2, hMLH1, hPMS1, hPMS2 and hMSH6 (711). Since MI in tumors of HNPCC patients is associated with deficient activity of one of these genes, such tumors are believed to have a replication error (RER) positive phenotype.
Many genes contain repeated sequences in their coding regions. In most cases they consist of 310 repeats of one nucleotide. They are shorter than microsatellite loci used in gene mapping, but still may be targets for deletions and insertions in cells with MI. For example, frameshift mutations of the APC gene (mostly deletions or insertions of one base) are highly prevalent in MI+ cases as compared with MI (12). Similarly, A repeats in the TGFß receptor II gene (TGFßRII) are often mutated by frameshifts in MI+ tumors and cell lines (13). Such mutations may liberate cells from negative growth control, leading to uncontrolled cell growth.
Frameshift alterations in tumors with MI are also found in simple repeats of other genes, including the mismatch repair genes hMSH3 and hMSH6 (14). The IGFIIR gene is involved in cell growth control (15). The repeated sequence of this gene was found to be changed in MI+ gastrointestinal tumors (16). Furthermore, the BAX gene, which promotes apoptosis, has also been found to be altered in a repeated sequence in 50% of MI+ colorectal tumors (17).
The conventional PCR method for microsatellites usually gives ambiguous bands in polyacrylamide gels and does not detect a small number of MI+ cells in a given tumor. We describe here a method that allows us to reveal a frequency of mutations as low as one altered repeated gene sequence among 1000 copies of the wild-type. Using this approach, we have analyzed alterations of simple repeats in coding regions of eight genes.
We developed a method allowing us to distinguish between a wild-type gene and a specific mutation at a simple repeat in this gene, by introduction of a restriction site in a repeat during PCR. This concept is depicted schematically in Figure 1A and B
, using the example of an (A)10
(A)9 mutation, which is applicable to the ATR or TGFßRII genes. Two non-complementary bases were introduced into the primer which covers the simple repeat. The introduced restriction site could distinguish between the wild-type (A)10 sequence (lacking a restriction site for HinfI) and the (A)9 mutated sequence (carrying the restriction site for HinfI). In order to check that we really could distinguish between the (A)10 and (A)9 sequences in the template, we constructed two plasmids by cloning the fragments of TGFßRII with the corresponding short repeat in Bluescript DNA. When such plasmids were used as templates for PCR, only the product synthesized on the template with (A)9 could be digested with HinfI (Figure 1C
). Thus, in this model experiment the method shown in Figure 1A and B
allowed us to distinguish between the templates containing (A)9 or (A)10.

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Fig. 1. Method to distinguish PCR products synthesized on wild-type and altered repeat templates. (A) Scheme illustrating the absence of a HinfI restriction site in the PCR product synthesized on DNA containing an (A)10 repeat. (B) Scheme illustrating the appearance of a HinfI restriction site in the PCR product synthesized on DNA containing an (A)9 repeat. These schemes are applicable to both the ATR and TGFßRII genes containing an (A)10 exonic repeat. (C) PCR products containing (A)10 or (A)9 repeats of the TGFßRII gene were cloned in Bluescript. The plasmids were then used as the templates in PCR using primers to introduce a restriction site: 5'-CTTTATTCTGGAAGATGCTGCTGC and 5'-GAAAGTCTCACCAGGCTTTTTGATT [restriction site for HinfI in the case of an (A)9 mutant]. After the reaction, the product was digested with HinfI and separated on an 8% polyacrylamide gel. The undigested and digested parts are indicated by and +, respectively. (D) Conventional PCR was done around an (A)10 repeat of the ATR gene in gastric cancer samples. For conventional PCR around an (A)10 repeat, primers for the ATR gene were 5'-CTTCTGTCTGCAAGGCCATT and 5'-AGCAAGTTTTACTGGACTAGG. (E) The same samples as indicated in (D) but processed by a novel method. For ATR containing (A)10, the primers were 5'-CTTCTGTCTGCAAGCCATT and 5'-ACTGGACTAGGTATTTTTGATT (restriction site for HinfI). PCR was performed in 25 µl of solution containing 0.2 mM dNTP, 3 pmol each primer, 0.22 ng Taq Start Antibody and 1 U of Taq polymerase (Boehringer) in the supplied buffer. Half of the PCR product was used for digestion with restriction enzyme. Samples were separated on an 8% polyacrylamide gel. The dried gel was examined by radioautography.
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Using conventional PCR techniques around a repeated sequence (Figure 1D
) in stomach tumor DNA, it was impossible to distinguish between a normal and an altered A repeat in the ATR gene. When the samples were analyzed by introduction of a restriction site into the repeated sequence, two of these stomach samples were found to be heterozygous for an (A)9 alteration (Figure 1E
).
In order to estimate the sensitivity of the method, we determined the level at which the altered repeat could be detected when diluted with wild-type template. We performed an experiment in which genomic DNA of the LS180 cell line [both alleles of the TGFßRII gene contain an (A)9 repeat] was mixed in various proportions with DNA from HeLa cells, containing the wild-type TGFßRII gene (Figure 2
). The results indicated that detection of the (A)9 sequence was still possible when the ratio between the altered and wild-type repeats was as low as 103.
Figure 3
shows the results for the analysis of (A)8 repeat alterations in the BRCA2 gene of colon tumor cell lines. Only two colon cell lines (both MI+) were found to be heterozygous at the A repeat. Both of them contained repeats which were partly digested by ScaI and MaeIII, indicating the presence in DNA of both (A)7 and (A)8 sequence. No alterations were found in the repeats of the genes Cx26 (Figure 3B
), PTCH, CTCF, NuMA and BRCA1 (not shown) in cell lines or studied human tumor samples with MI+ status.

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Fig. 3. Detection of alterations in exonic repeats. For BRCA2 containing an (A)8 repeat the primers were 5'-CATACAGTTAGCAGCGACAAAAGTA [restriction site for MaeIII in the case of the wild-type and for ScaI in the case of an (A)7 mutant] and 5'-AGTGAAGGGGCTCCCG. The PCR product was divided into two parts and adjusted with a buffer corresponding to the restriction enzyme. U, undigested; M, MaeIII [for an (A)8 in BRCA2]; S, ScaI [for an (A)7 in BRCA2]; D, DdeI [for a wild-type (G)6 in Cx26]. Separation was performed on an 8% polyacrylamide gel. For the other exonic repeats, where alterations were not found, the primers can be provided on request. They include: an (A)8 in BRCA1 [restriction enzyme DdeI for the wild-type sequence and SpeI for an (A)7 mutant]; a (G)6 in Cx26 (restriction enzyme DdeI for the wild-type sequence); a (C)6 in NuMA [restriction enzyme StuI for (C)5]; a (C)7 in PTCH (patched) [restriction enzyme DdeI for (C)6]; an (A)7 in CTCF (restriction enzyme DdeI for the wild-type repeat).
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Finally, only ATR, BRCA2 and TGFßRII were found to be altered at exonic repeats in tumor samples or cell lines (Table I
). ATR and BRCA2 were altered in 23 and 20%, respectively. TGFßRII had the highest frequency of mutated repeats, which were seen in 10 out of 13 MI+ cases (77%). No alteration was found in samples and cell lines without MI; this suggests that the mutator phenotype was responsible for these alterations.
Thus, we found alterations in simple exonic repeats only in MI+ cases and the frequency of alteration varied from 0 (for most of the genes studied) to 77% for TGFßRII. Moreover, all positive samples showed similar band intensities between mutant and wild-type alleles, suggesting that mutations arose before clonal expansion of the tumors. Mutations of TGFßRII were not found in the entire coding sequence of 36 sporadic breast cancers (18). Using our approach, we found frameshift mutations in one sample out of 19 (Table I
) and this frequency is compatible with the published MI+ frequency for such cancers (19,20). We did not find any exonic alterations of A repeats in melanoma. The frequency of alteration of TGFßRII in skin cancer was higher than the frequency of published MI (21).
Depending on the primers for PCR, the method allows us to search for any deletion or insertion in a repeat (Figure 3
) or to search for only one specific alteration (Figure 1
). The most frequent alteration found in a simple exonic repeat of several genes was a deletion of one nucleotide (22).
The results reported here clearly indicate that the introduction of a restriction site allows unambiguous distinction of the altered template from the wild-type (Figures 1 and 3
). This may be partly explained by the idea that the introduction of an artificial restriction site shortens the exonic repeat, decreasing the possibility of slippage and, subsequently, the appearance of a PCR product that does not correspond to the template.
Only three genes (ATR, BRCA2 and TGFßRII) out of the eight we studied were found to have an altered exonic A repeat in some tumor samples or cell lines. BRCA2 is a tumor suppressor gene which is involved in control of recombination through binding to RAD51 (23). ATR plays a critical role in cell response to ionizing radiation and cell cycle checkpoint control (24,25).
Except for cell lines HCT116, LS174T and LS180, where TGFßRII was altered in both alleles, all samples with alterations also contained exonic repeats of the normal length. It is unlikely that only half of the cells in the analyzed samples contained a deletion of one nucleotide of the A repeat in both alleles. However, recently a tumor in Msh2+/, APC/Min mice was found to be polyclonal, part of the tumor having no wild-type APC, while the other part had no wild-type Msh2 (26).
Colon tumor cell lines DLD1 and HCT15 originated from the same patient. They could have some differences due to different conditions of culture, however, we did not detect any difference in the exonic repeats studied.
Several specific mutations are necessary for tumor development (27). The presence of a mutator phenotype is necessary for accumulation of such a quantity of meaningful genetic alterations (1) and MI represents one such phenotype. During the accumulation of specific mutations, many alterations that are meaningless for tumorigenesis appear at different microsatellites, such as in CA repeats. Those which appear in a cell during accumulation of the necessary specific genetic alterations could be found in tumors as markers of MI, because they will be present in all (or in many) tumor cells.
In colon and stomach tissues a frameshift mutation in an exonic repeat of the TGFßRII gene may be relevant to tumor development (28,29). Although repeat sequences of the ATR and BRCA2 genes could also be targets in some specific tissues, the small number of samples we studied does not allow us to be certain. A low frequency of BRCA2 alterations at (A)8 repeats was found in colon tumors (two samples out of 42) and one sample out of 25 endometrial carcinomas (30,31).
No alteration was found at exonic repeats in five out of the eight genes studied. These were BRCA1, Cx26, NuMA, PTCH and CTCF. Thus, although MI could be detected at random loci in MI+ samples and cell lines, we found no evidence for accumulation of mutated repeat sequences in these genes. Our method is highly sensitive and this result means that the frequency of cells with altered repeats of these genes is less than 1 in 103 of the wild-type. This suggests that the frequency of alterations in exonic repeats in genes that are not involved in tumorigenesis is much lower in cells with MI than that in microsatellites used as markers of instability. This implies that these genes were not changed before clonal expansion occurred or that if changes did occur, they had no effect on the origin of the cancers studied.
Thus, some exonic repeat changes can be considered meaningless for the genesis of certain types of cancer. It is also possible that mutations in the exonic repeat sequences of such genes adversely affect cell viability and are therefore selected against (32). One of the potential applications of our method could be in experiments with chemical carcinogens in mice having defects in the mismatch repair system. This method may detect alterations at repeats before tumor development. The method could also be used in screening for residual disease in patients with gastric and a subset of colorectal cancers.
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Acknowledgments
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We are grateful to Mrs C.Déchaux for secretarial assistance and Dr J.Cheney for editing the manuscript. This work was partially supported by a European Commission grant, no. ENV4/CT97/0469.
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Notes
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1 To whom correspondence should be addressed Email: mironov{at}iarc.fr 
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Received March 16, 1999;
revised June 3, 1999;
accepted June 25, 1999.