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[Cancer Research 65, 4607-4613, June 1, 2005]
© 2005 American Association for Cancer Research

Molecular Biology, Pathobiology, and Genetics

Mutations in Two Short Noncoding Mononucleotide Repeats in Most Microsatellite-Unstable Colorectal Cancers

Tuija Hienonen1, Heli Sammalkorpi1, Susa Enholm1, Pia Alhopuro1, Thomas D. Barber5, Rainer Lehtonen1, Nina N. Nupponen1, Heli Lehtonen1, Reijo Salovaara1,2, Jukka-Pekka Mecklin6, Heikki Järvinen3, Riitta Koistinen4, Diego Arango1, Virpi Launonen1, Bert Vogelstein5, Auli Karhu1 and Lauri A. Aaltonen1

1 Department of Medical Genetics, Biomedicum Helsinki and 2 Department of Pathology, Haartman Institute, University of Helsinki; 3 Second Department of Surgery and 4 Department of Obstetrics and Gynecology and Clinical Chemistry, Helsinki University Central Hospital, Helsinki, Finland; 5 The Howard Hughes Medical Institute, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins Medical Institutions, Baltimore, Maryland; and 6 Department of Surgery, Jyväskylä Central Hospital, Jyväskylä, Finland

Requests for reprints: Lauri A. Aaltonen, Department of Medical Genetics, Biomedicum Helsinki, University of Helsinki, P.O. Box 63, FIN-00014 Helsinki, Finland. Phone: 358-9-1912-5595; Fax: 358-9-1912-5105; E-mail: lauri.aaltonen{at}helsinki.fi.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
DNA mismatch repair (MMR)–deficient cells typically accumulate mutations in short repetitive DNA tracts. This microsatellite instability (MSI) facilitates malignant transformation when affecting genes with growth-related and caretaker functions. To date, several putative MSI target genes have been proposed mainly based on high mutation frequency within their coding regions. However, some intronic repeat mutations have also been suggested to associate with MSI tumorigenesis, indicating the need for additional analyses on noncoding repeats. Here we have analyzed an intronic T9 repeat of semenogelin I (SEMG1) and report mutation frequencies of 51% (75 of 146) and 62% (8 of 13) in MMR-deficient primary colorectal cancers and cell lines, respectively. The putative effect of the SEMG1 mutations was assessed by RNA and protein level analyses, but no differences were detected between colorectal cancer cell lines with different SEMG1 status. Subsequently, the general background mutation frequency of MSI colorectal cancers was assessed by screening for intergenic T9 repeat alterations. One of 10 examined repeats was mutated in 70% (102 of 145) of the colorectal cancers evaluated. The frequencies observed here are notably higher than previously published in noncoding repeats shorter than 10 bp in MMR-deficient primary tumors. Our results indicate that high mutation frequencies, similar or higher than those observed in proposed and approved target genes, can be detected in repeat tracts of MSI tumors without any apparent selection pressure. These data call for urgent and thorough large-scale evaluation of mutation frequencies in neutral short repetitive sequences in MMR-deficient tumors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Colorectal cancers display two major types of genomic instability: chromosomal instability (CIN) and microsatellite instability (MSI). Approximately 85% of colorectal cancers harbor prominent karyotypic aberrations indicative of CIN, whereas the remaining 15% display MSI with more subtle nucleotide changes. Cells with MSI accumulate frame-shift mutations in both coding and noncoding microsatellite tracts. Thus, malignant transformation is promoted due to altered functions of cancer-related genes. The accumulation of frame-shift mutations is caused by defective mismatch repair (MMR) system underlying hereditary nonpolyposis colorectal cancer syndrome (1–7) and also a subset of nonsyndromic colorectal cancer (8), endometrial, and gastric cancer cases (9).

Most of the microsatellite mutations observed in MMR-deficient cells are background (bystander) events and in the absence of selection pressure, mutation frequencies in clonal tumors are generally considered to be low. However, high mutation frequencies can be found in MSI target genes with growth-related functions (here denoted as "real"). To date, substantial efforts have been directed towards identifying these real MSI target genes, mainly based on mutation frequency data. By 2002, 177 genes with 245 coding and noncoding mononucleotide repeats had been screened for mutations in MSI cancers (10). Since then, at least 84 new coding and noncoding repeats have been investigated, indicating a persistent interest in this field of research (11–22). In colorectal cancers, mutations have been found in a number of genes with key cellular roles, such as growth factor receptors (e.g., TGFßRII), DNA MMR genes (MSH3), transcription factors (TCF-4), cell cycle regulators (PTEN), and genes involved in apoptosis (BAX; ref. 10). Especially high mutation frequencies have been detected in, for example, TGFßRII and BAX with around 80% and 40% mutation frequencies, respectively (10, 23, 24).

In addition to high mutation frequency, functional analyses can support the role of a given gene in MSI tumorigenesis (9). For example, the tumorigenic effect of TGFßRII and BAX mutations is supported by cogent functional evidence (24, 25). Most of the MSI target gene studies have relied on mutation frequency data and functional studies have not been pursued due to their laborious nature. Furthermore, interpretation of results from such experiments is not necessarily straightforward, and some mutations may only have a minor effect on their own but a more pronounced effect when other genes are also affected (26–28).

The noncoding repeat mutations occurring in MMR-deficient tumors are generally not considered to promote tumor growth unless found in regulatory regions of cancer-related genes. Consequently, the mutation frequencies are considered to be low and indeed a maximum of 12% of the studied primary tumors taken together have harbored mutations in mononucleotide repeats shorter than 10 bp (10, 11, 14, 29). However, higher mutation frequencies have been found in some longer intronic repeats indicative of a possible selection pressure. For example, 93% of primary colorectal cancers harbored MRE11 deletions in a T11 repeat of intron 4 splice acceptor site. The mutations cause a partial splicing defect, reduced mRNA and protein levels, and impaired S-phase checkpoint (12). Similarly, polythymidine mutations in splice acceptor site of ATM introns 8 and 12 result in aberrant splice variants alongside with the wild-type transcripts. The mutation frequencies in the MSI colorectal cancer cell lines studied were 80% and 100% in introns 8 and 12, respectively (30). These studies indicate the potential importance of noncoding repeats in MSI tumorigenesis and call for additional analyses on noncoding repeats of the genome.

As new putative MSI target genes are being proposed largely based on mutation frequency data, it is important to extend our knowledge of the general background mutation rate in MSI tumors. In this study, we report the genetic analyses of semenogelin I (SEMG1) containing a T9 repeat starting at position +5 in intron 2 [IVS2+5(T)9]. SEMG1 was chosen as a focus of this study because it was mutated more frequently than the other intronic repeats analyzed in our pilot study. Here we have evaluated the mutation frequency of SEMG1 IVS2+5(T)9 in an extensive set of MSI colorectal cancers and cell lines. As controls, we screened a large set of MSS colorectal cancers and cell lines, normal tissue samples from patients with MSI colorectal cancer, and samples from anonymous cancer-free controls. Furthermore, a putative RNA or protein level effect of SEMG1 mutations was evaluated. Subsequently, we analyzed MSI colorectal cancers for the occurrence of frame-shift mutations in T9 repeats in other genomic regions distant from known genes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Sample selection. The sample set analyzed for SEMG1 IVS2+5(T)9 mutations included 146 MSI colorectal cancers and respective normal tissue DNA, 238 MSS colorectal cancers, and 31 cancer cell lines. As normal controls, we used samples from 50 anonymous cancer-free blood donors from the Finnish Red Cross Blood Transfusion Centre (Tables 1 and 2). To extend the study, a subset of 29 MSI colorectal cancers was evaluated for mutations in 10 intergenic T9 repeats. The colorectal adenocarcinoma and corresponding normal tissue samples were chosen from sample series collected since 1994 (31, 32).7 MSI status has been previously determined and of the MSI cases analyzed in this study, 20% (29 of 146 cases) harbor germ line MLH1 or MSH2 mutations (31–33).8 Most (92%, 280 of 304) of the tumor samples used in this study contained ≥50% carcinoma cells according to pathologist's evaluation (data not available on all samples). The respective normal tissue DNA was extracted from blood or normal colonic epithelium distant from the tumor margins. Patient information and samples were obtained with full informed consent and Ethical Review Board approval. Of the cell lines studied, 13 displayed MSI and originated from colon (10 cell lines), prostate (n = 2), or endometrium (n = 1). The remaining 18 cell lines were MSS colorectal cancer (Table 2).


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  		Table 1. Number of samples analyzed in this study

 

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  		Table 2. Cell lines analyzed for the SEMG1 IVS2+5(T)9 mutation

 
SEMG1 IVS2+5(T)9. To determine the mutation frequency of SEMG1 IVS2+5(T)9, we analyzed 121 MSI colorectal cancers, 21 MSS colorectal cancers, and 31 cell lines by sequencing. To exclude polymorphisms, we sequenced the normal tissue DNA of patients with MSI tumors and 50 anonymous cancer-free blood donor samples. In some of the MSI colorectal cancers sequenced, only a low mutant allele signal was seen. To exclude any possibility of a technical artifact, one plate (n = 93) of the samples were analyzed using denaturing high-performance liquid chromatography (DHPLC) after reamplification. In addition to 93 MSI colorectal cancers, we analyzed 230 MSS colorectal cancers by DHPLC. In total, SEMG1 IVS2+5(T)9 mutations were screened in 146 MSI colorectal cancers, 238 MSS colorectal cancers, 31 cell lines, 144 normal tissue samples from patients with MSI colorectal cancer, and 50 samples from cancer-free controls (Tables 1 and 2). Forward primer 5'-CATGAACAAAACGGCAGACA-3' and reverse primer 5'-TGGAGAGTCCCCAACAATTC-3' were used for sequencing and DHPLC. Protocols are available upon request.

SEMG1 coding region. A possible second hit in SEMG1 coding region and exon-intron borders was screened in MSI samples including 20 colorectal cancers, five colorectal cancer cell lines, and one endometrial cancer cell line. Twenty MSS colorectal cancers and one MSS colorectal cancer cell line were also analyzed (Tables 1 and 2). The three exons of SEMG1 were screened by sequencing or DHPLC. Protocols and primer sequences are available upon request.

SEMG1 1145del180. A presumably neutral SEMG1 variant lacking 60 amino acids (1145del180) has been detected at an allele frequency of 3% in the Swedish population (34). The possible association of this deletion and colorectal cancer was studied in 90 Finnish colorectal cancers (15 MSI and 75 MSS) of which 10 MSI tumors harbored a SEMG1 IVS2+5(T)9 mutation. The corresponding normal tissue samples from patients harboring the deletion were also analyzed. The population frequency of this variant was determined by analyzing samples from 93 Finnish cancer-free blood donors. The variant was screened by PCR and agarose gel electrophoresis using the previously published primers (34).

SEMG1 mRNA levels. To examine the possible effect of the SEMG1 IVS2+5(T)9 mutation on the RNA level, we did quantitative real-time PCR (QPCR) on six MSI colorectal cancer cell lines including two homozygous mutant (HCT 116 and LS 174T), two heterozygous mutant (SW48 and RKO), and two wild-type cell lines (SW480 and DLD-1; Table 2). Total RNA was extracted using TRIzol Reagent (Life Technologies, Paisley, Scotland). Total RNA was DNase treated and purified with RNase-Free DNase Set (50) and RNeasy Mini Kit (50; Qiagen GmbH, Hilden, Germany). cDNA was generated using M-MLV reverse transcriptase (Promega, Madison, WI) and RNase inhibitor (Promega). cDNA of all comparable samples were produced together, and negative controls were included to verify the specificity of the reactions. The amplification was done with cDNA-specific primers spanning SEMG1 intron 1. The expression level of GAPDH was used as a control. Amplifications were carried out using GeneAmp 5700 Sequence Detection System (Applied Biosystems, Foster City, CA). Product formation was monitored by detecting the fluorescence emitted by the incorporating SYBR Green (Applied Biosystems) between dsDNA. The reactions were produced in duplicates for SEMG1 and GAPDH in a single run and the run was repeated to verify the results. The relative copy numbers in each cell line were determined using the formula 2–{Delta}{Delta}Ct (ABI PRISM 7700 Sequence Detection System User Bulletin #2, Applied Biosystems). PCR conditions and primer sequences are available upon request.

SEMG1 protein levels. SEMG1 protein expression was analyzed by Western blotting. The culture medium and cell lysates were collected for protein analysis from MSI colorectal cancer cell lines HCT 116, LS 174T, SW48, RKO, SW480, and DLD-1 (Table 2). Cell lysates were obtained using PLCLB buffer supplemented with a protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Cell lysates and media were concentrated using Vivaspin columns (Vivascience AG, Hannover, Germany) and the concentration of each sample was measured by Bicinchoninic Acid Protein Assay Reagent Kit (Pierce, Rockford, IL). From each cell line, 160 µg of proteins from cell lysates and 15 µg of proteins from the medium were separated on 10% Tris-HCl gel (Bio-Rad Laboratories, Hercules, CA) and blotted onto polyvinylidene fluoride membrane (Millipore, Bedford, MA). The membranes were probed with a polyclonal SEMG1 antibody (rabbit anti-SVS; Biocarta, San Diego, CA) at a 1:3,000 dilution. Polyclonal rabbit GAPDH antibody (Abcam, Cambridge, United Kingdom) was used as a running control at a 1:8,000 dilution. In addition, standard Ponceau S staining protocols were used to control the amount of protein loaded. SEMG1 was detected by Western Breeze Chemiluminescent Immunodetection System (Invitrogen Life Technologies, Carlsbad, CA), and GAPDH detection was done according to Arango et al. (35). The protein band intensities were measured using a FluorChem8800 imaging system (Alpha Innotech, San Leandro, CA).

Intergenic T9 repeats. We examined the background mutation frequency of MSI colorectal cancers by screening a set of 29 colorectal cancers for alterations in 10 T9 repeats in intergenic regions distant from known genes (Table 1). The repeats analyzed were from genomic contigs AL161657, AC103870, AL445240, AC108706, AP002759, AP002801, AC008163, AC004006, and AC027013. Two repeats were analyzed from contig AC008163. Primers for the intergenic repeats were designed based on Ensembl database 9 Once a notable somatic mutation frequency in AC027013 was observed, we extended our analysis to include a total of 145 MSI colorectal cancers and the respective normal tissue samples. For AC027013, the forward primer 5'-CAGCACACATCAAGACACGTT-3' and reverse primer 5'-TTGAGATCCAGAGGGAGCTG-3' were used. Primer information for the other intergenic repeats is available upon request.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
SEMG1 IVS2+5(T)9. The mutation frequency of SEMG1 IVS2+5(T)9 was analyzed in primary colorectal cancers, cancer cell lines, normal tissue samples from MSI colorectal cancer patients, and anonymous cancer-free blood donors (Table 1). In the MSI colorectal cancers analyzed, a mutation frequency of 51% (75 of 146) was detected (Table 3; Fig. 1). Most cases displayed a heterozygous deletion of one T, but in two tumors, a biallelic deletion of one T was observed. Of the cell lines studied, 7 of 10 (70%) MSI colorectal cancer cell lines displayed IVS2+5(T)9 deletion (Tables 2 and 3). Five of these (KM12, LIM1215, LoVo, RKO, and SW48) had a heterozygous deletion of one T, whereas the remaining two (HCT 116 and LS174T) had a deletion in both alleles. Of the other MSI cell lines, the endometrial HEC59 showed a heterozygous deletion but the prostate cell lines LNCaP and PC-3 were wild type. None of the 18 MSS colorectal cancer cell lines or 238 MSS tumors had alterations (Tables 2 and 3). Normal tissue DNA was available from 144 patients with MSI tumors, and no frame-shift mutations were observed in the 141 samples successfully analyzed. Furthermore, no alterations were observed in the 50 cancer-free controls (Table 3).


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  		Table 3. Frequency of SEMG1, AC027013, and TCF-4 T9 mutations observed in this study

 


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  		Figure 1. Sequencing (left) and DHPLC (right) graphs of a SEMG1 wild-type (A) and mutant (B) sample.

 
SEMG1 coding region. To search for a second hit in SEMG1, the coding region and exon-intron borders were analyzed in MSI primary colorectal cancers and cancer cell lines. MSS primary colorectal cancers and cell lines were also analyzed (Tables 1 and 2). We observed no pathogenic changes but identified five neutral variants in MSI and MSS colorectal cancers: C1386T, Arg447His, Arg457Gln, C1417T, and IVS2+30A -> G. The variants were within the same PCR fragment as IVS2+5(T)9; thus, the frequency of the variants could be determined in the whole set of 146 MSI and 238 MSS colorectal cancers. The previously unpublished missense changes Arg447His and Arg457Gln were present also in the respective normal tissues and according to the sequence data, there was no loss of heterozygosity in the tumors. The missense variants were also found in cancer-free controls. The allele frequencies of the missense variants were low (≤2%) in colorectal cancers and controls.

SEMG1 1145del180. A previously identified presumably neutral 60-amino-acid deletion variant of SEMG1 was assessed in primary colorectal cancers and cancer-free controls. Of the 15 MSI and 75 MSS colorectal cancers analyzed, three MSS colorectal cancers harbored the deletion variant (allele frequency 2%). The deletion was present also in the respective normal tissue and in four cancer-free control samples (allele frequency 2.2%). These results are in accordance with the allele frequencies reported by Lundwall et al. (3.1%).

Genotype-phenotype correlations. The MSI colorectal cancer patients with SEMG1 IVS2+5(T)9 mutations (n = 75) and wild-type SEMG1 (n = 71) were compared to find possible differences in the clinicopathologic characteristics of the patients. No significant differences were detected in age of onset, sex distribution, location or staging of the tumor, or presence of germ line MMR mutation (Table 4).


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  		Table 4. Clinicopathologic characteristics of the MSI colorectal cancer patients with or without SEMG1 IVS2+5(T)9 mutations

 
SEMG1 mRNA levels. To assess the putative effect of the SEMG1 IVS2+5(T)9 mutation on the RNA level, we did QPCR experiments on six colorectal cancer cell lines with different SEMG1 status. The SEMG1 PCR product was sequenced to verify the specificity of the reactions. We observed a low level of SEMG1 expression and no differences in the expression levels of HCT 116, LS 174T, SW48, and DLD-1 (Fig. 2). Data from cell lines SW480 and RKO were not analyzed due to primer dimer formation.



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  		Figure 2. Relative SEMG1 expression levels in MSI colorectal cancer cell lines as determined using the formula 2–{Delta}{Delta}Ct. The SEMG1 status of each cell line is depicted.

 
SEMG1 protein levels. To assess the putative effect of the SEMG1 IVS2+5(T)9 mutation at the protein level, protein extracts were examined from six colorectal cancer cell lines with different SEMG1 status. Possible differences in the size or amount of the protein products between these cell lines were assessed by Western blotting. Consistent with the RNA experiments, only low levels of SEMG1 were detected and there were no differences in the protein sizes or amounts among the cell lines evaluated. Protein bands of ~45 and 60 kDa were detected in the cell lysates and media, respectively, indicating a possible modification prior transport out of the cell (Fig. 3). Bands of the same size were detected also using a monoclonal semenogelin antibody F91-11E6 (36).



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  		Figure 3. Western blot analysis of SEMG1 levels in MSI colorectal cancer cell lines. A and B, SEMG1 detected from medium and cell lysates, respectively. C, GAPDH running control. 1 and 8, size standards; 2, HCT 116; 3, LS 174T; 4, RKO; 5, SW48; 6, DLD-1; and 7, SW480.

 
Intergenic T9 repeats. We analyzed a sample set of 29 MSI colorectal cancers for mutations in 10 intergenic T9 repeats to obtain information on the background mutation frequency of MSI colorectal cancers. After successful analysis of at least 72% (91% on an average) of the samples, mutation frequencies varying between 0% and 86% were detected (0% for AL161657, 7% for AC103870, 10% for AL445240, 10% for AC108706, 27% for AP002759, 28% for AP002801, 29% for AC008163/1, 30% for AC008163/2, 54% for AC004006, and 86% for AC027013). To exclude polymorphisms, we analyzed the respective normal tissue samples of patients with frame-shift mutations in the tumors. Polymorphisms were found only in AC004006 where 7 of 14 (50%) normal tissue samples displayed the frame-shift also. Thus, the mutation frequency of AC004006 was in fact 27%. Because of the extremely high mutation frequency in AC027013, we analyzed the complete set of 145 MSI tumor and respective normal tissue samples. In total, 102 of 145 (70%) of the tumors harbored AC027013 mutations in contrast to 0 of 143 of the respective normal tissue samples (Table 3).

Verification of sequencing results. To compare the mutation frequency of SEMG1 with the mutation frequency of a respective suggested MSI target gene, we sequenced the T9 repeat of TCF-4 exon 17 in 123 MSI colorectal cancers (Table 1). Altogether 73 of 123 (59%) of the tumors harbored a frame-shift mutation in the T9 repeat. No mutations were observed in the 15 normal tissue samples from patients with TCF-4 mutations (Table 3).

To exclude the possibility of sequencing artifacts or miscalling of mutant samples, some of the tumor samples with low mutation peaks in SEMG1 IVS2+5(T)9 were reanalyzed by DHPLC; the results were in perfect agreement. Furthermore, an independent analysis of SEMG1 and AC027013 T9 repeats was done in 47 normal/tumor sample pairs. The mutation status of the samples was determined by using different amplification and sequencing instrumentation as well as different primers. Sample calling was done blindly, without knowledge of the previously determined mutation analyses. The two independent analyses showed excellent concordance; 100% and 98% for SEMG1 and AC027013, respectively. Both laboratories independently chose not to score mutation signals <10% (compared with wild type signal) as mutations.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Here we have analyzed primary colorectal cancers and cancer cell lines for mutations in SEMG1 IVS2+5(T)9. Previous studies have shown that SEMG1 together with a highly homologous SEMG2 are the major proteins of human seminal plasma (37, 38). In addition to male reproductive tract, SEMG1 is expressed in a variety of other tissue types (e.g., skeletal muscle and gastrointestinal tract; ref. 39). Despite the wide expression pattern, other cellular functions of SEMG1 have not been confirmed, though a role in cell adhesion has been suggested (40). Additionally, increased expression of SEMG1 has been detected in chronic leukemia and in small cell lung carcinoma (40, 41).

Our analyses on an extensive set of primary colorectal cancers and cell lines show that SEMG1 IVS2+5(T)9 mutations are found in a large proportion of MSI colorectal cancers (51%) and cell lines (62%). The mutations seem associated with MMR deficiency as no mutations were detected in normal tissues or in MSS cancer cells. Of the MSI samples analyzed here, two primary colorectal cancers and two colorectal cancer cell lines had a homozygous deletion of one T in the IVS2+5(T)9. In the heterozygous samples, no second hit was found in the coding region or exon-intron borders of SEMG1. Thus, the frequency of biallelic mutations observed here was low, being 1.4% and 15% in the MSI colorectal cancers and MSI cell lines, respectively. However, the necessity of biallelic mutations can be questioned because haploinsufficiency could also contribute to MSI tumorigenesis (42).

To evaluate the possible effects of the SEMG1 IVS2+5(T)9 mutations, we analyzed the SEMG1 RNA and protein levels in colorectal cancer cell lines with different SEMG1 status. No evidence of RNA or protein level differences were obtained although the low level of SEMG1 expression might hinder the detection of slight differences between the cell lines. Due to the low level of expression and possible secondary structures of cDNA, we were not able to amplify the whole SEMG1 cDNA. Thus, a possible partial splicing defect could not be excluded although to our knowledge, there are no reports of donor splice site microsatellite mutations causing altered splicing. Our observations indicate either that SEMG1 mutations have consequences that escaped our analyses, or that the high mutation frequency observed here is a bystander event with no tumorigenic implications.

When analyzing the general background mutation frequency of MSI colorectal cancers, frequent alterations in the 10 intergenic T9 repeats were observed. The 70% mutation frequency in AC023017 is notably higher than previously identified (5.7%) in the six studied intronic mononucleotide repeats of the same size (10, 14). This result calls for reevaluation of previous views. The importance of analyzing a sufficient number of repeats of a certain length to obtain reliable estimation on the general mutation frequencies needs to be emphasized. Furthermore, the mutation detection methods have improved during the past few years. However, the mutations were easy to score, as shown by the almost 100% concordance between the two laboratories.

Our results indicate large variability in short repeat tracts under MMR deficiency and emphasize the occurrence of high frequency of mutations without apparent selection pressure. It is well known that apart from selection pressure, mutation frequency of a given microsatellite is controlled by the repeat length, type, and furthermore, a possible effect from the surrounding sequence or chromatin structure (43). The repeats analyzed here are of the same composition and size; thus, the surrounding sequence might explain the substantial variation in the mutation frequencies. At present, little is known about the possible sequence elements affecting replication fidelity but the information produced here and other MSI target gene studies provide valuable material for such sequence comparisons. Of note, although the intergenic T9 repeats were chosen from genomic regions distant from known genes, possible new transcripts might be discovered in the future and could explain the high frequency of mutations observed. Indeed, new putative transcripts in the vicinity of AC027013 T9 repeat were presented in the updated Celera Human Genome database. The location of the T9 repeat with respect to these transcripts, however, speaks against a functional significance: the location of the T9 repeat in the first putative transcript was in intron 2, 2.7 and 16.9 kb apart from the exon-intron borders. In relation to transcript 2, the T9 was 1.1 kb upstream of the transcription start site. Prediction of a possible promoter region of transcript 2 was pursued with several programs but none were obtained.

Our analyses on intronic and intergenic T9 repeats show that the mutability of repeat regions is highly variable under MMR deficiency, possibly because of flanking sequence elements. The mutation frequencies observed here are high even though the alterations are not expected to causally associate with malignant transformation. Therefore, the previous analyses on putative MSI target genes need to be carefully evaluated, and in the absence of supporting functional evidence the role of these genes in MSI tumorigenesis should be interpreted with caution. Our results emphasize the need for extensive evaluation of mutation frequencies in neutral short repetitive sequences in MSI cancers.


   Acknowledgments
 
Grant support: Academy of Finland (44870, Finnish Center of Excellence Program 2000-2005), Finnish Cancer Society, Sigrid Juselius Foundation, and European Commission grant GLG2-CT-2001-01861.

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.

We thank Mikko Aho, Anniina Leskinen, Inga-Lill Svedberg, Annika Korvenpää, and Sini Marttinen for technical assistance.


   Footnotes
 
7 Unpublished data. Back

8 Unpublished data. Back

9 http://www.ensembl.org/. Back

Received 1/18/05. Revised 3/ 8/05. Accepted 3/ 9/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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