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Within the past decade microsatellites have developed into one of the most popular genetic markers. Despite the widespread use of microsatellite analysis, an integral picture of the mutational dynamics of microsatellite DNA is just beginning to emerge. Here, I review both generally agreed and controversial results about the mutational dynamics of microsatellite DNA. Microsatellites are short DNA sequence stretches in which a motif of one to six bases is tandemly repeated. It has been known for some time that these sequences can differ in repeat number among individuals. With the advent of polymerase chain reaction (PCR) technology this property of microsatellite DNA was converted into a highly versatile genetic marker (Litt and Luty 1989; Tautz 1989; Weber and May 1989). Polymerase chain reaction products of different length can be amplified with primers flanking the variable microsatellite region. Due to the availability of high-throughput capillary sequencers or mass spectrography the sizing of alleles is no longer a bottleneck in microsatellite analysis. The almost random distribution of microsatellites and their high level of polymorphism greatly facilitated the construction of genetic maps (Dietrich et al. 1994; Dib et al. 1996) and enabled subsequent positional cloning of several genes. Almost at the same time, microsatellites were established as the marker of choice for the identification of individuals and paternity testing. The high sensitivity of PCR-based microsatellite analysis was not only of great benefit for forensics, but opened completely new research areas, such as the analysis of samples with limited DNA amounts (e.g., many social insects) or degraded DNA (e.g., feces, museum material) (Schlötterer and Pemberton 1998). More recently, microsatellite analysis has also been employed in population genetics (Goldstein and Schlötterer 1999). Compared with allozymes, microsatellites offer the advantage that, in principle, several thousand potentially polymorphic markers are available. Nevertheless, the application of microsatellites to population genetic questions requires a more detailed understanding of the mutation processes of microsatellite DNA as the evolutionary time frames covered in population genetics are often too long to allow novel microsatellite mutations to be ignored. Additional interest in the evolution of microsatellite DNA comes from the discovery that trinucleotide repeats, a special class of microsatellites, are involved in human neurodegenerative diseases (e.g., fragile X and Huntington's disease). A detailed understanding of the processes underlying microsatellite instability is therefore an important contribution toward a better understanding of these human neurodegenerative diseases.
BACKGROUND & AIMS: 5-Fluorouracil improves mortality in stage III colorectal cancer patients. In vitro studies suggest that microsatellite instability influences cell survival after 5-fluorouracil treatment. We investigated the survival influence of 5-fluorouracil in patients with microsatellite instability-high tumors. METHODS: We collected data and tumors on 204 consecutive stage II and III colorectal cancer patients from registries at the University of California and Veterans Administration hospitals in San Diego, California, from 1982 to 1999. Archival DNA was extracted, and microsatellite instability was assessed by National Cancer Institute-recommended markers. Cox proportional hazard modeling was used to determine survival associations for microsatellite instability and 5-fluorouracil treatment status. RESULTS: We identified 36 microsatellite instability-high (17.6%) and 168 non-microsatellite instability-high tumors (82.4%). Microsatellite instability-high tumors were significantly associated with proximal colon location, presence of mucin, and surrounding lymphoid reaction. Univariate and multivariate analyses showed no survival difference between microsatellite instability-high and non-microsatellite instability-high groups (hazard ratio, 1.04; P = 0.88). Dichotomized by use of 5-fluorouracil, there was increased risk of death in patients who received no adjuvant chemotherapy (hazard ratio, 2.02; P = 0.02). However, the benefit of 5-fluorouracil was different between microsatellite instability-high and non-microsatellite instability-high groups. Patients with non-microsatellite instability-high tumors who received 5-fluorouracil had better survival compared with patients who were not treated (P < 0.05). Conversely, patients with microsatellite instability-high tumors who were treated with 5-fluorouracil had no survival difference compared with patients without treatment (P = 0.52). CONCLUSIONS: There is improved survival in patients with non-microsatellite instability-high tumors after 5-fluorouracil-based chemotherapy that does not extend to patients with microsatellite instability-high tumors. The microsatellite instability status of a patient's colorectal cancer may indicate differences in 5-fluorouracil-based chemosensitivity; this is consistent with in vitro studies.
BACKGROUND: Colon cancers with high-frequency microsatellite instability have clinical and pathological features that distinguish them from microsatellite-stable tumors. We investigated the usefulness of microsatellite-instability status as a predictor of the benefit of adjuvant chemotherapy with fluorouracil in stage II and stage III colon cancer. METHODS: Tumor specimens were collected from patients with colon cancer who were enrolled in randomized trials of fluorouracil-based adjuvant chemotherapy. Microsatellite instability was assessed with the use of mononucleotide and dinucleotide markers. RESULTS: Of 570 tissue specimens, 95 (16.7 percent) exhibited high-frequency microsatellite instability. Among 287 patients who did not receive adjuvant therapy, those with tumors displaying high-frequency microsatellite instability had a better five-year rate of overall survival than patients with tumors exhibiting microsatellite stability or low-frequency instability (hazard ratio for death, 0.31 [95 percent confidence interval, 0.14 to 0.72]; P=0.004). Among patients who received adjuvant chemotherapy, high-frequency microsatellite instability was not correlated with increased overall survival (hazard ratio for death, 1.07 [95 percent confidence interval, 0.62 to 1.86]; P=0.80). The benefit of treatment differed significantly according to the microsatellite-instability status (P=0.01). Adjuvant chemotherapy improved overall survival among patients with microsatellite-stable tumors or tumors exhibiting low-frequency microsatellite instability, according to a multivariate analysis adjusted for stage and grade (hazard ratio for death, 0.72 [95 percent confidence interval, 0.53 to 0.99]; P=0.04). By contrast, there was no benefit of adjuvant chemotherapy in the group with high-frequency microsatellite instability. CONCLUSIONS: Fluorouracil-based adjuvant chemotherapy benefited patients with stage II or stage III colon cancer with microsatellite-stable tumors or tumors exhibiting low-frequency microsatellite instability but not those with tumors exhibiting high-frequency microsatellite instability.
Intraductal papillary neoplasms of the biliary tree are unusual lesions characterized by solitary or diffuse growth along the intra- and/or extrahepatic biliary tract. Biliary papillary neoplasms bear some clinicopathologic similarity to intraductal papillary mucinous neoplasms of the pancreas. Like intraductal papillary mucinous neoplasms of the pancreas, biliary papillary neoplasms can be purely intraductal lesions or can give rise to invasive adenocarcinomas. We recently studied the genetic alterations present in a series of biliary papillary neoplasms and noted the presence of allelic shifts in some biliary tumors during allelic loss assays on chromosomes 5q and 18q. This suggested that microsatellite instability might play a role in the molecular pathogenesis of biliary papillary neoplasms. Genomic DNA was extracted from 17 intraductal papillary neoplasms, 6 associated invasive cholangiocarcinomas, and corresponding normal tissues, and microsatellite instability testing was performed using the 5 microsatellite loci recommended by the 1997 National Cancer Institute-sponsored consensus conference (D2S123, D5S346, D17S250, Bat-25, and Bat-26). High-level microsatellite instability was considered to be present when at least two of five microsatellite loci showed allelic shifts, and low-level microsatellite instability, when only one locus was shifted, as per the National Cancer Institute criteria. We also determined the methylation status of the DNA mismatch repair gene hMLH1 by bisulfite treatment of genomic DNA, followed by methylation-specific PCR. High-level microsatellite instability was present in 2 of 17 (11.8%) biliary papillary neoplasms, including 1 case of purely intraductal tumor and 1 case with both intraductal and invasive cholangiocarcinoma components. In both cases there was extensive microsatellite instability, with allelic shifts in five of five and four of five microsatellite markers, respectively. Low-level microsatellite instability was present in 6 of 17 (35.3%) biliary papillary neoplasms, including 2 cases of purely intraductal tumor and 4 cases with both intraductal and invasive cholangiocarcinoma components. Interestingly, the pattern of allelic shifts was frequently not identical between the intraductal and invasive cholangiocarcinoma components; although the same microsatellite markers were shifted, alleles of differing lengths were generated in the intraductal and invasive components of the neoplasms with high-level microsatellite instability and of two neoplasms with low-level microsatellite instability. None of the biliary papillary neoplasms (0 of 10 cases with adequate DNA for evaluation) showed methylation of hMLH1. These results indicate that microsatellite instability is a relatively frequent event in papillary neoplasms of the biliary tree but is not associated with hMLH1 promoter hypermethylation. The finding that alleles of differing lengths were frequently generated between the intraductal and invasive components of those tumors with microsatellite instability suggests that there is significant genetic heterogeneity within these neoplasms.
Colorectal cancer with microsatellite instability has a characteristic clinicopathologic profile, featuring right-sided, lymphocyte-rich tumors with a better prognosis than microsatellite stable (MSS) carcinoma. Mucinous and signet ring cell carcinomas are both over-represented among microsatellite instability-high cancers. The clinicopathologic features of mucinous microsatellite instability-high cancer parallel those of the overall microsatellite instability-high set, but it is not known whether the same is true for signet ring cell carcinoma, particularly given the fact that signet ring histology is a well-documented adverse prognostic factor. We recorded age, sex, tumor size, site, grade, stage, histologic pattern, growth pattern, Crohn-like reaction, vascular invasion and tumor-infiltrating lymphocytes in 72 resected signet ring cell carcinomas of the colorectum. Microsatellite instability was determined by a combination of polymerase chain reaction and immunohistochemical stains for hMLH1, hMSH2 and hMSH6. Tumors with instability at >30% of informative markers and/or loss of hMLH1 or hMSH2 expression were designated microsatellite instability-high; all others were classified as MSS. A total of 22 (31%) signet ring cell carcinomas were microsatellite instability-high. Compared to MSS signet ring cell carcinoma, they were more likely to be right-sided (81 vs 45%, P=0.005) and to affect older patients (68 vs 26%, P=0.0007) of female sex (59 vs 28%, P=0.03). Crohn-like reaction (45 vs 16%, P=0.02) and high tumor infiltrating lymphocyte counts (32 vs 8%, P=0.03) were more common in the microsatellite instability-high setting. There was no significant difference in 5-year survival in microsatellite instability-high vs MSS patients (41 vs 34%, P=0.3). In conclusion, approximately one-third of signet ring carcinomas of the colorectum are microsatellite instability-high. Microsatellite instability-high signet ring carcinomas share clinicopathologic features with other microsatellite instability-high cancers: older age group, female preponderance, right-sided location, Crohn-like reaction and numerous tumor-infiltrating lymphocytes. Microsatellite instability status does not appear to be a significant predictor of survival in signet ring cell carcinoma of the colorectum.
We analyze published data from 592 AC microsatellite loci from 98 species in five vertebrate classes including fish, reptiles, amphibians, birds, and mammals. We use these data to address nine major questions about microsatellite evolution. First, we find that larger genomes do not have more microsatellite loci and therefore reject the hypothesis that microsatellites function primarily to package DNA into chromosomes. Second, we confirm that microsatellite loci are relatively rare in avian genomes, but reject the hypothesis that this is due to physical constraints imposed by flight. Third, we find that microsatellite variation differs among species within classes, possibly relating to population dynamics. Fourth, we reject the hypothesis that microsatellite structure (length, number of alleles, allele dispersion, range in allele sizes) differs between poikilotherms and homeotherms. The difference is found only in fish, which have longer microsatellites and more alleles than the other classes. Fifth, we find that the range in microsatellite allele size at a locus is largely due to the number of alleles and secondarily to allele dispersion. Sixth, length is a major factor influencing mutation rate. Seventh, there is a directional mutation toward an increase in microsatellite length. Eighth, at the species level, microsatellite and allozyme heterozygosity covary and therefore inferences based on large-scale studies of allozyme variation may also reflect microsatellite genetic diversity. Finally, published microsatellite loci (isolated using conventional hybridization methods) provide a biased estimate of the actual mean repeat length of microsatellites in the genome.
BACKGROUND: The moss Physcomitrella patens is an emerging model in comparative plant science. At present, the Physcomitrella genome is sequenced at the Joint Genome Institute (USA). In this study we present our results on the development of expressed sequence tag-derived microsatellite markers for Physcomitrella patens, their classification and applicability as genetic markers on the intra- as well as on the interspecies level. We experienced severe restrictions to compare our results on Physcomitrella with earlier studies for other plant species due to varying microsatellite search criteria and a limited selection of analysed species. As a consequence, we performed a side by side analysis of expressed sequence tag-derived microsatellites among 24 plant species covering a broad phylogenetic range and present our results on the observed frequencies. RESULTS: We identified 3,723 microsatellites using the software MISA in a non-redundant Physcomitrella expressed sequence tag database comprising more than 37 megabases of nucleotide information. For 2,951 microsatellites appendant primer sequences have been derived. PCR of 376 microsatellites yielded 88 % successful amplicons and over 30 % polymorphisms between two Physcomitrella accessions. The polymorphism information content of 64 microsatellites based on 21 different Physcomitrella accessions was comparably high with a mean of 0.47 +/- 0.17. Of the 64 Physcomitrella microsatellite markers, 34 % respectively 79.7 % revealed cross-species applicability in two closely related moss species. In our survey of two green algae, two mosses, a fern, a fern palm, the ginkgo tree, two conifers, ten dicots and five monocots we detected an up to sevenfold variation in the overall frequency with a minimum of 37 up to maximal 258 microsatellites per megabase and a high variability among the different microsatellite class and motif frequencies. Numerous species-specific microsatellite frequencies became evident and several deviations to earlier reports were ascertained. CONCLUSION: With the Physcomitrella microsatellite marker set a valuable tool has been made available for further genetic and genomic applications on the intra- as well as on the interspecies level. The comparative survey of expressed sequence tag-derived microsatellites among the plant kingdom is well suited for a classification of future studies on plant microsatellites.
Human cancers with a high frequency microsatellite instability phenotype develop due to defects in DNA mismatch repair genes. Silencing of a DNA mismatch repair gene, hMLH1 gene, by promoter hypermethylation is a frequent cause of the microsatellite instability-H phenotype. Using methylation specific PCR we investigated the methylation status of the hMLH1 gene promoter in 17 solitary gastric cancers (12 microsatellite instability-H and five microsatellite stable tumours from 17 patients), and 13 multiple gastric cancers (eight microsatellite instability-H, one low frequency microsatellite instability-L and four microsatellite stable tumours from five patients) and also examined non-cancerous gastric mucosa both adjacent to and distant from each tumour. Expression of hMLH1 protein was evaluated by immunohistochemistry. All microsatellite instability-H tumours (20 out of 20) had evidence of methylation of hMLH1 promoter, whereas only one out of 10 microsatellite instability-L and microsatellite stable tumours did (P<0.0000005), and the methylation status correlated with hMLH1 protein expression (P<0.000003). Furthermore, methylation of the hMLH1 promoter was detected in 50% (6 out of 12) and 63% (5 out of 8) of non-cancerous gastric mucosa samples adjacent to, and in 33% (4 out of 12) and 40% (2 out of 5) of those obtained from distant portion of, solitary and multiple cancers with microsatellite instability-H. Thus both solitary and multiple gastric cancers with microsatellite instability-H have evidence of similar high levels of hMLH1 promoter hypermethylation in the surrounding non-cancerous tissue. Hypermethylation of the hMLH1 promoter occurs in non-cancerous gastric mucosa of microsatellite instability-H tumours and may increase the risk of subsequent neoplasia.
Microsatellite lengths change over evolutionary time through a process of replication slippage. A recently proposed model of this process holds that the expansionary tendencies of slippage mutation are balanced by point mutations breaking longer microsatellites into smaller units and that this process gives rise to the observed frequency distributions of uninterrupted microsatellite lengths. We refer to this as the slippage/point-mutation theory. Here we derive the theory's predictions for interrupted microsatellites comprising regions of perfect repeats, labeled segments, separated by dinucleotide interruptions containing point mutations. These predictions are tested by reference to the frequency distributions of segments of AC microsatellite in the human genome, and several predictions are shown not to be supported by the data, as follows. The estimated slippage rates are relatively low for the first four repeats, and then rise initially linearly with length, in accordance with previous work. However, contrary to expectation and the experimental evidence, the inferred slippage rates decline in segments above 10 repeats. Point mutation rates are also found to be higher within microsatellites than elsewhere. The theory provides an excellent fit to the frequency distribution of peripheral segment lengths but fails to explain why internal segments are shorter. Furthermore, there are fewer microsatellites with many segments than predicted. The frequencies of interrupted microsatellites decline geometrically with microsatellite size measured in number of segments, so that for each additional segment, the number of microsatellites is 33.6% less. Overall we conclude that the detailed structure of interrupted microsatellites cannot be reconciled with the existing slippage/point-mutation theory of microsatellite evolution, and we suggest that microsatellites are stabilized by processes acting on interior rather than on peripheral segments.
Surveys of variability of homologous microsatellite loci among species reveal an ascertainment bias for microsatellite length where microsatellite loci isolated in one species tend to be longer than homologous loci in related species. Here, we take advantage of the availability of aligned human and chimpanzee genome sequences to compare length difference of homologous microsatellites for loci identified in humans to length difference for loci identified in chimpanzees. We are able to quantify ascertainment bias for a range of motifs and microsatellite lengths. Because ascertainment bias should not exist if a microsatellite selected in one species is as likely to be longer as it is to be shorter than its homologue, we propose that the nature of ascertainment bias can provide evidence for understanding how microsatellites evolve. We show that bias is greater for longer microsatellites but also that many long microsatellites have short homologues. These results are consistent with the notion that growth of long microsatellites is constrained by an upper length boundary that, when reached, sometimes results in large deletions. By evaluating ascertainment bias separately for interrupted and uninterrupted repeats we also show that long microsatellites tend to become interrupted, thereby contributing a second component of ascertainment bias. Having accounted for ascertainment bias, in agreement with results published elsewhere, we find that microsatellites in humans are longer on average than those in chimpanzees. This length difference is similar among repeat motifs but surprisingly comprises two roughly equal components, one associated with the repeats themselves and one with the flanking sequences. The differences we find can only be explained if microsatellites are both evolving directionally under a biased mutation process and are doing so at different rates in different closely related species.
BACKGROUND: Despite the great advances in genomic technology observed in several crop species, the availability of molecular tools such as microsatellite markers has been limited in melon (Cucumis melo L.) and cucurbit species. The development of microsatellite markers will have a major impact on genetic analysis and breeding of melon, especially on the generation of marker saturated genetic maps and implementation of marker assisted breeding programs. Genomic microsatellite enriched libraries can be an efficient alternative for marker development in such species. RESULTS: Seven hundred clones containing microsatellite sequences from a Tsp-AG/TC microsatellite enriched library were identified and one-hundred and forty-four primer pairs designed and synthesized. When 67 microsatellite markers were tested on a panel of melon and other cucurbit accessions, 65 revealed DNA polymorphisms among the melon accessions. For some cucurbit species, such as Cucumis sativus, up to 50% of the melon microsatellite markers could be readily used for DNA polymophism assessment, representing a significant reduction of marker development costs. A random sample of 25 microsatellite markers was extracted from the new microsatellite marker set and characterized on 40 accessions of melon, generating an allelic frequency database for the species. The average expected heterozygosity was 0.52, varying from 0.45 to 0.70, indicating that a small set of selected markers should be sufficient to solve questions regarding genotype identity and variety protection. Genetic distances based on microsatellite polymorphism were congruent with data obtained from RAPD marker analysis. Mapping analysis was initiated with 55 newly developed markers and most primers showed segregation according to Mendelian expectations. Linkage analysis detected linkage between 56% of the markers, distributed in nine linkage groups. CONCLUSIONS: Genomic library microsatellite enrichment is an efficient procedure for marker development in melon. One-hundred and forty-four new markers were developed from Tsp-AG/TC genomic library. This is the first reported attempt of successfully using enriched library for microsatellite marker development in the species. A sample of the microsatellite markers tested proved efficient for genetic analysis of melon, including genetic distance estimates and identity tests. Linkage analysis indicated that the markers developed are dispersed throughout the genome and should be very useful for genetic analysis of melon.
The patterns of mutation and evolution at 13 microsatellite loci were studied in the filamentous fungal genus Neurospora. First, a detailed investigation was performed on five microsatellite loci by sequencing each microsatellite, together with its nonrepetitive flanking regions, from a set of 147 individuals from eight species of Neurospora. To elucidate the genealogical relationships among microsatellite alleles, repeat number was mapped onto trees constructed from flanking-sequence data. This approach allowed the potentially convergent microsatellite mutations to be placed in the evolutionary context of the less rapidly evolving flanking regions, revealing the complexities of the mutational processes that have generated the allelic diversity conventionally assessed in population genetic studies. In addition to changes in repeat number, frequent substitution mutations within the microsatellites were detected, as were substitutions and insertion/deletions within the flanking regions. By comparing microsatellite and flanking-sequence divergence, clear evidence of interspecific allele length homoplasy and microsatellite mutational saturation was observed, suggesting that these loci are not appropriate for inferring phylogenetic relationships among species. In contrast, little evidence of intraspecific mutational saturation was observed, confirming the utility of these loci for population-level analyses. Frequency distributions of alleles within species were generally consistent with the stepwise mutational model. By comparing variation within species at the microsatellites and the flanking-sequence, estimated microsatellite mutation rates were approximately 2500 times greater than mutation rates of flanking DNA and were consistent with estimates from yeast and fruit flies. A positive relationship between repeat number and variance in repeat number was significant across three genealogical depths, suggesting that longer microsatellite alleles are more mutable than shorter alleles. To test if the observed patterns of microsatellite variation and mutation could be generalized, an additional eight microsatellite loci were characterized and sequenced from a subset of the same Neurospora individuals.
BACKGROUND: Recent studies have shown that microsatellites are unstable in various types of cancers, and such genetic instability at the microsatellite loci (microsatellite instability) has been considered to play an important role in the development of cancer. However, the clinicopathologic significance of microsatellite instability in gastric cancer has not been clarified. METHODS: To elucidate the role of genetic instability in the development of gastric cancer, the presence of microsatellite instability was examined in 25 cases of gastric cancer using fresh-frozen tumor-normal paired samples using a polymerase chain reaction (PCR)-based method. Microsatellite instability was defined as tumors that showed altered banding patterns at two or more microsatellite loci. RESULTS: The incidence of microsatellite instability in gastric cancer cases was 4 of 25 patients (16%) and 4 of 26 cancers (15%). A significantly high incidence of microsatellite instability was observed in both the elderly (P < 0.01) and in lymph node metastasis-negative patients (P < 0.05). All patients with gastric cancer showing microsatellite instability were negative for lymphatic or venous permeation. A statistically significant association of microsatellite instability with no lymphatic permeation was thus observed (P < 0.05). CONCLUSIONS: This study revealed infrequent lymph node metastasis and lymph vessel invasion in the patients with gastric cancer demonstrating microsatellite instability. Although the number of examined cases was small, these findings suggest that gastric cancer that shows microsatellite instability may thus behave in a less malignant manner.
BACKGROUND & AIMS: Microsatellite instability was first described in hereditary nonpolyposis colorectal cancers and sporadic colorectal cancers, in which it was associated with a good prognosis. The aim of this study was to assess the advantages of a novel fluorescent assay for detecting microsatellite instability. METHODS: Eleven fluorescently tagged microsatellites and an automated DNA sequencer were used to investigate 54 sporadic colorectal adenocarcinomas. RESULTS: This fluorescent assay combined accurate allele sizing with cross-sectional data display and allowed improved assessment of microsatellite instability. Twenty-two percent of cancers (12 of 54) showed microsatellite instability with at least one marker. For tumors showing microsatellite instability, results were obtained for a minimum of eight markers. Six tumors showed microsatellite instability at high frequency (at least 63% of markers affected), and 42% of the patients who had a tumor showing microsatellite instability had a synchronous and/or metachronous colorectal tumor (vs. 7% of patients whose tumor did not show microsatellite instability). Patients with a microsatellite instability-positive tumor had an improved prognosis (P = 0.03). CONCLUSIONS: The use of this fluorescent assay improved the assessment of microsatellite instability with the automated analysis and cross-sectional data display. The assay identified a subgroup of patients who showed microsatellite instability and who also showed clinical features that differed from the microsatellite instability-negative cases.
Chromosomal instability in colon tumors implies the presence of numerical and structural chromosome aberrations and is further characterized by the absence of microsatellite instability and the occurrence of KRAS and/or TP53 mutations. In a previous screening of 194 colon tumors for both microsatellite instability and TP53 mutation, we found 25 microsatellite-unstable tumors, in 9 (36%) of which, presumed to be chromosomally stable, there were TP53 mutations. This prompted us to investigate whether a TP53 mutation in these microsatellite-unstable tumors would be an indicator of chromosomal instability, that is, whether this would be a category of tumors showing both microsatellite and chromosomal instability. For chromosomal instability assessment, we performed array-comparative genomic hybridization analysis of tumor and control DNA extracted from formalin-fixed, paraffin-embedded stage III colon tumor specimens. The array consisted of 435 subtelomere-specific BACs. We compared all but one (whose DNA was of bad quality) of the microsatellite-unstable TP53 mutation-containing tumors (8) with a similarly sized group of microsatellite-unstable tumors without TP53 mutation (11). Microsatellite-unstable tumors with a TP53 mutation showed on average 0.9 aberrations (range 0-3) when assessed with this array system. Those without a TP53 mutation showed on average 0.7 aberrations (range 0-2). Thus, microsatellite-unstable tumors showed few chromosomal abnormalities regardless of TP53 mutation status. Because, in our study, the microsatellite-stable tumors had on average 3.4chromosomal abnormalities (range 0-7), a clear difference exists between microsatellite-unstable and -stable tumors. Because a substantial proportion of microsatellite-unstable colon tumors carry a TP53 mutation while showing relativelyfewchromosomal aberrations, a TP53 mutation in these tumors cannot be considered to be an indicator of chromosomal instability.
Fugu rubripes (Fugu) has one of the smallest recorded vertebrate genomes and is an economic tool for comparative DNA sequence analysis. Initial characterization of 128 kb of Fugu DNA attributed the compactness of this genome, in part, to a sparseness of repetitive DNA sequence compared with mammalian genomic sequences. This paper describes a new and comprehensive analysis in which 501 theoretically possible microsatellites with a repeat unit of one to six bases were used to query two orders of magnitude more Fugu DNA (i.e. 11.338 Mb). A total of 6042 microsatellites were identified and categorized. In decreasing order, the 20 most frequently occurring microsatellites are AC, A, C, AGG, AG, AGC, AAT, AAAT, ACAG, ACGC, ATCC, AAC, ATC, AGGG, AAAG, AAG, AAAC, AT, CCG and TTAGGG. The 20 most frequently occurring microsatellites represent 81.79% of all microsatellites identified. Our results indicate that one microsatellite occurs every 1.876 kb of DNA in Fugu, 11.55% of the microsatellites are detected in open reading frames that are predicted protein coding regions. With respect to the proportion of microsatellites present in open reading frames and the total abundance (bp) of all microsatellites, the genome of Fugu is similar to the genome of many other vertebrate species. Previous estimates performed indicate that approximately 1% of many vertebrate genomes are comprized of microsatellite sequences. However, many differences prevail in the abundance and frequency of the individual microsatellite classes. Many of the frequently occurring microsatellites in Fugu are known to code in other species for regions in proteins such as transcription factors, whilst others are associated with known functions, such as transcription factor binding sites and form part of promoter regions in DNA sequences of genes. Therefore, it is likely that such repeats in genomes have a role in the evolution of genes, regulation of gene expression and consequently the evolution of species.
PURPOSE: In case-control studies, it was reported that microsatellite instability might be helpful in predicting the development of metachronous multiple colorectal cancers. The purpose of this cohort-like study was to determine whether microsatellite instability is a novel independent marker in predicting metachronous colorectal carcinomas after colorectal cancer surgery. METHODS: Three hundred twenty-eight colorectal carcinoma patients were surveyed by periodic colonoscopy for at least three years after surgery. Among these, DNA from paraffin-embedded sections was available for 272 cases. DNA of these cases was studied for six microsatellite markers (five dinucleotide repeats, one mononucleotide repeat). Microsatellite instability phenotype was defined as alterations in one or more loci. RESULTS: Median follow-up period was 74 months, and the median number of colonoscopies was 4.6. The percentage of microsatellite instability-positive cases was 26.4 percent (72/272). Seventeen metachronous colorectal carcinomas were detected during the follow-up period. Incidences of metachronous colorectal carcinomas in microsatellite instability-positive and microsatellite instability-negative cases were 15.3 and 3 percent, respectively (P < 0.001). The cumulative five-year incidence of metachronous colorectal carcinomas was significantly higher in microsatellite instability-positive cases than in microsatellite instability-negative cases (12.5 vs. 2.5 percent, P < 0.0001). Logistic regression analysis of the relationship between incidence of metachronous colorectal carcinomas and possible risk factors (namely, coexistence of adenoma at the time of surgery, family history of colorectal carcinoma, history of extracolonic malignancy, and microsatellite instability status) showed that microsatellite instability and coexistence of adenoma were significant independent risk factors for the occurrence of metachronous colorectal carcinomas, with values of P = 0.001 and 0.02, respectively. CONCLUSION: These data indicate that microsatellite instability can be regarded as a novel independent and important marker for predicting the development of metachronous colorectal carcinoma after surgery.