Mismatch repair genes and mononucleotide tracts as mutation targets in colorectal tumors with dierent degrees of microsatellite instability Antonio Percesepe 1,2 , Paula Kristo 1 , Lauri A Aaltonen 1 , Maurizio Ponz de Leon 2 , Albert de la Chapelle 1,3 and PaÈivi PeltomaÈki 1,3 1 Department of Medical Genetics, Haartman Institute, University of Helsinki, PO Box 21, Haartmaninkatu 3, FIN-00014 Helsinki, Finland; 2 Department of Internal Medicine, University of Modena, Via del Pozzo, 71, 41100 Modena, Italy; 3 Division of Human Cancer Genetics, Comprehensive Cancer Center, Ohio State University, 420 W 12th Avenue, Columbus, Ohio 43210, USA Microsatellite instability occurs in 15% of colorectal carcinomas and may be due to replication errors (RER). The pattern of instability ± `severe' vs `mild' ± and the tumorigenic pathway, as re¯ected by the involvement of functionally important genes, may vary according to the underlying gene(s). We de®ned `mild' RER as mono- or tetranucleotide repeat instability in the absence of widespread instability at dinucleotide repeats and studied 15 colorectal tumors with this phenotype for mutations in the DNA mismatch repair genes MSH2, MLH1, MSH3, and MSH6. No mutations were found, suggest- ing that these genes were not implicated. We then compared colorectal cancers with `mild' RER (n=15), and those with `severe' RER without (n=11) or with (n=22) detectable mutations in MSH2 or MLH1 to assess the involvement of mononucleotide repeats contained in the coding regions of MSH3, MSH6, BAX, and TGFb RII. The combined mutation rates of the above mentioned loci varied signi®cantly between the three groups of tumors, being 0%, 25% and 52%, respectively. Furthermore, the individual genes showed speci®c patterns of involvement; for example, among tumors with `severe' RER, TGFb RII displayed uniformly high mutation rates while MSH3, MSH6, and BAX were more frequently altered in tumors that also showed MSH2 or MLH1 mutations. Our ®ndings suggest that dierent subcategories exist among unstable tumors, de®ned by the RER pattern on the one hand and tumorigenic pathway on the other, and structural changes of MSH2 and MLH1 are likely to explain only a proportion of these cases. Keywords: mismatch repair; microsatellite instability; colorectal cancer; mutation analysis; HNPCC Introduction Current models of cancer development are based on the experimental evidence that mutations accumulate in a stepwise fashion, leading to abnormalities in speci®c genes and resulting in clonal expansion of neoplastic cells (Fearon and Vogelstein, 1990). It has been argued that the number of modi®cations in oncogenes and tumor suppressor genes is too high to be accounted for only by the spontaneous mutation rate, but a genetically unstable phenotype is required for multi- step carcinogenesis (Loeb, 1991). A novel mechanism of genetic instability has been elucidated in neoplasms from dierent organs, consisting of alterations in length (insertions or deletions) of repetitive sequences, microsatellites, interspersed along the whole genome (Aaltonen et al., 1993; Ionov et al., 1993; Thibodeau et al., 1993). Such instability is a characteristic conse- quence of mutations in DNA mismatch repair genes that result in a failure to correct replication errors (RER) (reviewed in PeltomaÈki and de la Chapelle, 1997). Homozygous mutations in `major' mismatch repair genes, such as MSH2 and MLH1 may give rise to secondary mutations in other genes which contain structural targets for mutations and are selected for during tumorigenesis (Malkhosyan et al., 1996). Potential targets that have been proposed to play a role in colorectal tumorigenesis include other mismatch repair genes, for example MSH3 and MSH6 (also called GTBP) (Malkhosyan et al., 1996), as well as other genes, such as the gene for transforming growth factor b receptor II (TGFb RII; Markowitz et al., 1995), BAX (Rampino et al., 1997), and the gene for insulin-like growth factor II receptor (IGFIIR; Souza et al., 1996). Since the earliest observations it became apparent that two types of microsatellite instability exist, depending on the number of the mutated markers, and on the degree of the increase or the decrease of the fragment size. Tumors with dramatic changes at a single locus often show a widespread instability at most of the studied microsatellite loci; they share certain clinical and pathological features like proximal location in the colon, poor dierentiation, and better prognosis (Thibodeau et al., 1993; Ionov et al., 1993; Aaltonen et al., 1993; Lothe et al., 1993; Kim et al., 1994). This pattern characterizes Hereditary Nonpolyposis Colo- rectal Carcinoma (HNPCC) tumors and around 10% of sporadic colorectal cancers, and is associated with mutations in MSH2, MLH1, or PMS2 in most tumors from the HNPCC group and in one-third of tumors from the sporadic group (Bùrresen et al., 1995; Liu et al., 1996; Konishi et al., 1996; Wu et al., 1997). Less clear are the role and the mechanisms underlying low- level microsatellite instability. Based on observations in yeast, MSH3 and MSH6 whose protein products complex with the MSH2 protein in a partially redundant fashion (Marsischky et al., 1996) have been implicated in the explanation of the phenomenon. MSH6 recognizes mainly single-base mispairs and one- or two-nucleotide loops, and MSH6 mutant human tumors and cell lines show microsatellite instability Correspondence: A Percesepe Received 2 December 1997; revised 18 February 1998; accepted 18 February 1998 Oncogene (1998) 17, 157 ± 163  1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc