SHORT REPORTS Searching for microsatellite mutations in coding regions in lung, breast, ovarian and colorectal cancers Eva Forgacs 1 , Jonathan D Wren 2 , Craig Kamibayashi 1 , Masashi Kondo 1 , Xie L Xu 1 , Sanford Markowitz 9 , Gail E Tomlinson 1 , Carolyn Y Muller 1 , Adi F Gazdar 1,3 , Harold R Garner 4,5,6,7 and John D Minna* ,1,4,8 1 The Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, TX 75390-8593, USA; 2 Program of Genetics and Development, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, TX 75390-8593, USA; 3 Department of Pathology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, TX 75390-8593, USA; 4 Department of Internal Medicine, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, TX 75390-8593, USA; 5 Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, TX 75390-8593, USA; 6 McDermott Center for Human Growth and Development, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, TX 75390-8593, USA; 7 Center for Biomedical Inventions, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, TX 75390-8593, USA; 8 Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, TX 75390-8593, USA; 9 Howard Hughes Medical Institute, Case Western Reserve University, Cleveland, Ohio, OH 44106, USA RepX represents a new informatics approach to probe the UniGene database for potentially polymorphic repeat sequences in the open reading frame (ORF) of genes, 56% of which were found to be actually polymorphic. We now have performed mutational analysis of 17 such sites in genes not found to be polymorphic (50.03 frequency) in a large panel of human cancer genomic DNAs derived from 31 lung, 21 breast, seven ovarian, 21 (13 microsatellite instability (MSI)+ and eight MSI7) colorectal cancer cell lines. In the lung, breast and ovarian tumor DNAs we found no mutations (50.03 ± 0.04 rate of tumor associated open reading frame mutations) in these sequences. By contrast, 18 MSI+ colorectal cancers (13 cancer cell lines and ®ve primary tumors) with mismatch repair defects exhibited six mutations in three of the 17 genes (SREBP-2, TAN-1, GR6) (P50.000003 compared to all other cancers tested). We conclude that coding region microsatellite alterations are rare in lung, breast, ovarian carcinomas and MSI (7) colorectal cancers, but are relatively frequent in MSI (+) colorectal cancers with mismatch repair de®cits. Oncogene (2001) 20, 1005 ± 1009. Keywords: microsatellite instability; mutation; repeti- tive DNA sequences Repetitive DNA sequences such as microsatellites consist of 1 to 10 nucleotides that are repeated a variable number of times. These repeat sequences can be polymorphic, and such polymorphic microsatellite markers have been used in a variety of genetic studies, such as linkage analysis. When the repeat sequences occur in the open reading frame, polymorphic amino acid sequences can be generated. Several inherited neurological disorders such as Friedreich's Ataxia and Huntington's Disease have been linked to intergenera- tional locus-speci®c expansion of tri-nucleotide tracts (Andrew et al., 1993; Bidichandani et al., 1998). The mechanism of expansion or contraction of these repetitive sequences is not fully understood, but is believed to be associated with slipped-strand mispair- ing, uneven recombination, or a combination of both (Jakupciak and Wells, 1999; Wells, 1996). Somatically acquired microsatellite alterations occur frequently in endometrial, gastrointestinal, testicular, colorectal and ovarian carcinomas leading to char- acterizing these tumors as microsatellite instable (MSI+) or not (MSI7) (Gurin et al., 1999; King et al., 1997; Liu et al., 1995; Orth et al., 1994; Yamamoto et al., 1997). These alterations are correlated with mutations in mismatch repair (MMR) system genes (hMSH2, hMLH1, hMSH6 etc.) (Bronner et al., 1994; Herman et al., 1998; Liu et al., 1996, 1999; Mal- khosyan et al., 1996; Nicolaides et al., 1994). Several studies have shown that mismatch repair mutant colorectal tumors also exhibit mutations in genes with nucleotide repeats such as TGF b II and BAX (Markowitz et al., 1995; Parsons et al., 1995; Rampino et al., 1997). We have developed informatics tools to search DNA databases for sequences such as repeats that could potentially be polymorphic (Fondon et al., 1998; Wren et al., 2000). Currently our RepX software has identi®ed over 30 000 expressed se- quences with repetitive DNA elements predicted to be polymorphic, providing a comprehensive list of UniGene predicted polymorphisms (Wren et al., Oncogene (2001) 20, 1005 ± 1009 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc *Correspondence: JD Minna, Hamon Center for Therapeutic Oncology, University of Texas Medical Center, 6000 Harry Hines Blvd., NB. 8.206, Dallas, Texas, TX 75390-8593, USA Received 25 September 2000; revised 28 December 2000; accepted 3 January 2001