GASTROENTEROLOGY1995;109:1368-1374 RAPID COMMUNICATIONS In Vitro Transcription/Translation Assay for the Screening of hMLH1 and hMSH2 Mutations in Familial Colon Cancer MICHAEL C. LUCE,* GIANCARLO MARRA, t DHARAM P. CHAUHAN,* LUIGI LAGHI,* JOHN M. CARETHERS,* SAJEEV P. CHERIAN, ~ MARY HAWN,* CAMERON G. BINNIE,* LAUREN N. W. KAM-MORGAN,* MATTHEW C. CAYOUETTE,* MINORU KOI, t and C. RICHARD BOLAND ¢ *Roche Biomedical Laboratories, ResearchTriangle Park, North Carolina; and ~Department of Internal Medicine, Universityof Michigan Medical School, Ann Arbor, Michigan (Drs. Luce and Marra contributed equally to this work.) Background & Aims: Hereditary nonpolyposis colo- rectal cancer (HNPCC) has been linked recently to a defect in repairing mismatched nucleotides in DNA. The aim of this study was to screen for germline mutations that result in prematurely truncated proteins in two of the mismatch repair genes identified at this time, hMLH1 and hMSH2, in a consecutive series of patients belonging to familial aggregations of colorectal cancer. Methods: Nineteen individuals with colorectal cancer from 19 families were consecutively referred because of a strong positive family history of colorectal cancer. Premature truncation mutations in hMLH1 and hMSH2 were sought from lymphocyte RNA by using an in vitro transcription/translation (IVTT) assay. Results: Protein truncating mutations in the hMLH1 or hMSH2 genes were found in 50% of families with HNPCC (6 of 12) but were not observed in any of the remaining familial aggregations that did not fulfill the standard criteria for HNPCC. In some of the IVTT-positive samples, the mutations were characterized by genomic sequencing. Conclusions: IVTT may be a practical method to accom- plish primary screening of germline mutations in DNA mismatch repair genes in HNPCC; however, a broader approach is necessary to obtain a more complete pic- ture of the mutational spectrum in HNPCC and other familial aggregations of colorectal cancer. C olorectal cancer is one of the most common malig- nant neoplasms in humans. Because 15%-20% of adenomatous polyps* and colorectal cancers 2 occur in fa- milial aggregations, this is probably the most frequent form of hereditary neoplasia. The risk of developing colo- rectal cancer increases proportionally with the number of affected relatives or early age of onset, 3-5 and an au- tosomal dominant pattern of inheritance has been re- ported in as many as 4% ofcolorectal cancers. 6 The classic form of syndromic familial colorectal cancer is known as hereditary nonpolyposis colorectal cancer (HNPCC). :-.5 Uniform criteria for this syndrome were developed by the International Collaborative Group on HNPCC in Amsterdam in 199013 and require the following: (1) three or more relatives have a histologically verified colorectal cancer, one of whom is a first-degree relative of the other two, and familial adenomatous polyposis is excluded; (2) colorectal cancers involve at least two successive genera- tions; and (3) one or more colorectal cancer cases is diag- nosed before 50 years of age. The hereditary susceptibility to colorectal cancer in patients with HNPCC has been found to be caused by a defect in the DNA mismatch repair system) 5'I6 At present, four genes involved in nucleotide mismatch re- pair have been identified that cosegregate with HNPCC susceptibility, i.e., hMHS2, hk4LHI, hPMS1, and hPk4S2.15-2o The predisposition to cancer is thought to be produced by an inactivating germline mutation in one allele of any one of the mismatch repair genes that, when followed by an acquired mutation or loss of the wild-type allele, 2. results in the loss of DNA repair func- tion and the emergence of the hypermutable phenotype in the target tissues (i.e., colon, endometrium, and oth- ers). Furthermore, the possibility of a dominant negative effect of some mutation types has been recently re- ported. 22 The genetic studies published to date have been per- formed on large, referred kindreds with HNPCC. Based on a group of selected families that were studied with several techniques, Liu et al. suggested that mutations Abbreviations used in this paper: HNPCC, hereditary nonpolyposis colorectal cancer; IVTT, in vitro transcription/translation; PCR, poly- merase chain reaction; SSCP, single-strand conformation polymor- phism. © 1995 by the American Gastroenterological Association 0016-5085/95/$3.00