1 Divisions of Oncology and Epidemiology, Departments of Medicine and Biological Chemistry, University of California, Irvine, Irvine, California 92697, USA. Departments of 2 Internal Medicine and 3 Epidemiology, University of Michigan, Ann Arbor, Michigan 48109, USA. 4 Department of Community Medicine and Epidemiology Carmel Medical Center and Technion Faculty of Medicine Haifa, Israel. 5 Molecular Structure Section, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892, USA. 6 The Institute for Genetic Medicine, University of Southern California, Los Angeles, California 90089, USA. 7 Affymetrix Corporation, Santa Clara, California 95051, USA. 8 BioCaptus, Bothell, Washington 99164, USA. Departments of 9 Pathology and 10 Human Genetics, University of Michigan, Ann Arbor, Michigan 48109, USA. 11 Hereditary Cancer Institute, Creighton University. Omaha, Nebraska 50035, USA. 12 Genome Technology Branch, National Human Genome Research Institute, Bethesda, Maryland 20892, USA. Correspondence should be addressed to S.M.L. (slipkin@uci.edu) or S.B.G. (sgruber@med.umich.edu). Published online 6 June 2004; doi:10.1038/ng1374 The MLH1 D132H variant is associated with susceptibility to sporadic colorectal cancer Steven M Lipkin 1 , Laura S Rozek 2,3 , Gad Rennert 4 , Wei Yang 5 , Peng-Chieh Chen 1 , Joseph Hacia 6 , Nathan Hunt 6 , Brian Shin 1 , Steve Fodor 7 , Mark Kokoris 8 , Joel K Greenson 9 , Eric Fearon 2,9,10 , Henry Lynch 11 , Francis Collins 12 & Stephen B Gruber 2 LETTERS 694 VOLUME 36 | NUMBER 7 | JULY 2004 NATURE GENETICS Most susceptibility to colorectal cancer (CRC) is not accounted for by known risk factors. Because MLH1, MSH2 and MSH6 mutations underlie high-penetrance CRC susceptibility in hereditary nonpolyposis colon cancer (HNPCC), we hypothesized that attenuated alleles might also underlie susceptibility to sporadic CRC. We looked for gene variants associated with HNPCC in Israeli probands with familial CRC unstratified with respect to the microsatellite instability (MSI) phenotype. Association studies identified a new MLH1 variant (415G→C, resulting in the amino acid substitution D132H) in ∼1.3% of Israeli individuals with CRC self-described as Jewish, Christian and Muslim. MLH1 415C confers clinically significant susceptibility to CRC. In contrast to classic HNPCC, CRCs associated with MLH1 415C usually do not have the MSI defect, which is important for clinical mutation screening. Structural and functional analyses showed that the normal ATPase function of MLH1 is attenuated, but not eliminated, by the MLH1 415G→C mutation. The new MLH1 variant confers a high risk of CRC and identifies a previously unrecognized mechanism in microsatellite-stable tumors. These studies suggest that variants of mismatch repair proteins with attenuated function may account for a higher proportion of susceptibility to sporadic microsatellite-stable CRC than previously assumed. To identify variant alleles in MLH1, MSH2 or MSH6 with high sensi- tivity and specificity, we designed a new high-density oligonu- cleotide array (HNPCC Chip) that uses improved bioinformatic Figure 1 Variant allele detection by the HNPCC Chip resequencing array. (a) Graph of HNPCC Chip. Loss of hybridization analysis for full MLH1 coding sequence and splice junctions. Data are the average of signals obtained for one sense and one antisense chip hybridization with the same individual’s genomic DNA. Nucleotide substitutions in individuals with CRC are graphically shown as a signal peak above baseline (ratio of signal for wild-type DNA to DNA from affected individual; WT/AI). Downward spikes below graph were inserted post hoc to facilitate identification of exon boundaries. This DNA was heterozygous with respect to a C→T base substitution in MLH1 exon 14. The boundaries of all 19 MLH1 coding exons are labeled on the ordinate axis. (b) Higher magnification of HNPCC Chip signal analysis for MLH1 exon 14 (108 bp). WT/AI, normalized ratio of chip hybridization signal for fluorescein- labeled wild-type reference DNA divided by the hybridization signal for streptavidin-biotin-phycoerythrin–labeled DNA from an affected individual. The exact position of the C→T substitution is indicated by the arrow in both panels. WT/AI ratio C T heterozygote C T heterozygote WT/AI ratio MLH1 exon MLH1 exon 14 a b © 2004 Nature Publishing Group http://www.nature.com/naturegenetics