Background: Hereditary non-polyposis colorectal cancer (HNPCC) is clinically defined by familial clustering of colorectal cancer and other associated tumours. Methods: By thorough molecular and clinical evaluation of 41 families, two different groups were characterised: group 1, 25 families with truncating mutations in MLH1 or MSH2 (12 novel mutations); and group 2, 16 Amsterdam positive families without mutations in these genes and without microsatellite instability in their corresponding tumours. Results: Significant clinical differences between these two groups were found. Firstly, earlier age of onset for all colorectal cancers (median 41 v 55 years; p,0.001) and all tumours (median 43 v 56 years; p = 0.022) was observed, comparing groups 1 and 2. Secondly, 68% of the index colorectal cancers were localised proximally of the splenic flexure in group 1 compared with 14% in group 2 (p,0.010). Thirdly, more synchronous and metachronous colorectal (p = 0.017) and extracolorectal tumours (p,0.001) were found in group 1. Fourthly, a higher colorectal adenoma/carcinoma ratio (p = 0.030) and a tendency towards more synchronous or metachronous adenomas in group 2 (p = 0.084) was observed, indicating a slower progression of adenomas to carcinomas. As three mutation negative tumours revealed chromosomal instability after comparative genomic hybridisation, these tumours may be caused by one or more highly penetrant disease alleles from the chromosomal instability pathway. Conclusion: These data show that HNPCC includes at least two entities with clinical and molecular differences. This will have implications for surveillance programmes and for cancer research.

Hereditary nonpolyposis colorectal cancer (HNPCC) is due to defects in DNA mismatch repair (MMR) genes MSH2, MLH1, MSH6, and to a lesser extent PMS2. Of 466 suspected HNPCC families, we defined 54 index patients with either tumors of high microsatellite instability (MSI-H) and/or loss of expression for either MLH1, MSH2, and/or MSH6, but without a detectable pathogenic point mutation in these genes. This study cohort was augmented to 64 patients by 10 mutation-negative index patients from Amsterdam families where no tumors were available. Deletion/duplication screening using the multiplex ligation-dependent probe amplification (MLPA) revealed 12 deletions in MSH2 and two deletions in MLH1. These deletions constitute 17% of pathogenic germline alterations but elucidate the susceptibility to HNPCC in only 22% of the mutation-negative study cohort, pointing towards other mutation mechanisms for an inherited inactivation of MLH1 or MSH2. We describe here four novel deletions. One novel and one known type of deletion were found for three and two unrelated families, respectively. MLPA analysis proved a reliable method for the detection of genomic deletions in MLH1 and MSH2; however, sequence variations in the ligation-probe binding site can mimic single exon deletions.