Incidence of germline hMLH1 and hMSH2 mutations (HNPCC patients) among newly diagnosed colorectal cancers in a Slovenian population

EDITOR—Hereditary non-polyposis colorectal cancer (HNPCC) syndrome is a common autosomal dominant predisposition to colorectal cancer. Clinical diagnostic features of sporadic and HNPCC associated colorectal cancer do not diVer significantly and until recently the identification of HNPCC patients was based mainly on their family history. Because of the importance for relatives of HNPCC patients to be clinically examined frequently in order to detect the disease at an early, curable stage, the International Collaborative Group (ICG) on HNPCC proposed criteria for identification of HNPCC families. According to the guidelines agreed by the ICG in Amsterdam in 1990, an HNPCC family has to fulfil the following criteria: there should be at least three relatives with colorectal cancer (one of whom is a first degree relative to the other two), at least two successive generations should be aVected, and one relative should be diagnosed under the age of 50. CRC patients with HNPCC syndrome can also develop cancer of the endometrium, stomach, ovary, and urinary and hepatobiliary tracts. In several epidemiological studies, the incidence of HNPCC has been estimated to be between 0.5% and 15% of all colorectal cancers. The identification of mismatch repair genes (MMR), of which at least five (hMLH1, 8 hMSH2, 10 PMS1, 10 PMS2, and hMSH6 (GTBP)) are associated with HNPCC, has enabled mutational analysis in families fulfilling complete or partial Amsterdam criteria. Carriers of germline MMR mutations have a higher than 80% risk for cancer by the age of 75. The great majority of germline mutations were found in approximately equal proportions in hMLH1 and hMSH2, while mutations in the other three MMR genes have been reported only in a limited number of cases. Germline hMLH1 and hMSH2 mutations were also found in a considerable proportion of colorectal cancer patients from families not fulfilling the Amsterdam criteria, especially if they were young, 15 indicating that some HNPCC families might be missed if they are preselected before mutational analysis. Mutations in MMR genes result in microsatellite instability (MSI), which is characteristic of more than 90% of tumours in HNPCC patients but was also found in 12-15% of sporadic colorectal tumours. In the absence of other diagnostic criteria, MSI analysis of tumours could be valuable markers in HNPCC identification. Here we report an eVective MSI analysis of CRC and subsequent mutational analysis of the hMLH1 and hMSH2 genes in tumours with considerable MSI for identification of HNPCC among randomly collected, newly diagnosed colorectal cancers. This approach allowed us to identify HNPCC families and to estimate the minimal incidence of HNPCC in a Slovenian population based solely on molecular genetic analysis. Primary colorectal adenocarcinomas and corresponding normal tissue samples were collected from patients who gave consent for testing of their DNA. Between 1996 and 1998, 300 newly diagnosed CRC patients from clinics all over Slovenia participated in this study. The sample of CRC is thus representative for the Slovenian population. All samples were gathered in a central institution where two physicians histologically evaluated each resection for a high proportion of tumour tissue. We also confirmed a high proportion of cancer cells versus normal cells in tumour tissue in many samples that exhibited LOH during MSI analysis. Normal colorectal mucosa taken from a site distant from the tumour was used as a normal control in the study. We isolated DNA after tissue digestion using standard phenol/chloroform extraction and ethanol precipitation from frozen colorectal tumours and corresponding normal tissue samples. For MSI analysis of tumour and control