Subjects

DNA was derived from fresh blood and/or frozen normal and tumor tissue taken surgically using standard protocols. A total of 150 patients were referred to the Queen Giovanna Hospital, Sofia, Bulgaria and analyzed for MSI. According to the family history, patients were sub grouped into four main categories: 1. The “HNPCC” group, including families accorded to Bethesda criteria [28]; 2. The “Family” group- represented by families with one or more first-degree relatives of the proband affected by tumors at any site; 3. "Sporadic” group included patients whose families did not show any other cancer cases among relatives or without sufficient family information.

Analysis of microsatellite instability (MSI)

            In a previous study, a set of five polymorphic markers- 1 mono- BAT26, 3 di- D2S123, D5S346, D18S35 and 1 tetranucleotide- FGA have been selected for detecting MSI and LOH in these regions. The repeat markers were amplified from both normal and tumor DNA samples and were resolved by 6% denaturing polyacrylamide gel electrophoresis and detected by automated fluorescence sequencer ALFExpress (Pharmacia Biotech, Uppsala, Sweden). Microsatellite instability was defined by the appearance of different alleles in the tumor DNA, when compared with the corresponding normal DNA. As for MSI, patients were classified in two groups: 1.MSI negative (-) with no MSI at any of the loci examined and MSI positive (+), divided in two sub groups: 1. MSI-L (low) with MSI at one to two loci examined, and 2. MSI-H (high) with more than three of the loci examined demonstrating MSI. Loss of heterozygosity (LOH) was defined by at least 50% reduction in the relative intensity of one allele in the tumor compared with normal DNA in at least 2 of the markers.

Promoter methylation assay

            In this study we investigated the MLH1 promoter methylation in a total of 25 (10 sporadic cases and 15 with family history) tumors with MSI, 13 with LOH (8 sporadic and 5 with family history) and 15 randomly selected sporadic MSI-negative cases.

The methylation status of the hMLH1 promoter was evaluated by a PCR-based assay according to the procedure detailed by Kane et al. [23] as follows: an aliquot of 100ng, both from normal and tumor genomic DNA was digested overnight with a 60-fold excess of methylation sensitive HpaII endonuclease (Promega) in a total volume of 10µl. A control restriction reaction was performed by the methylation insensitive isoschizomer MspI (New England Biolabs) using the same template. The whole restriction reaction was then amplified in a total volume of 50µl using primers from the MLH1 promoter region that flank the four CCGG recognition sites upstream of the ATG initiator codon- forward primer 5’- CGCTCGTAGTATTCGTGC- 3’ and reverse primer 5’- ACCTCAGTGCCTCGTGCTCACGTTC- 3’. Thirty rounds of PCR were performed using the following conditions: 94^oC, 60sec.; 55^oC, 60 sec.; 72^oC, 60 sec.

The expected 603 bp. PCR products were size sorted by 2% agarose gel electrophoresis and visualized by staining with ethidium bromide. All restriction digestions and PCR reactions were repeated to verify the results.

Statistical calculations were performed with WinStat 4.3 program package (StatSoft Inc., 1993).