MLH1 PROMOTER HYPERMETHYLATION IN
BULGARIAN PATIENTS WITH COLORECTAL CANCER
Kadiyska T1,*, Tzancheva M1, Nedin D2, Alexandrova A2,
Marinov M2, Kaneva R1, Damyanov D2, Mitev V3, Kremensky I1
*Corresponding Author: Dr. Tanya Kadiyska, Laboratory of Molecular Pathology, University Hospital of Obstetrics and Gynaecology “Maichin Dom”, 2 Zdrave str., Sofia 1431, Bulgaria; Tel: +359-2-9520124; Fax: +359-2-9520490; E-mail: alextanya@excite.com page: 3
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MATERIALS AND METHODS
Subjects. DNA was derived from fresh blood and/or frozen normal and micro-dissected 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 according to the 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) the 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: one mono- BAT26, three D2S123, D5S346, D18S35 and one tetranucleotide-FGA, have been selected for detecting MSI and loss of heterozygosity (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 (PAGE) and detected by an automated fluorescence sequencer (ALFExpress; Pharmacia Biotech, Uppsala, Sweden). Micro-satellite 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: MSI negative (–) with no MSI at any of the loci examined, and MSI positive (+), which were then divided in two sub groups: MSI-L (low) with MSI at one-to-two loci examined, and MSI-H (high) with more than three of the loci examined demonstrating MSI. In contrast to MSI, where new alleles are generated, LOH is correlated with the loss of the wild type allele in tumor DNA (i.e., in certain subpopulation of analyzed cells). Using a fluorescence-based method of detection, the loss of alleles can be precisely determined by calculation of the ratio of the peak heights of normal and tumor alleles. The LOH is indicated by ratios of less than 0.5 or higher than 2.0 [29].
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 (eight sporadic and five with family history) and 15 randomly selected sporadic MSI(–) 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 100 ng, from both normal and tumor genomic DNA was digested overnight with a 60-fold excess of methylation sensitive HpaII endonuclease (Promega, Madison, WI, USA) in a total volume of 10 μl. A control restriction reaction was performed by the methylation insensitive isoschizomer MspI (New England Biolabs, Inc., Beverly, MA, USA) using the same template. The whole restriction reaction was then amplified in a total volume of 50 L using primers from the MLH1 promoter region that flank the four CCGG recognition sites upstream of the ATG initiator codon: forward primer 5’-CGC TCG TAG TAT TCG TGC-3’ and reverse primer 5’-ACC TCA GTG CCT CGT GCT CAC GTT C-3’. Thirty cycles of PCR were performed using the following conditions: denaturation at 94°C for 60 seconds; annealing at 55°C for 60 seconds, and extension at 72°C for 60 seconds. 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).
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