The present report supports the role of MLH1 promoter methylation as a major mechanism of MLH1 gene inactivation in the MSI (+) sporadic cases. Fifty percent (5/10) of the sporadic cases showed methylation, compared with only one case from the “Family” group and none from the “HNPCC” cases. Using MSI (+) cell lines Wheeler et al. also found 50% to be fully methylated, although others have reported up to 84% of sporadic MSI (+) tumors showing promoter methylation [30]. This difference could be explained by the use of different methods for detection. The first one (bisulfite treatment) detects both “partially” and “fully” methylated samples. The second one, based on the HpaII enzyme digestion detects definitively only the “fully” methylated samples.

After HpaII digestion of genome DNA, only the fully methylated tumor samples gave a positive amplification. For three of the tumors we supposed a partial methylation due to the weak PCR product. However we did not include them in the group of “methylated”, because only fully methylation of the MLH1 promoter in the cases without mutation detected was related to the lack of gene expression [29].

All patients with MLH1 promoter methylation developed colorectal cancer in the age between 54 and 78, supporting the concept that this is an age- related event.

The presence of hypermethylation in one tumor from the “Family” cases confers the possibility for a selective advantage to subsequent methylation of a both wild- type and mutant sequences.

Table 1. Veriables and MLH1 promoter status in the MSI(+) and LOH cases analyzed

Jones and Laird [22] have recently pointed out that Knudson’s two- hit hypothesis should extend to include epigenetic mechanisms of gene inactivation, such as methylation. In the case of methylation, it is not clear which event occurs first. Presumably, a missense change precedes methylation or one allele is methylated before the other. On the other hand the treatment of cell lines derived from such tumors with the DNA methyltransferase inhibitor 5-aza-2`-deoxycytidine (5-azad-C) leads to demethylation of the MLH1 promoter, reaccumulation of the MLH1 protein and restoration of mismatch- repair activity. [24]. It should therefore be possible to reverse the cancer phenotype by the use of methylation inhibitors [31]. Recently, many clinical approaches are trying to demonstrate target gene demethylation and probably the success in this area of pharmacogenetics is forthcoming.

Our method for detection of the MLH1 promoter region hypermethylation is a simple and applicable method in the diagnostic algorythm for colorectal cancer. We propose it as an obligatory step following the analysis for MSI and before screening for mutations in the MMR genes, especially in sporadic cases, because in most of the cases these patients are mutation “negative”.