IDENTIFICATION OF DNA METHYLATION MARKERS FOR HUMAN BREAST CARCINOMASUSING THE METHYLATION-SENSITIVE RESTRICTION FINGERPRINTING TECHNIQUE

We have developed a PCR-based method, called methylation-sensitive res triction fingerprinting (MSRF), to screen changes in DNA methylation i n breast carcinomas, Two hypermethylation-containing fragments, HBC-1 (for ''hypermethylation in breast cancer'') and HBC-2, were identified in the amplified breast tumor DNA relative to the amplified normal br east DNA of a patient. Nucleotide sequence analysis revealed no signif icant matches between the sequence of HBC-1 and the known sequences in the GenBank database, whereas the sequence of HBC-2 matched the upstr eam region of an antisense WT1 (Wilms' tumor suppressor gene) promoter , The methylation status in the breast tumor DNA from this patient was confirmed by Southern hybridization using HBC-1 and HBC-2 as probes, respectively, Further analysis showed that HBC-I was methylated aberra ntly in 90% (17 of 19 patients) of the primary breast carcinomas exami ned, This study demonstrates that MSRF provides a useful means for scr eening aberrant changes in DNA methylation during tumorigenesis, The c ommonly methylated fragments identified by MSRF could potentially supp lement pathological markers currently used for cancers and additionall y lead to the discovery of novel methylated tumor suppressor genes.