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Department of Pathology and Anatomical Sciences, Ellis Fischel Cancer Center [T. H-M. H., B.C.H., P.T., H.T.], and Departments of Biochemistry and Child Health [D.E.L., D.B.L.], School of Medicine, University of Missouri, Columbia, Missouri 65203
We have developed a PCR-based method, called methylation-sensitive restriction fingerprinting (MSRF), to screen changes in DNA methylation in 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 breast DNA of a patient. Nucleotide sequence analysis revealed no significant matches between the sequence of HBC-1 and the known sequences in the GenBank database, whereas the sequence of HBC-2 matched the upstream 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-1 was methylated aberrantly in 90% (17 of 19 patients) of the primary breast carcinomas examined. This study demonstrates that MSRF provides a useful means for screening aberrant changes in DNA methylation during tumorigenesis. The commonly methylated fragments identified by MSRF could potentially supplement pathological markers currently used for cancers and additionally lead to the discovery of novel methylated tumor suppressor genes.
1 This work was supported by NIH grant R29-CA-69065 (to T. H-M. H.) and by United States Army Medical Research Command Grant DAMD17-96-1-6055 (to D. B. L.).
2 To whom requests for reprints should be addressed, at the Department of Pathology and Anatomical Sciences, Ellis Fischel Cancer Center, University of Missouri, 115 Business Loop I-70 West, Columbia, MO 65203. Phone: 573-882-1283; Fax: 573-884-5206.
Received 10/18/96. Accepted 1/27/97.
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