Combination of different methodologies elucidates tumor specific methylation in medulloblastoma

Introduction: The methylation status of the CpG Islands of p16, p14, CDH1, TIMP3 and MGMT plays a role in the origin and spread of malignancies. We examined the methylation status of their CpG Islands in medulloblastomas and other common malignant brain tumors of childhood, by Methylation Specific PCR (MSP), COmbined Bisulfit Restriction Analysis (COBRA) and Bisulfit Sequencing.

Results: MSP revealed aberrant methylation in 24/57 (MGMT), 11/53 (p14), 16/58 (p16), 6/60 (TIMP3) and 38/56 (CDH1) medulloblastomas (MB), while normal cerebella showed only limited methylation for CDH1 (3/7) and MGMT (3/9). The reexamination of the methylated islands using COBRA confirmed aberrant methylation in 12/27 (MGMT), 0/1 (p14), 8/16 (p16), 1/4 (TIMP3) and 0/42 (CDH1) of the samples under study. Apart from MGMT (2/2) there was no methylation confirmed for normal cerebella. Comparing the level of detection for these methods, MSP showed a 1000 fold higher sensitivity than COBRA. The methylation status in several tumors might be below the detection limit of this method.

Conclusion: The partial confirmation of methylation detected by MSP using COBRA demonstrates that these genes are methylated at the detection limit of COBRA. This hints to an overestimation of aberrant methylation caused by the higher sensitivity of the MSP. Methylation of p14, p16 and TIMP3, missing completely from normal cerebella, was only seen in MB, but rarely or not at all in anaplastic ependymoma and stPNETs.

Supported by Deutsche Krebshilfe 10–2091-Fr 2 and the Karl Bröcker Stiftung