DNA promoter methylation of CCM genes in human cerebral cavernous malformations: Importance of confirming MSP data through sequencing

Background: Cerebral cavernous malformations (CCMs) is the second most common cerebrovascular disease and is classified as familial (20%) and sporadic (80%) forms. Loss of function mutation of three CCM genes results in the familial CCM. Considering the similar clinic presentation of familial and sporadic CCMs, and based on enriched CpG islands in the DNA promoter region of three CCM genes, we hypothesized that DNA methylation of the CpG islands of the CCM genes is involved in human CCM, thereby leading to loss of CCM genes. Material and methods: 69 human CCMs including sporadic (n = 40), multiple (n = 15) and familial (n = 14) cases. DNA was extracted from the surgical specimens of CCMs followed by bisulfite conversion. The methylation status of the promoter regions of three CCM genes was detected by methylation specific PCR (MSP). To confirm the results of MSP, four MSP-positive probes showing CCM3 methylation underwent deep bisulfite sequencing (DBS). Results: MSP mostly excluded methylation of CCM1 and CCM2 promotor regions (data not shown). In the case of CCM3, 12 out of 55 sporadic cases showed positivity for MSP (21.8%). Deep bisulfite sequencing revealed that four CCM3 MSP positive cases were all negative for DNA methylation. Conclusion: The present study suggests that DNA promotor methylation of CCM1-3 genes is not involved in human family CCMs and that it is important to confirm MSP data with DBS. Further study with higher number of sporadic CCM patients is required for better understanding whether this epigenetic mechanism is involved in the pathology of CCM.