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.

中文翻译：

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人脑海绵状畸形中CCM基因的DNA启动子甲基化：通过测序确认MSP数据的重要性

背景

脑海绵状畸形（CCM）是第二常见的脑血管疾病，分为家族性（20％）和散发性（80％）。三个CCM基因的功能缺失会导致家族性CCM。考虑到家庭和零星CCM的类似诊所的介绍，并根据在三个DNA启动子区域富集的CpG岛CCM基因，我们推测在的CpG岛的DNA甲基CCM基因参与人体CCM，从而导致损失的CCM基因。

材料与方法

69个人类CCM，包括零星（n = 40），多发（n = 15）和家族（n = 14）病例。从CCM的手术标本中提取DNA，然后进行亚硫酸氢盐转化。通过甲基化特异性PCR（MSP）检测了三个CCM基因启动子区域的甲基化状态。为了确认MSP的结果，对显示CCM3甲基化的4个MSP阳性探针进行了深亚硫酸氢盐测序（DBS）。

结果

MSP大多排除了CCM1和CCM2启动子区域的甲基化（数据未显示）。在CCM3病例中，55例散发病例中有12例显示MSP阳性（21.8％）。亚硫酸氢盐的深层测序表明，有4例CCM3 MSP阳性病例均对DNA甲基化阴性。

结论

本研究表明，CCM1-3基因的DNA启动子甲基化不参与人类家族CCM，因此与DBS确认MSP数据非常重要。为了更好地了解这种表观遗传机制是否与CCM病理学相关，需要对更多的散发性CCM患者进行进一步研究。