Exp Dermatol. 2010 Oct;19(10):926-8. doi: 10.1111/j.1600-0625.2010.01120.x. Epub 2010 Aug 31.
Basal cell carcinoma (BCC) is the most common form of skin cancer. Mutations of the PTCH hallmark gene are detected in about 50-60% of BCCs, which raises the question whether other mechanisms such as promoter methylation can inactivate PTCH. Therefore, we performed methylation analysis of the PTCH promoter in a total of 56 BCCs. The sensitivity of three different methods, including direct bisulphite sequencing PCR, MethyLight and high-resolution melting (HRM), was applied and compared. We found that HRM analysis of DNA from fresh tissue [rather than formalin-fixed and paraffin-embedded tissue (FFPE)] was the most sensitive method to detect methylation. Low-level methylation of the PTCH promoter was detected in five out of 16 analysed BCCs (31%) on DNA from fresh tissue but only in two (13%) samples on short-time stored FFPE DNA from the very same tumors. In contrast, we were unable to detect methylation by HRM on long-time stored DNA in any of the remaining 40 BCC samples. Our data suggest that (i) HRM on DNA extracted from fresh tissue is the most sensitive method to detect methylation and (ii) methylation of the PTCH promoter may only play a minor role in BCC carcinogenesis.
基底细胞癌（BCC）是最常见的皮肤癌。大约 50-60%的 BCC 中检测到 PTCH 标志性基因的突变，这引发了一个问题，即其他机制（如启动子甲基化）是否可以使 PTCH 失活。因此，我们对总共 56 个 BCC 中的 PTCH 启动子进行了甲基化分析。我们应用并比较了三种不同方法的敏感性，包括直接亚硫酸氢盐测序 PCR、MethyLight 和高分辨率熔解（HRM）。我们发现，HRM 分析新鲜组织中的 DNA [而不是福尔马林固定和石蜡包埋组织（FFPE）]是检测甲基化的最敏感方法。在新鲜组织的 DNA 中检测到五个分析的 BCC 中有五个（31%）存在 PTCH 启动子低水平甲基化，但在非常相同的肿瘤的短时间保存的 FFPE DNA 中只有两个（13%）样本。相比之下，我们无法通过 HRM 在其余 40 个 BCC 样本中的任何一个长时间保存的 DNA 中检测到甲基化。我们的数据表明：（i）HRM 在新鲜组织提取的 DNA 上是检测甲基化的最敏感方法；（ii）PTCH 启动子的甲基化可能在 BCC 癌变中只起次要作用。
