Shaw Richard J, Hall Gillian L, Lowe Derek, Liloglou Triantafillos, Field John K, Sloan Phillip, Risk Janet M
Molecular Genetics & Oncology Group, School of Dental Sciences, University of Liverpool, Liverpool, England.
Arch Otolaryngol Head Neck Surg. 2008 Mar;134(3):251-6. doi: 10.1001/archoto.2007.50.
To evaluate promoter methylation quantitation using recently described pyrosequencing techniques by correlation with messenger RNA (mRNA) expression.
DNA was extracted from tissue samples and was subjected to bisulphite conversion. Quantitative methylation data for multiple CpG sites in each of 9 gene promoters were obtained for tumors using pyrosequencing. RNA was extracted and converted to complementary DNA, and this formed the template for relative quantitation assays of the expression of each gene by real-time reverse transcription-polymerase chain reaction.
Academic research.
Thirty-seven patients with head and neck squamous cell carcinoma.
The genes studied were P16 (OMIM 600160), cyclin A1 (OMIM 604036), RARB (OMIM 180220), E-cadherin (OMIM 192090), MGMT (OMIM 156569), STAT1 (OMIM 600555), ATM (OMIM 607585), hMLH1 (OMIM 120436), and TIMP3 (OMIM 188826). Immunohistochemistry was also performed for p16.
STAT1, TIMP3, ATM, and hMLH1 promoters were essentially unmethylated in all cases. The data for cyclin A1 (Spearman rank correlation, rho = -0.53; P < .001), MGMT (rho = -0.53, P < .001), and RARB (rho = -0.34, P =.02) showed the expected negative correlation between levels of methylation and mRNA expression. The data relating to E-cadherin were inconclusive. Surprisingly, P16 expression was statistically significantly greater in those cases with higher levels of methylation (rho = 0.57, P < .001), a finding at odds with assumptions usually made in the literature relating gene promoter methylation to reduced gene expression. The results from p16 immunohistochemistry were in keeping with the mRNA data, but the number of positive staining samples proved too few for statistical analysis.
These data present a novel perspective on head and neck cancer epigenetics and reveal new and some unexpected associations and findings. The advantages of pyrosequencing over nonquantitative techniques are discussed in analyses of this nature.
通过与信使核糖核酸（mRNA）表达进行相关性分析，评估使用最近描述的焦磷酸测序技术进行启动子甲基化定量分析的情况。
从组织样本中提取DNA，并进行亚硫酸氢盐转化。使用焦磷酸测序技术获取9个基因启动子中每个启动子多个CpG位点的肿瘤定量甲基化数据。提取RNA并转化为互补DNA，以此作为通过实时逆转录 - 聚合酶链反应对每个基因表达进行相对定量分析的模板。
学术研究机构。
37名头颈部鳞状细胞癌患者。
所研究的基因包括P16（在线人类孟德尔遗传数据库编号600160）、细胞周期蛋白A1（在线人类孟德尔遗传数据库编号604036）、视黄酸受体β（在线人类孟德尔遗传数据库编号180220）、E - 钙黏蛋白（在线人类孟德尔遗传数据库编号192090）、O6 - 甲基鸟嘌呤 - DNA甲基转移酶（在线人类孟德尔遗传数据库编号156569）、信号转导和转录激活因子1（在线人类孟德尔遗传数据库编号600555）、共济失调毛细血管扩张症突变基因（在线人类孟德尔遗传数据库编号607585）、人错配修复蛋白1（在线人类孟德尔遗传数据库编号120436）和金属蛋白酶组织抑制因子3（在线人类孟德尔遗传数据库编号188826）。还对p16进行了免疫组织化学检测。
在所有病例中，信号转导和转录激活因子1、金属蛋白酶组织抑制因子3、共济失调毛细血管扩张症突变基因和人错配修复蛋白1启动子基本未甲基化。细胞周期蛋白A1（斯皮尔曼等级相关系数，rho = -0.53；P <.001）、O6 - 甲基鸟嘌呤 - DNA甲基转移酶（rho = -0.53，P <.001）和视黄酸受体β（rho = -0.34，P =.02）的数据显示甲基化水平与mRNA表达之间存在预期的负相关。与E - 钙黏蛋白相关的数据尚无定论。令人惊讶的是，在甲基化水平较高的病例中，P16表达在统计学上显著更高（rho = 0.57，P <.001），这一发现与文献中通常将基因启动子甲基化与基因表达降低相关联的假设相悖。p16免疫组织化学结果与mRNA数据一致，但阳性染色样本数量过少，无法进行统计学分析。
这些数据为头颈部癌表观遗传学提供了新的视角，揭示了新的和一些意想不到的关联及发现。在这类分析中讨论了焦磷酸测序相对于非定量技术的优势。
