Quantitative high-resolution CpG island mapping with Pyrosequencing™ reveals disease-specific methylation patterns of the CDKN2B gene in Myelodysplastic syndrome and myeloid leukemia
Quantitative high-resolution CpG island mapping with Pyrosequencing™ reveals disease-specific methylation patterns of the CDKN2B gene in Myelodysplastic syndrome and myeloid leukemia
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DOI：
10.1373/clinchem.2007.072629
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发表时间：
2007-01-01
影响因子：
9.3
通讯作者：
Lehmann, Ulrich
中科院分区：
文献类型：
--
作者：
Brakensiek, Kai;Wingen, Luzie U.;Lehmann, Ulrich
背景资料：通过异常CpG岛甲基化的基因沉默是人类肿瘤发生中最广泛分析的表观遗传事件，并且具有巨大的诊断和预后潜力。然而，甲基化模式通常是非常异质的，这对开发用于诊断目的的甲基化测定提出了严峻的挑战。我们使用焦磷酸测序（TM）技术来确定CDKN 2B基因[细胞周期蛋白依赖性激酶抑制剂213]的CpG岛中68个CpG位点的甲基化状态。（p15，抑制CDK 4）]，在骨髓恶性肿瘤中经常高甲基化，在一系列骨髓样品中，来自骨髓增生异常和骨髓白血病患者（n=82）和来自32个对照。总共对7762个单独的甲基化位点进行了定量评价。结果：使用优化的测序引物和新的Pyro Q-CpG（TM）软件，可以精确和重复地定量，使用一个单一的测序引物，最多15个CpG位点分布在100 bp左右。对整个CpG岛的广泛统计分析首次揭示了骨髓恶性肿瘤中CDKN 2B基因的疾病特异性甲基化模式和具有高区分能力的差异甲基化小区域，即使是低度骨髓增生异常综合征样本也能与对照组区分开来，这一结果在9个对照组和36个患者样本的独立组中得到证实。现在可以使用焦磷酸测序软件结合优化的测序引物对整个CpG岛进行精确的定量甲基化作图。该方法揭示了疾病特异性甲基化模式，并能够开发特异性诊断测定。(c)2007年美国临床化学协会
Background: Gene silencing through aberrant CpG island methylation is the most extensively analyzed epigenetic event in human tumorigenesis and has huge diagnostic and prognostic potential. Methylation patterns are often very heterogeneous, however, presenting a serious challenge for the development of methylation assays for diagnostic purposes.Methods: We used Pyrosequencing (TM) technology to determine the methylation status of 68 CpG sites in the CpG island of the CDKN2B gene [cyclin-dependent kinase inhibitor 213 (p15, inhibits CDK4)], frequently hypermethylated in myeloid malignancies, in a series of bone marrow samples from patients with myelodysplasia and myeloid leukemia (n=82) and from 32 controls. A total of 7762 individual methylation sites were quantitatively evaluated. Precision and reproducibility of the quantification was evaluated with several overlapping primers.Results: The use of optimized sequencing primers and the new Pyro Q-CpG (TM) software enabled precise and reproducible quantification with a single sequencing primer of up to 15 CpG sites distributed over similar to 100 bp. Extensive statistical analyses of the whole CpG island revealed for the first time disease-specific methylation patterns of the CDKN2B gene in myeloid malignancies and small regions of differential methylation with high discriminatory power that enabled differentiation of even low-grade myelodysplastic syndrome samples from the controls, a result that was confirmed in an independent group of 9 control and 36 patient samples.Conclusion: The precise quantitative methylation mapping of whole CpG islands is now possible with Pyrosequencing software in combination with optimized sequencing primers. This method reveals disease-specific methylation patterns and enables the development of specific diagnostic assays. (c) 2007 American Association for Clinical Chemistry