Analysis of DNA methylation pattern of promoter CpG islands in idiopathic pulmonary fibrosis.

CpG二核苷酸胞嘧啶残基的甲基化构成了哺乳动物基因表达的表观遗传控制的基础。基因组甲基化模式在发育、印记和肿瘤发生等各种生物学过程中起着至关重要的作用。CpG二核苷酸中的大多数胞嘧啶在人类基因组中是甲基化的，但一些在特定的GC丰富的座位上仍然没有甲基化，称为CpG岛。这些小片段的DNA序列位于60%的人类基因的启动子和第一外显子区域，这些CpG岛上胞嘧啶的甲基化与基因表达的丧失有关。虽然已有大量证据表明癌症中存在异常的从头甲基化，但正常和其他疾病中维持和从头甲基化的调节尚不清楚。在这项研究中，我们开发了一种新的基于微阵列的方法来检测包括转录因子和信号分子在内的360个基因启动子CpG的甲基化模式的差异。利用这个微阵列，我们分析了来自特发性肺纤维化(IPF)患者的基因组样本。通过甲基化扩增方法从患者和正常捐赠者的基因组样本中制备荧光探针。杂交后，分析阵列数据以比较两组的甲基化状态。在~25个位点检测到差异，提示IPF患者的甲基化状态不同。然后，我们用亚硫酸氢盐测序法分析了这些座位上每个CpG位点的甲基化情况，并在IPF中检测到了几个启动子CpG低甲基化的基因。这些结果提示表观遗传因素可能参与了IPF的发病机制，我们现在检测这些基因的蛋白水平。

Methylation of cytosine residues of the CpG dinucleotides constitutes the basis of an epigenetic control of gene expression in mammals. Genomic methylation patterns are of critical importance in various biological processes such as development, imprinting, and tumorigenesis. Most cytosines within CpG dinucleotides are methylated in the human genome, but some remain unmethylated in specific-GC rich loci, called CpG islands. These small stretches of DNA sequences are located in the promoter and first exon regions of 60% of human genes, and methylation of cytosine in these CpG islands is associated with loss of gene expression. Although much evidence has been accumulated to show aberrant de novo methylation in cancer, regulation of maintenance and de novo methylation in normal and other diseases is obscure. In this study, we developed a new microarray-based method to detect differences in methylation patterns of the promoter CpG of 360 genes including transcription factors and signaling molecules. Using this microarray, we analyzed genome samples derived from patients with idiopathic pulmonary fibrosis (IPF). Fluorescent probes were prepared from genome samples obtained from patients and normal donors by a methylation amplification method. After hybridization, array data was analyzed to compare the methylation states of two groups. Differences were detected at 〜25 loci, suggesting differential methylation states in patients with IPF. We, then, analyzed methylation of each CpG site of these loci by a bisulfite sequencing method, and detected several genes whose promoter CpG is hypomethylated in IPF. These results suggest a possible involvement of epigenetic factors in pathogenesis of IPF, and we examine the protein levels of these genes now.