2010-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/647991摘要：隨著人類基因圖譜的解密，科學家發現在基因序列之上的基因調控 (Epigentics) 益發重要，其中，DNA 的甲基化調控著許多基因的活化與否，並控制許多基因的表現。DNA 的甲基化，尤其是在基因的啟動子區域中的 CpG island 位置，會阻礙轉錄因子與DNA 結合，進而導致基因沈默 (silencing)。異常的DNA 甲基化和許多人類疾病與癌症的發生息息相關，因此，本計劃著重於甲基化相關基因之探討。然而，DNA 甲基化的檢測便是一項關鍵性的工作。因此，此第一年計劃將發展許多分析技術檢測DNA 的甲基化，包括序列分析、聚合酶連鎖反應、高解析度熔點分析儀、DNA 突變分析儀等。在此第二年計畫中，我們將進一步以此技術運用於DNA 異常甲基化所造成的人類疾病研究，包含小胖威力症、天使症候群、貝克威思-威德曼綜合症、羅素-西弗氏症以及癌症相關基因，建立高解析度與高效能的DNA 甲基化分析平台，提供臨床應用與發展。然而，許多癌症的發生和惡化能仍無法被理解，因此，此計劃第三年將發展以微陣列為基礎 (microarray-based) 的基因甲基化分析技術，建立DNA 甲基化的全基因分析平台，可以更了解癌症的發生原因與掌握病程變化，發展早期偵測癌症發生的基因標記，和癌症預後相關的指標。此計劃將有助於 Epigenome 的研究進展並且可以更深入研究腫瘤的發生學與致病機轉。<br> Abstract: This three years project focuses on the epigenetic changes investigation because it isheritable modifications that do not involve alterations in the primary DNA sequence. Theyregulate crucial cellular functions such as genome stability, X-chromosome inactivation, andgene imprinting. Epidemiological and experimental observations now suggest that suchchanges may also explain the fetal basis of adult diseases such as cancer, cardiovasculardisorders, neurological diseases, and behavioral modifications. The main molecular eventsknown to initiate and sustain epigenetic modifications are histone modification and DNAmethylation.In the first year, we will specifically focus on existing and emerging technologies used instudying DNA methylation, which occurs primarily at CpG dinucleotides in the genome.These include standard exploratory tools used for global profiling of DNA methylation andtargeted gene investigation: bisulfite sequencing,methylation sensitive PCR (MSP), methylation-sensitive restriction enzymes, denaturinghigh-performance liquid chromatography (DHPLC), methylation-specific multiplex PCR,methylation-specific high-resolution melting-curve analysis (MS-HRM), methylation-specificmultiplex ligation-dependent probe amplification (MS-MLPA). Moreover, the basic operatingprincipals, resource requirements, applications, and benefits and limitations of eachmethodology are investigated in the project.In the second year, we will focus on detecting DNA methylation in clinical samples.Carcinogenesis is also accompanied by changes in methylation of genomic DNA. Thesechanges include an aberrant local hypermethylation of promotors of various tumor suppressorgenes, which results in their silencing, and whole-genome hypomethylation accompanied byactivation of oncogenes, retrotransposons, and genomic instability. Thus, detect DNAmethylation that may be useful in early detection of cancer screening. In part, this is becausealterations in DNA methylation – both hypermethylation and hypomethylation – are earlyevents in carcinogenesis. In addition, DNA methylation is a tumor-specific change in DNAthat can be detected in blood, sputa, and biopsy specimens using highly sensitive, low-costmethods we investigated in the first year. It may be possible to use DNA methylation profilesto distinguish promoter hypermethylation profiles between different types of tumors from thesame organ site as well as between tumors from different tissues. Aberrant methylation ofpromoter CpG islands has been implicated in a number of inherited disorders, including thefragile X syndrome, Beckwith-Wiedemann syndrome, Rett syndrome, Prader-Willi syndrome(PWS) and Angelman syndrome (AS).In the third year, because this still leaves some cases with an unknown genetic aetiology,we will use advanced arrays including CGH array, bead array and short oligonucleotide arrayas tool and robust data to identify novel and candidate cancer-specific methylated genes.Therefore, further studies are needed to elucidate the underlying cause of the epigeneticdefects in imprinting disorders. Identification of all gene promoters methylated would greatlyadvance our understanding of gene regulatory networks in cancer genetics. We then usebisulfite DNA sequencing, methylation-specific PCR (MSP), methylation-specific multiplexPCR, and others (methods set up in the first year) to confirm cancer-specificmethylation in alarge number of novel genes. Finally, this approach will greatly expand our knowledge ofmethylated promoters, sets the stage for rapid and full elucidation of methylated gene targetsand pathways in human imprinting disorders and cancers.甲基化基因印記微陣列癌症DNA methylationgenetic imprintingmicroarraycancer