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Methodological development for the identification of disease related genes by taking advantage of chromosome abnormalities

利用染色体异常鉴定疾病相关基因的方法学开发

基本信息

批准号：
12204005
负责人：
ISOBE Masaharu
金额：
$ 28.03万
依托单位：
Toyama University, Faculty of Engineering
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12204005/
关键词：
chromosome abnormality adult T-cell leukemia HTLV-1 chromosome translocation PCR BAC SNP 14番染色体 ATL1

项目摘要

Chromosomal translocations are frequently found to be associated with various malignant disorders as well as congenital abnormalities. The characterization of chromosomal breakpoint greatly helps to identify disease related genes. To improve the step of structural characterization of a breakpoint, we have developed new method based on the adaptor-ligated polymerase chain reaction (AL-PCR). This method allowed us to complete the isolation and characterization of a breakpoint within a few days using just 100ng of patient's genomic DNA.To prove the validity of this method, we applied this for the isolation of HTLV-1 viral integration sites in adult T-cell leukemia (ATL) patients. We isolated a total of 58 HTLV-1 integration sites using AL-PCR from 33 ATL patients and five ATL cell lines. The chromosomal target for integration was selected at random, but the integration favourably occurred within the transcription units; more than 59.5% of total integration was observed within the transcriptional unit. All inserted genes by HTLV-1 integration were expressed in normal T-cells. Upregulation of genes due to viral integration was found in two out of nine ATLL cases; about 4.4-and 102-fold elevated ankyrin-1 (ANK-1) and gephyrin ( GPHN) gene expressions were observed, respectively.The integration of HTLV-1 is not enough to give rise a tumor. The change in expression of cellular genes is required for leukemogenesis of ATL. Thus, we applied AL-PCR for the isolation of breakpoints found in several ATL patients. From characterization of breakpoints, we found a gene named ATL1 nearby breakpoint of recurrent chromosome translocation. The ATL1 was often down regulated in ATL patient. The forced expression of ATL1 gene revealed the tumor suppressor activity in Hela cells. This suggests that the ATLI is a causative gene commonly involved in leukemogenesis of ATL.

染色体易位经常被发现与各种恶性疾病以及先天性异常有关。染色体断点的表征有助于疾病相关基因的鉴定。为了提高断点结构表征的步骤，我们开发了基于接头连接聚合酶链反应（AL-PCR）的新方法。这种方法使我们能够在几天内完成断点的分离和表征，仅使用100ng的患者基因组DNA。为了证明该方法的有效性，我们将该方法应用于成人t细胞白血病（ATL）患者HTLV-1病毒整合位点的分离。我们利用AL-PCR从33例ATL患者和5个ATL细胞系中分离出58个HTLV-1整合位点。整合的染色体靶点是随机选择的，但在转录单位内进行整合是有利的；在转录单位内观察到超过59.5%的总整合。HTLV-1整合后插入的所有基因均在正常t细胞中表达。9例ATLL病例中有2例因病毒整合而出现基因上调；锚蛋白-1 （ankyin -1）和格phyrin （GPHN）基因表达分别升高约4.4倍和102倍。HTLV-1的整合并不足以产生肿瘤。细胞基因表达的改变是ATL白血病发生所必需的。因此，我们应用AL-PCR分离了几个ATL患者的断点。从断点的特征中，我们发现一个名为ATL1的基因位于复发性染色体易位的断点附近。ATL患者的ATL1常出现下调。ATL1基因的强制表达揭示了Hela细胞的抑瘤活性。这表明ATLI是一个致病基因，通常参与ATL的白血病发生。