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

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。染色体断裂点的鉴定有助于疾病相关基因的鉴定。为了改进断裂点结构鉴定的步骤，我们发展了一种基于衔接子连接聚合酶链反应（AL-PCR）的新方法。这种方法使我们能够在几天内完成一个断点的分离和表征，只需100 ng的患者的基因组DNA。为了证明这种方法的有效性，我们将其应用于成人T细胞白血病（ATL）患者中HTLV-1病毒整合位点的分离。我们使用AL-PCR从33名ATL患者和5个ATL细胞系中分离出总共58个HTLV-1整合位点。随机选择用于整合的染色体靶标，但整合有利地发生在转录单位内;在转录单位内观察到超过59.5%的总整合。通过HTLV-1整合的所有插入基因在正常T细胞中表达。在9例ATLL病例中，有2例发现由于病毒整合引起的基因上调;分别观察到锚蛋白-1（ANK-1）和桥蛋白（GPHN）基因表达升高约4.4倍和102倍。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分离在几个ATL患者中发现的断点。通过对断裂点的分析，我们发现了一个位于染色体易位断裂点附近的基因，命名为ATL 1。ATL 1在ATL患者中表达下调。ATL 1基因在Hela细胞中的强制表达揭示了其抑癌活性。提示ATLI是ATL白血病发生的一个常见致病基因。

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.