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

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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.