建立偵測急性骨髓白血病WT1基因突變的檢驗流程

Abstract

Wilm’s tumor 1, discovered to be important in kidney tumor of child, plays the role in regulating gene expression. For instance, WT1 in breast cancer acts as an oncogene, forming tumors or causing metastasis through activating c-myc, Bcl-2 and E-cadherin. On the other hand, WT1 in kidney tumor could inactivates c-myc, Bcl-2 and E-cadherin.Similar contradict results was found in patients with AML. CD34+ cell, from AML patients tended to have higher WT1 expression than those from normal patients. On the other hand, 15% of AML patients was with mutant WT1. Both of them demonstrated poor prognosis with standard chemotherapy. In clinical practice, WT1 could be as a biomarker for AML patients to monitor disease status. The aim of this study is to develop an effective process to detect WT1 mutation, providing references for clinical diagnosis, follow-up management and prognostic evaluation. We coupled real-time PCR with the theorem of high resolution melting to set up a standard operation protocol(SOP) for differentiating mutant from normal WT1. In this protocol, LCGreen dye was used to intercalate double strand DNA and show fluorescence, but the fluorescence would disapper when double strand DNA dissociated into single strand DNA with increasing temperature. Comparing the difference of melting peaks by analytic software, we could differentiate the mutants from the wild types. Afterward, we confirmed the mutation type by direct sequencing or GeneScan. We believe this detecting process can decrease the cost effectively, and increase the sensitivity, compared to direct sequencing detecting method.