Study On WT1 Gene Mutation And Its Promoter Region DNA Methylation Status In Hematology Neoplasms

Objective: To analyze mutations of WT1 exon 7, 8,9 and 10 in acute leukemia cases. To study the DNA methylation pattern of WTl gene promoter region within hematologic neoplastic cell lines and its correlation with WTl gene expression by the PCR-based methods. And to make a preliminary discussion on the mechanism of WTl gene abnormality in hematologic malignancies.Methods: â‘ By comparison with 100 control cases, 68 cases of acute leukemia at diagnosis (52 acute myeloid leukemia [AML], 16 acute lymphoblastic leukemia [ALL]) were screened for WT1 gene mutations in exon 7, 8, 9 and 10 by single-strand conformational polymorphism (PCR-SSCP) , and then confirmed by direct sequencing. â‘¡RT-PCR and methylation-specific PCR (MSP) were performed to study WT1 gene expression and the DNA methylation status in its promoter region within hematologic neoplastic cell lines 8226, HL-60, Jurkat, K562, KG-1, NB4, SHI-1, Raji, U266 and U937. â‘¢After 5-aza-CdR demethylation treatment of U937 cell line, where high DNA methylation level of WT1 gene promoter region and low mRNA expression level were identified, the changes of WT1 gene expression level, including mRNA and protein, and its promoter region methylation status were detected.Results: â‘ Using PCR-SSCP and direct sequencing, we found no gene mutations in WT1 exon 8 and 10 in those 68 acute leukemia cases, and found a polymorphism change in exon 7 in 26.5% cases (18 out of 68 cases). Only in one AML-M5b case with chromosomal rearrangement, a point mutation was observed at position 61 from the start of exon 9, resulting in an A to G transition and a change from Lys to Glu. In those 100 control cases, we found no gene mutations in exon 7,8,9 and 10, meanwhile an identicalpolymorphism change was observed in 21% cases(21 out of 100 cases) in exon 7. ?By using RT-PCR, HL-60, K562, KG-1, NB4 and SHI-1 cell lines demonstrated higher levels of WTl expression, while extremely low levels were found in 8226, Jurkat, Raji, U266 and U937 cell lines. In MSP analysis, the DNA hypermethylation in WTl gene promoter region were identified in 8226, Jurkat, Raji, U266 and U937 cell lines with low WTl expression level. But no such DNA hypermethylation were observed in the same region of HL-60^ K562^ KG-1 ^ NB4 and SHI-1 cell lines. ?At day 6 and day 9 after demethylation respectively, the WTl gene mRNA and protein expression level in U937 line cell were enhanced, in comparision with those without demethylation treatment At the same time, DNA methylated levels decreased, but unmethylated levels increased in WTl gene promoter region. At day 9 of demethylation, WTl gene mRNA levels are obviously higher than that at day 6, in company with the decreased methylated level and increased unmethylated level.Conclusion: (DThe mutations of WTl gene exon 7, 8,9 and 10 are infrequent in acute leukemia at diagnosis, providing inadequate evidence for the role of WTl gene mutation in leukemogenesis. ?Modulation of the DNA methylation status in WTl promoter region is one of the epigenetic mechanisms to regulate its expression.