DIFFERENTIAL DNA METHYLATION IN AML PATIENTS WITH WILD-TYPE AND MUTATED DNMT3A AND PROGNOSTIC IMPACT OF DNA METHYLATION

Sborník

Introduction. DNA methylation has been established as a mechanism playing an important role during the process of leukemogenesis. Mutations in DNA methyltransferase 3A (DNMT3A) gene have been found in patients with acute myeloid leukemia (AML) and have been shown to be associated with adverse clinical outcome. Aims. We studied the relationship between DNA methylation status of selected genes and DNMT3A mutational status and also the impact of DNA methylation on the patients’ prognosis. Patients and Methods. We examined 79 AML patients at diagnosis (intermediate and high cytogenetic risk) and 20 healthy donors for the presence of aberrant DNA methylation of 12 tumor suppressor genes (TSG) (CDKN2B, CALCA, CDH1, ESR1, SOCS1, MYOD1, DAPK1, TIMP3, ICAM1, TERT, CTNNA1, EGR1) by MethyLight and for mutations in the gene DNMT3A by direct sequencing. We also studied methylation status of 24 HOX genes using methylation-restriction endonucleases followed by RQ-PCR arrays in 10 AML samples compared to 4 healthy donor samples. Results. Sequencing of cDNA between amino acids 300 and 930 revealed that 32 of 79 AML patients had DNMT3A mutation. MethyLight assessment of 12 TSG showed the following frequencies of hypermethylation: CDKN2B (47%), CALCA (43%), CDH1 (22%), SOCS1 (24%), MYOD1 (18%), ESR1 (14%). The remaining 6 genes were weakly methylated in less than 10 % AML patients at diagnosis and were therefore excluded from further analysis. Comparing overall cumulative DNA methylation levels and numbers of simultaneously hypermethylated (HM) genes to mutational status of DNMT3A gene, we found lower levels of DNA methylation (P<0.0001) as well as lower numbers of concurrently hypermethylated genes (P<0.0001) in patients with DNMT3a mutations. We observed the same trend also in DNA methylation levels of HOX genes when comparing AML patients with mutated (n=4) versus wild-type (n=6) DNMT3A. Furthermore, DNA methylation levels or numbers of HM genes were independent of age, gender, percentage of blasts (PB and BM) or cytogenetic risk group (intermediate versus high risk). When assessing DNA methylation impact on prognosis we found a correlation between higher DNA methylation (cumulative levels and numbers of HM genes, respectively) and increased overall survival (OS) (P=0.03 and P=0.01, respectively) as well as lower incidence of relapse (P=0.05 and P=0.01, respectively). For individual genes, SOCS1 was found to be significantly linked with better OS (P=0.04). Conclusions. Our results show that numbers of simultaneously hypermethylated genes and DNA methylation levels of selected TSG as well as HOX genes differs between AML patients with wild-type and mutated DNMT3A. Increased DNA methylation of these TSG is linked with better prognosis in AML patients.This study is a part of the COST Action BM0801 (EuGESMA) and is supported by the Czech Ministry of Education, Youth and Sports (MŠMT) OC10042 grant.

Haematologica, 2012; 97(s1):  16