Aberrant Methylation of Multiple Tumor Suppressor genes and Expression Level of DNA Methyl Transferases(DNMT1, 3A, 3B) in Acute Myeloid Leukemia.
Authors: Ekmekci Cumhur Gokhan , Sirma Sema , Gutierrez Marina , Siraj Khalid , Ustek Duran , Bhatia Kishor , Ozbek Ugur 
Published: April 2020

Global DNA hypomethylation is followed by regional hypermethylation occur in tumorigenesis and these hypermethylation is often associated with the inactivation of the tumor supresser genes. DNA methyltransferases are known to be responsible for the methylation of CpG island in human. In this study, we were interested in determining the extent and pattern of concurrent methylation of the various genes, SOCS1, DAP-Kinase, p15, E-Cadherin, RARβ, HIC1, p16, GSTP1, ER and p73 and also expression level of DNA methyltransferases genes in pediatric and adult AML samples to determine their roles in tumorogenesis. Genomic DNA was extracted from mononuclear cells of 50 AML patients(25 pediatric and 25 adult) and treated with sodium bisulfite for methylation spesific PCR(p15, p16, p73, SOCS1, RARβ, E-Cad, GSTP1, DAP-Kinaz, 5-HIC) and COBRA analysis (for ER). The frequency of methylation was shown in table 1. Among these patients samples DNA methyltransferases genes expression of 25(12 pediatric and 13 adult) patient and 6 control samples from healthy volenteers were determined by Q-PCR technique and β2 microglobulin used for normalisation as an internal control. Expression levels were given in table 2. Statistical analysis made by SPSS software for windows.

Methylation was very common in AML. The frequency of methylation was shown for DAP-Kinase, ER, p15, SOCS1, E-cadherin, RARβ and p73 in table 1. Three other genes, p16, GSTP1 and HIC1 were unmethylated. All of 10 genes were unmethylated in 5 pediatric and in 2 adult samples on the other hand methylation at least in one gene for the rest of the 43 samples. DAP-Kinase was the most frequently methylated gene for both group(both for 56%). Unlike to previous studies ER methylation seems to frequently methylated in pediatric group than in adults(pediatric 42%, adult 28%). Just the opposite was valid for the RARβ methylation (pediatric 4%, adult 20%). Based on the 10 genes calculated MI for pediatric and adult group average were 0.11 (mean=0,1946) and 0,2 (mean=0,2112) respectively.

Expression analysis of DNA methyltransferases (DNMT1, 3A, 3B) genes; for DNMT1 we couldn’t find any statistical difference in between control group and in pediatric AML samples (p= 0,833) While in adult group we found higher expression level of DNMT1 when we compare with the controls (p=0,027). Furthermore we observed slight significant difference in between adult and pediatric group (p=0,044). For the DNMT3A, both in pediatric and adult group expression level of the gene was significantly higher than the controls (p= 0,06; p= 0,03 respectively) on the other hand no differences in between adult and pediatric groups(p=0.228).

ER hypermethylation in pediatrics and RARβ hypermethylation in adults and DAP-Kinase hypermethylation in both groups is a frequent events in AML. Hypermethylation and imbalanced expression of DNMTs in AML could suggest a potantial mechanism of epigenetic modulation in leukomogenesis.

Table 1

Table 2

p15 (%) SOCS-1(%) E-Caderin(%) RAR β(%) Dap-Kinase(%) ER(%) p73(%) Pediatric 32 36 8 4 56 42 17 Adult 36 32 20 20 56 28 18 Total 34 34 14 12 56 35 17,5 DNMT1 DNMT3A DNMT3B Mean 6.62±8.29 x10−2 7.82±26.31 x10−2 0.57±1.82 x10−2 Median 3.95±4.33 x10−2 1.08 ±1.42 x10−2 0.19 ±0.26 x10−2 Range 0.01–36.07 x10−2 0.24–131.30 x10−2 0.002–9.10 x10−2