Methylation of microRNA Promoters in Myelodysplastic Syndromes
Begum Erdogan, Dunfa Peng, Leng Han, Zhongming Zhao, Wael El-Rifai, Annette S Kim. Vanderbilt University Medical Center, Nashville, TN; Stanford University, Palo Alto, CA
Background: Myelodysplastic syndromes (MDS) are diseases predominantly of the elderly characterized by ineffective hematopoiesis. We have shown that eight intragenic microRNAs (miRNAs) are down-regulated in MDS and may contribute to the ineffective hematopoietic maturation seen in this disease. miRNAs are small non-coding RNAs that regulate protein expression and are critical to cell differentiation. We hypothesize that hypermethylation of the miRNA promoters contributes to the decreased expression of these miRNAs in MDS patients.
Design: Genomic DNA was isolated from fresh frozen or paraffin embedded bone marrow (BM) aspirates from 41 normal controls and 36 MDS patients and processed using the methylSEQr Bisulfite Conversion Kit (Applied Biosystems, CA). PCR amplification of the CpG-rich regions in the promoters of the miRNA host genes was performed with subsequent pyrosequencing by the Biotage PyroMark MD (Qiagen, CA). These results were compared to Robust Multichip Analysis (RMA) of the data derived from Microarray Innovations in Leukemia (MILE) study as well as to published global methylation differences between MDS samples and controls.
Results: We confirmed the feasibility of using archival paraffin tissues for the study by comparing the methylation patterns of paired fresh frozen and paraffin embedded BM samples (R2 = 0.98). Statistical analysis of the data collected from the paraffin-embedded samples of 22 normal individuals and 26 MDS patients showed increased promoter methylation (p < 0.05) in several miRNA host genes in MDS patients not taking DNMTIs. These CpG sites included loci in the promoters of WWP2 (host gene for miR-140), ZNF207 (host gene for miR-632) and PPARGC1B (host gene for miR-378). Several other CpG sites in IGF2 (miR-483) and SFRS2 (miR-636) trended toward significance. There was no difference in methylation levels for the other host gene promoters. Of note, the 6 MDS patients on therapy with DNMTIs demonstrated similar methylation patterns to those of normal controls. Of the differentially methylated promoters, IGF2 was identified in other published MDS methylation studies. As confirmation of the coregulation of the miRNAs and their host genes at the promoter level, WWP2 and ZNF207 were found to be underexpressed in MDS compared to controls in the MILE study data.
Conclusions: Increased methylation of several miRNA promoters is found in MDS compared to normal controls. This may explain the underexpression of diagnostic miRNAs in MDS, as well as of their host genes. Treatment with DNMTIs results in normalization of the methylation at those sites.
Tuesday, March 20, 2012 2:15 PM
Platform Session: Section G, Tuesday Afternoon