Quantitative Analysis of Tissue and Developmental Stage-Specific Differences in DNA Methylation throughout the β-Globin Gene Locus by DNA MassARRAY,

Abstract Abstract 3196 DNA methylation plays a fundamental role in developmental regulation of γ-globin gene expression. The baboon is an excellent animal model to study the role of DNA methylation in developmental globin gene regulation because 1) the sequence and arrangement of genes within the β-globin locus are highly conserved between baboon and man, 2) only simian primates exhibit true fetal-stage γ-globin expression, 3) large numbers of highly purified erythroid cells can be isolated from baboon fetuses derived from timed pregnancies at varying stages of gestation and from the bone marrows of adult baboons, and 4) treatment of baboons with DNA methyltransferase inhibitors induce abundant γ-globin expression in adult baboons. Bisulfite sequence analysis data has shown that DNA hypomethylation within the 5’ promoter regions of the e- and γ-globin genes is associated with expression during the embryonic and fetal stages of development (Lavelle et al Blood Cell Mol Dis 36:269, 2006), and developmentally regulated, large extended domains of DNA hypomethylation surrounding expressed genes have been also reported (Lathrop et al Exp Hematol 37:807, 2009). Although analysis of DNA methylation by bisulfite sequence analysis is considered to be the “gold standard”, quantitative analysis by this method is laborious and limited by the ability to perform DNA sequence analysis of large numbers of clones isolated following PCR amplification and bisulfite modification. Recently, a new quantitative method, MassARRAY, has been developed that allows direct quantitative analysis of cytosine methylation within defined PCR amplicons, thus eliminating the need for the laborious sequence analysis of large numbers of clones and allowing more precise quantitative analysis. To develop MassARRAY technology as a tool for studies of DNA methylation of the β-globin gene locus we compared the ability of MassARRAY and bisulfite sequencing to quantitate differences in DNA methylation at 13 specific CpG sites spanning the baboon β-globin gene cluster from HS4 within the LCR to the 5’ β-globin gene region in peripheral WBC, primitive nucleated erythrocytes isolated from a baboon fetus at 40 d gestation, purified erythroid precursors from pre-switch fetal liver, post-switch fetal bone marrow (BM) and adult BM pre- and post-treatment with decitabine administered by either subcutaneous injection (0.5mg/ml sc; 10d) or orally (0.25mg/kg/2d per week; 8 weeks) in combination with tetrahydrouridine. PCR amplification of specific regions of the baboon β-globin gene locus from bisulfite-converted DNA was performed using primers containing the 5’ T7 and 10mer tags selected using Sequenom Epidesigner software. PCR conditions were optimized to yield single bands by agarose gel analysis in all reactions. PCR products were treated with shrimp alkaline phosphatase and in vitro transcription and RNase digestion performed, followed by analysis using the Maldi-TOF MassARRAY system (Sequenom). Quantitation of DNA methylation of specific CpG residues was performed using EpiTYPER software. To compare results obtained by MassARRAY and traditional bisulfite sequence analysis, PCR products from bisulfite-converted DNA were also cloned in the PCR4 vector and sequence analysis of at least 10 clones performed for each amplicon. Results of MassARRAY and bisulfite sequence analysis were in close agreement. Analysis of all sites in all samples yielded a mean difference of 12.3 + 9.2% dmC between these two techniques. Four CpG sites within the LCR region were highly methylated in WBC DNA compared to erythroid tissues. Developmental differences in DNA methylation in the 5’ e- and γ-globin regions were associated with stage-specific expression of these genes. Modest differences (10–15% dmC) in DNA methylation of four CpG sites in the 5’ γ-globin gene region in BM erythroid cells isolated from baboons treated with oral low-dose decitabine-tetrahydrouridine in combination detected by DNA MassARRAY were consistent with the modest 15–20% elevations of fetal hemoglobin (HbF). In conclusion, quantitative analysis of DNA methylation by MassARRAY technology provides an alternative, less laborious, sensitive quantitative methodology for analysis of differences in DNA methylation throughout the β-globin gene locus during development and following treatment with HbF-inducing agents. Disclosures: No relevant conflicts of interest to declare.