DNA was extracted from peripheral blood lymphocytes and percent cytosine methylation was determined as previously described (Nielsen et al. 2009). Briefly, genomic DNA (300 ng) was sodium bisulfite treated using the EZ-96 DNA Methylation Kit D5004 (Zymo Research, Orange, CA) and amplified (Nielsen et al. 2009). DNA was sequenced at GENEWIZ, Inc. (South Plainfield, NJ) on an ABI 3730 XL sequencer (Applied Biosystems, Foster City, CA). Trace files (.ab1) were analyzed using the ESME version 3.2.1 software from Epigenomics AG (Berlin, Germany) (Lewin et al. 2004). The percent methylation calls by the ESME were reviewed by two independent researchers who visually inspected all the methylation calls using the associated electropherograms generated by the ESME software.
