Genomic DNA was extracted (DNeasy, Qiagen) according to the manufacturer’s protocol. For PCR, we used a 5′-GGG TTC TGC TTT GCA ACT TC-3′ sense primer and a 5′-CCT TTT TCC TGG GGA GTT G-3′ antisense primer that were directed to the NR3C1 promoter (Genebank accession number AY436590). Primers were selected that covered a 536-bp region that included the region for sodium bisulfite analyses. The resulting PCR products for each subject were sequenced bidirectionally using the forward and the reverse primer on an ABI 3100 genetic analyzer (Applied Biosystems) following the manufacturer’s instructions. Genetic variation was assessed throughout the NR3C1 promoter region used for bisulfite analysis by alignment of genomic DNA with the previously published NR3C1 promoter sequence19 using freely available software (CLC Workbench, CLC bio).
