The same 5-HTTLPR/rs25531 genotyping protocol was applied to DNA samples from all three cohorts. Primer sequences for genotyping 5-HTTLPR are described previously28, the forward primer having the sequence (5′- ATGCCAGCACCTAACCCCTAATGT-3′) and the reverse (5′-GGACCGCAAGGTGGGCGGGA-3′). PCR was conducted using the following cycling conditions: initial 15-min denaturing step at 95°C, followed by 35 cycles of 94°C for 30 sec, 66°C for 30 sec and 72°C for 40 sec, and a final extension phase of 72°C for 15 min. Reactions were performed in 10X reaction Buffer IV (ABgene), 1.5mM MgCl2, 50ng of genomic DNA, 5pmols of each primer, 0.3mM dNTPs and 1 unit of Native Taq (Promega). PCR products were subsequently digested by MspI restriction enzyme for 4 hours at 37°C. The digestion products were separated on a 3% agarose gel (MultiABgarose, ABgene) supplemented with Ethidium bromide (0.03%, BDH) and visualized by ultraviolet transillumination. Genotype calls were made by three independent raters, who reached consensus on 100% of the Discovery and Replication cohort samples. Genotype could not be determined accurately for one postmortem sample. Thus, it was removed from analysis, leaving a final sample of 34 individuals (10 women, mean age 49.44 ± 12.02).
