Genotyping of SLC6A4 VNTR Variants

VNTR polymorphisms in the SLC6A4 promoter (5HTTLPR) and intron 2 (STin2) regions were genotyped as described in our previous study [39 (link)]. Simultaneous genotyping of rs25531 (NG_011747.2: 3609A/G) and 5HTTLPR was based on restriction fragment length polymorphism (RFLP) analysis of PCR products obtained using 50 ng genomic DNA, HotStart Taq DNA Polymerase (Qiagen) and the following primers (0.6 μM) each: forward, 5'-CTCCCTGTACCCCTCCTAGG-3’ (NG_011747.2: 3527–3546) and reverse, 5’-TGCAAGGAGAATGCTGGAG-3' (NG_011747.2: 3801–3819). The cycling conditions were: 95°C for 15 min; 40 cycles of 95°C for 30 s, 60°C for 45 s, 72°C for 45; 72°C for 7 min. PCR products were digested overnight at 37°C with MspI (New England Biolab), electrophoresed using the QIAxcel system, and sized by QIAxcel ScreenGel Software (both from Qiagen). Fragments of 211 and 38 bp corresponded to the S allele, fragments of 245 and 38 bp corresponded to the La allele, while fragments of 162, 83 and 38 bp corresponded to the Lg allele. The concordance of 5HTTLPR genotypes obtained by this protocol and the previously mentioned protocol [39 (link)] was 100%. In analogy with other studies, 5HTTLPR/rs25531 haplotypes were dichotomized into high-expressing (La/La) and low-expressing (other) group [40 (link),41 (link)].