Association of Anxiety-Related Traits with a Polymorphism in the Serotonin Transporter Gene Regulatory Region

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Blood for DNA isolation and analysis was obtained from healthy human volunteers. Oligonucleotide primers flanking the 5-HTTLPR and corresponding to the nucleotide positions -1416 to -1397 (stpr5, 5′-GGCGTTGCCGCTCTGAATGC) and -910 to -888 (stpr3, 5′-GAGGGACTGAGCTGGACAACCAC) of the 5-HTT gene 5′-flanking regulatory region were used to generate 484- or 528-bp fragments. PCR amplification was carried out in a final volume of 30 µl consisting of 50 ng of genomic DNA, 2.5 mM deoxyribonucleotides (dGTP/7-deaza-2′-dGTP = l/l), 0.1 µg of sense and antisense primers, 10 mM tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, and 1 U of Taq DNA polymerase. Annealing was carried out at 61°C for 30 s, extension at 72°C for 1 min, and denaturation at 95°C for 30 s for 35 cycles

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Epstein-Barr virus-transformed lymphoblasts with the genotypes l/l (n = 4), l/s (n = 3), and s/s (n = 3) were grown in RPMI 1640 supplemented with 10% newborn calf serum at 37°C in a humidified atmosphere at 5% CO₂. For induction of 5-HTT expression, lymphoblasts were treated with 50 to 200 µM forskolin or 0.5 to 2 µM PMA and grown for an additional 24 hours

The human 5-HTT gene 5′ regulatory sequence (promoter) is derived from the ~1.7-kb clone λHG5-HTT/P-HB (EMBL-GenBank accession number X76753) that was isolated from a human genomic library in λZAP express (Stratagene) as described (6). The long variant of the 5-HTT regulatory sequence (l 5-HTTP, base pairs -1440 to +22 with respect to the transcription initiation site) was ligated into the promoterless luciferase (luc+) expression vector pGL3 basic (Promega). The short variant of the 5-HTT gene promoter (s 5-HTTP, base pairs 1396 to +22) was generated by cleaving the short, deletion-containing 484-bp PCR product with Pst I and ligating it into the Pst I site of the 5-HTTP luc+ construct after the fragments flanked by the Pst I sites at nucleotide positions 1366 and 1192 had been removed. Inserts and insert-vector boundaries were verified by sequence analysis. Long and short human 5-HTT luc+ constructs and controls were transiently expressed in lymphoblasts with different genotypes (13), and luc+ gene expression was studied relative to the pGL3 basic and pGL3 control vectors. Transfection efficiency was assessed by cotransfection with pSV-βGal (Promega). For transient expression, lymphoblasts (2 × 10⁵ cells) were exposed for 24 hours to 5 µg of construct DNA complexed with 5 µl of Transfectam lipofectin reagent (Promega) in 5 ml of RPMI 1640. Cells were grown for an additional 24 hours before harvest in 1 ml of luciferase lysis buffer. Extracts were assayed for luciferase activity by addition of 10 µl of cell lysate samples at 15-s intervals to 100 ml of luciferin reagent. Chemiluminescence was counted for 15 s at a constant time (90 s) after reagent mixing in a liquid scintillation spectrometer

Total RNA was isolated from lymphoblasts (13) by guanidine thiocyanate column purification (Qiagen). The 28S/18S bands of ribosomal RNA were analyzed by densitometry to control for variations in RNA concentration, and single-stranded cDNA (37°C, 60 min) was synthesized with random primer. 5-HTT mRNA was measured by semiquantitative competitive reverse transcription PCR with a 5-HTT cDNA-derived template containing a 172-bp deletion (base pairs 1635 to 1806) as internal standard. The PCR amplification (30 s at 95°C, 30 s at 61°C, 1 min at 72°C for 35 cycles) of 355- or 527-bp fragments was carried out with the amplimers se3 (5′-ATGCAGAAGCGATAGCCAACATG, base pairs 1437 to 1459 with respect to the transcription initiation site) and 3re (5′-AGATGAGGTTCCTATGCAGTAAC, base pairs 2147 to 2167). 5-HTT mRNA concentrations of lymphoblast cell lines with the l/l genotype were first titrated against incremental concentrations of competitive template ranging from 0.01 to 1.0 ng. The concentration of the competitive template at target/template equilibrium was then used to compare mRNA concentrations semiquantitatively in lymphoblast cell lines with different genotypes (13) before and after induction of 5-HTT gene transcription. To control for differences in the efficiency of reverse transcription of mRNA, we performed cDNA synthesis and subsequent competitive PCR in quadruplicate. The reaction products were electrophoresed through 2% agarose, visualized by ultraviolet illumination in the presence of ethidium bromide, and quantified by densitometric analysis

Inhibitor binding to the 5-HTT protein was assayed by incubating membranes from different lymphoblast cell lines (13) with [¹²⁵I]RTI-55 (0.05 to 1 nM) for 1 hour at 37°C as described [Ramamoorthy J. D., et al., J. Biol. Chem. 270, 17189 (1995). Nonspecific binding was determined in the presence of 5 µM paroxetine. RTI-55 [3β-(4-iodophenyl)tropan-2β-carboxylic acid methyl ester tartrate] is a cocaine analog that potently inhibits 5-HT uptake and binds to 5-HTT with high sensitivity (8) [Boja J. W., et al., in Dopamine Receptors and Transporters, Niznik H. B., Ed. (Dekker, New York, 1994), pp. 611–644]. We determined 5-HT uptake by incubating 1 × 107 suspended lymphoblasts with 0.1 to 1 µM [³H]5-HT for 30 min at 25°C in the absence or presence of 0.1 mM imipramine

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We thank M. Schad, G. Ortega, and S. Jatzke for technical assistance, W. Davis and D. Drake for editorial assistance, and M. Altemus, J. Mizrahi, and A. Jaffe for logistical support. Supported by the Deutsche Forschungsgemeinschaft, the Bundesministerium für Bildung und Forschung, the European Commission, and the Intramural Research Programs of NIMH and NCI. K.P.L. is supported by the Hermann and Lilly Schilling Foundation