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Abstract

Transporter-facilitated uptake of serotonin (5-hydroxytryptamine or 5-HT) has been implicated in anxiety in humans and animal models and is the site of action of widely used uptake-inhibiting antidepressant and antianxiety drugs. Human 5-HT transporter (5-HTT) gene transcription is modulated by a common polymorphism in its upstream regulatory region. The short variant of the polymorphism reduces the transcriptional efficiency of the 5-HTT gene promoter, resulting in decreased 5-HTT expression and 5-HT uptake in lymphoblasts. Association studies in two independent samples totaling 505 individuals revealed that the 5-HTT polymorphism accounts for 3 to 4 percent of total variation and 7 to 9 percent of inherited variance in anxiety-related personality traits in individuals as well as sibships.

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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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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
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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
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34.
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

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Published In

Science
Volume 274 | Issue 5292
29 November 1996

Submission history

Received: 13 June 1996
Accepted: 22 October 1996
Published in print: 29 November 1996

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Authors

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Klaus-Peter Lesch*
K.-P. Lesch, A. Heils, S. Petri, Department of Psychiatry, University of Würzburg, Füchsleinstrasse 15, 97080 Würzburg, Germany.
Dietmar Bengel
D. Bengel, B. D. Greenberg, J. Benjamin, D. L. Murphy, Laboratory of Clinical Science, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892, USA.
Armin Heils
K.-P. Lesch, A. Heils, S. Petri, Department of Psychiatry, University of Würzburg, Füchsleinstrasse 15, 97080 Würzburg, Germany.
Sue Z. Sabol
S. Z. Sabol and D. H. Hamer, Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Benjamin D. Greenberg
D. Bengel, B. D. Greenberg, J. Benjamin, D. L. Murphy, Laboratory of Clinical Science, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892, USA.
Susanne Petri
K.-P. Lesch, A. Heils, S. Petri, Department of Psychiatry, University of Würzburg, Füchsleinstrasse 15, 97080 Würzburg, Germany.
Jonathan Benjamin
D. Bengel, B. D. Greenberg, J. Benjamin, D. L. Murphy, Laboratory of Clinical Science, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892, USA.
Clemens R. Müller
C. R. Müller, Institute of Human Genetics, University of Würzburg, Am Hubland, 97074 Würzburg, Germany.
Dean H. Hamer
S. Z. Sabol and D. H. Hamer, Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Dennis L. Murphy
D. Bengel, B. D. Greenberg, J. Benjamin, D. L. Murphy, Laboratory of Clinical Science, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892, USA.

Notes

* To whom correspondence should be addressed. E-mail: [email protected]

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