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

Gray J. A., The Neuropsychology of Anxiety: An Inquiry into the Functions of the Septo-Hippocampal System (Oxford Science, New York, 1982);

Cloninger C. R., Psychiatr. Dev. 4, 167 (1986);

Eysenck H. J., in Handbook of Abnormal Psychology, , Eysenck H. J., Ed. (Pitman, London, 1957), pp. 131–156

Cloninger C. R., Arch. Gen. Psychiatry 44, 573 (1987).

Cloninger C. R., Psychiatr. Dev. 6, 83 (1988).

Loehlin J. C., Am. Psychol. 44, 1285 (1989);

Heath A. C., Cloninger C. R., Martin N. G., J. Pers. Soc. Psychol. 66, 762 (1994);

Plomin R., Owen M. J., McGuffin P., Science 264, 1733 (1994);

Bergeman C. S., et al., Psychol. Aging 3, 399 (1988);

Pedersen N. L., et al., J. Pers. Soc. Psychol. 55, 950 (1988);

Lander E. S., Schork N. J., Science 265, 2037 (1994).

Chen H. T., Clark M., Goldman D., J. Pharmacol. Toxicol. Methods 27, 209 (1992);

Hensler J. G., et al., Synapse 17, 1 (1994);

Whitaker-Azmitia P., Peroutka S., Ann. N.Y. Acad. Sci. 600, 4 (1990);

Westenberg H. G., Murphy D. L., Den Boer J. A., Eds., Advances in the Neurobiology of Anxiety Disorders (Wiley, New York, 1996).

Lesch K. P., et al., J. Neural Transm. 91, 67 (1993);

Lesch K. P., et al., ibid. 95, 157 (1994).

Gelernter J., Pakstis A. J., Kidd K. K., Hum. Genet. 95, 677 (1995).

Ramamoorthy S., et al., Proc. Natl. Acad. Sci. U.S.A. 90, 2542 (1993).

Lesch K. P., et al., Biol. Psychiatry 37, 215 (1995);

Altemus M., et al., Am. J. Med. Genet. Neuropsychiatr. Genet. 67, 409 (1996);

Di Bella D., et al., Psychiatr. Genet. 5, S100 (1996);

Ogillvie A. D., et al., Lancet 347, 731 (1996);

Collier D., et al., Neuroreport 7, 1675 (1996).

Heils A., et al., J. Neural Transm. 102, 247 (1995);

Heils A., et al., J. Neurochem. 66, 2621 (1996).

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

Iken K., et al., Cell. Immunol. 163, 1 (1995);

Faraj B. A., Olkowski Z. L., Jackson R. T., Int. J. Immunopharmacol. 16, 561 (1994).

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

Benjamin J., et al., Nature Genet. 12, 81 (1996).

Two independent groups of predominantly male siblings, other family members, and unrelated individuals were studied: (i) The NIMH sample (17) was recruited from the NIH and local college campuses by advertising for pairs of brothers and pairs of sisters for a study of personality traits and chromosomes. The sample consisted of 221 subjects, of whom 93% were male and 7% were female. The average age was 23.3 ± 6.8 years (range 18 to 64 years), the average educational level was 15.6 ± 2.1 years (range 12 to 20 years), and the average Kinsey score was 0.2 ± 0.7 (range 0 to 5.6, where 0 is exclusively heterosexual and 6 is exclusively homosexual). The ethnic composition was 79.1% white non-Hispanic, 10.0% Asian/Pacific Islander, 4.1% Hispanic/Latino, 4.1% African American/Black, and 2.7% other. The family structure of the NIMH sample was 208 siblings from 104 families and 13 unrelated individuals. (ii) The NCI sample [Hamer D. H., et al., Science 261, 321 (1993);Hu S., et al., Nature Genet. 11, 248 (1995) was collected from NIH clinics and local and national homophile organizations for a study of sexual orientation, HIV progression, and psychological traits. The sample consisted of 284 subjects of whom 92% were male and 8% were female. The average age was 37.6 + 9.7 years (range 18 to 72 years), the average educational level was 17.3 ± 2.6 years (range 12 to 20 years), and the average Kinsey score was 4.8 ± 2.0 (range 0 to 6). The ethnic composition was 93.6% white non-Hispanic, 5.3% Hispanic/Latino, 0.7% African American/Black, 0.4% Native American/Alaskan, and 0.4% other. The family structure of the NCI sample was 251 siblings from 106 families, 9 parents, and 24 unrelated individuals

McCrae R. R., Costa P. T. J., Personality in Adulthood (Guilford, New York, 1990).

Costa P. T. J., McCrae R. R., in Handbook of Personality Inventories, , Cheek J., Donahue E. M., Eds. (Plenum, New York, in press).

Heninger G. R., Psychopharmacology: The Fourth Generation of Progress, , Bloom F. E., Kupfer D. J., Eds. (Raven, New York, 1995), p. 471;

Murphy D. L., et al., in Serotonin: From Cell Biology to Pharmacology and Therapeutics II (Kluwer Academic, Dordrecht, Netherlands, 1993), pp. 223–230;

Benkert O., Wetzel H., Szegedi A., Int. Clin. Psychopharmacol. 8 (suppl. 1), 3 (1993).

Griebel G., Pharmacol. Ther. 65, 319 (1995).

Handley S. L., ibid. 66, 103 (1995).

A complete table of the S - L differences and F values for all of the NEO Neuroticism facets, 16PF second-order and primary factors, estimated TPQ factors, and TPQ Harm Avoidance subscales is available from the authors by request

Cattell R. B., J. Abnorm. Soc. Psychol. 38, 476 (1943);

Cattell R. B., The Description and Measurement of Personality (World Book, Yonkers, NY, 1946);

Wiggins J. S., Pincus A. L., Annu. Rev. Psychol. 48, 473 (1992).

Population association was assessed by one-way analysis of variance (ANOVA) using SPSS Statistical Software

George V. T., Elston R. C., Genet. Epidemiol. 4, 193 (1987). Across-pedigrees association was assessed using the ASSOC program of the S.A.G.E. package [;

Elston R. C., George V. T., Sorant A. J. M., in S.A.G.E. Users Guide, Release 2.2 (Department of Biometry and Genetics, Louisiana State University Medical Center, New Orleans, 1994);

Nick T. G., et al., Genet. Epidemiol. 12, 145 (1995). Within-pedigrees association was determined by a paired t test using genetically discordant sib-pairs and was conservatively corrected for the nonindependence of siblings from the same family (17).

Den Boer J. A., et al., Int. Clin. Psychopharmacol. 9 (suppl. 4), 47 (1995).

Owens M. J., Nemeroff C. B., Clin. Chem. 40, 288 (1994);

Iny L. J., et al., Biol. Psychiatry 36, 281 (1994);

Faludi G., Tekes K., Tothfalusi L., J. Psychiatry Neurosci. 19, 109 (1994).

Fuxe K., et al., Neuropharmacology 22, 389 (1983);

Beasley C. M., Masica D. N., Potvin J. H., Psychopharmacology 107, 1 (1992);

Blier P., de Montigny C., Trends Pharmacol. Sci. 15, 220 (1994);

Cook E. H., et al., Neuropharmacol. Neurotoxicol. 5, 1745 (1994);

Maj J., Moryl E., J. Neural Transm. 88, 143 (1992).

Shuey D. L., Sadler T. W., Lauder J. M., Teratology 46, 367 (1992).

Paterson A. H., et al., Genetics 127, 181 (1991);

Stuber C. W., et al., ibid. 132, 823 (1992);

Flint J., et al., Science 269, 1432 (1995);

Ebstein R. P., et al., Nature Genet. 12, 78 (1996);

Ohno T., Kawazu S., Tomono S., Metabolism 45, 218 (1996);

Cambien F., et al., Nature 359, 641 (1992);

Arbustini E., et al., Br. Heart J. 74, 584 (1995);

Cambien F., Clin. Genet. 46, 94 (1994);

Gardemann A., et al., Circulation 92, 2796 (1995).

Mulder R. T., Joyce P. R., Cloninger C. R., Compr. Psychiatry 35, 225 (1994);

Svrakic D. M., et al., Arch. Gen. Psychiatry 50, 991 (1993);

Andrews G., et al., J. Affective Disord. 19, 23 (1990);

Kendler K. S., et al., Arch. Gen. Psychiatry 50, 863 (1993);

MacKinnon A., Mitchell P. M., in Handbook of Depression and Anxiety, , Den Boer J., Ad Sitsen J. M., Eds. (Dekker, New York, 1994), pp. 71–119;

Kendler K. S., et al., Arch. Gen. Psychiatry 44, 451 (1987);

Collier D., et al., Mol. Psychiatry, in press

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