Serotonin transporter, tryptophan hydroxylase, and monoamine oxidase A gene polymorphisms in premenstrual dysphoric disorder

American Journal of Obstetrics and Gynecology (2006) 195, 1254–9

www.ajog.org

Serotonin transporter, tryptophan hydroxylase, and monoamine oxidase A gene polymorphisms in premenstrual dysphoric disorder Julia L. Magnay, MSc,a,* Khaled M. K. Ismail, MRCOG,b Gail Chapman, MPhil,b Leanne Cioni, BSc,a Peter W. Jones, PhD,c Shaughn O’Brien, FRCOGb Institute of Science and Technology in Medicine,a Keele University, Staffordshire, UK; Academic Unit of Obstetrics and Gynecology, University Hospital of North Staffordshire,b Trent, UK; Mathematics Department,c Keele University, Staffordshire, UK Received for publication January 25, 2006; revised May 9, 2006; accepted June 29, 2006

KEY WORDS Premenstrual dysphoric disorder Serotonin transporter Tryptophan hydroxylase Monoamine oxidase Polymorphism

Objective: The purpose of this study was to investigate whether common polymorphisms of key genes that control the serotonin (5-hydroxytryptamine) pathway are associated with premenstrual dysphoric disorder. Study design: The study sample comprised 53 women with clinically diagnosed premenstrual dysphoric disorder (age range, 27-46 years; mean age, 37.7 years) and 52 healthy control subjects (age range, 22-48 years; mean age, 36.2 years). Eight polymorphisms that encode the 5-hydroxytryptamine transporter (LPR, VNTR-2, and 30 UTR G/T), tryptophan hydroxylase 1 (TPH1 G-6526A, G-5806T, and A218C), and monoamine oxidase A (monoamine oxidase A promoter VNTR-1 and exon 8 Fnu 4H1) were genotyped. Genotype and allelic frequencies were analyzed by chi-square test and stepwise logistic regression analysis. Results: There was no significant association between any genotype and clinical category and no significant allelic distribution profiles in either the premenstrual dysphoric disorder group or the control group. Conclusion: These findings do not support a major role for common 5-hydroxytryptamine transporter, TPH1, and monoamine oxidase A polymorphisms in contributing to susceptibility to premenstrual dysphoric disorder. Ó 2006 Mosby, Inc. All rights reserved.

Supported by a research grant awarded by the North Staffordshire Medical Institute. * Reprint requests: Julia Lindsey Magnay, MSc, Keele University Medical School, Institute for Science and Technology in Medicine, Thornburrow Drive Hartshill, Stoke-on-Trent, Staffordshire ST4 7QB, UK. E-mail: [email protected] 0002-9378/$ - see front matter Ó 2006 Mosby, Inc. All rights reserved. doi:10.1016/j.ajog.2006.06.087

Premenstrual syndrome (PMS) is characterized by recurrent psychologic and/or somatic symptoms that occur specifically during the luteal phase of the menstrual cycle and resolve during menstruation. Premenstrual dysphoric disorder (PMDD) is the extreme, predominantly psychologic end of the PMS spectrum

Magnay et al and is estimated to occur in 3% to 8% of women with PMS.1 PMDD requires precise diagnostic criteria that were outlined in the Fourth Edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV).2 The precise cause of PMDD is unknown. It is unlikely that an ovarian hormonal imbalance per se is a major factor, because circulating levels have been shown repeatedly to be normal throughout the menstrual cycle. Instead, these normal cyclic hormonal changes appear to trigger PMDD in susceptible individuals. Several lines of evidence suggest that an underlying dysregulation of serotonergic neurotransmission plays a pivotal role in PMDD.3-5 Studies indicate that serotonin (5-hydroxytryptamine) exerts an inhibitory effect on symptoms, such as irritability, affect lability and depression, which are core features of PMDD.6 Ovarian steroids have been found to influence profoundly the activity of the serotonergic system.7 Falling levels of ovarian hormones have been associated with decreased serotonergic activity8; low luteal-phase whole blood and platelet serotonin levels have been found in women with PMS.3,9 In addition, selective serotonin reuptake inhibitors, which specifically block serotonin reuptake into the presynaptic terminal, act as an effective treatment for PMDD.10 Thus, the current predominant theory proposes that women who have PMDD have an underlying dysregulation of the serotonergic system and that the normal hormonal changes that occur during the luteal phase amplify this condition, which precipitates symptoms in susceptible individuals. Several lines of evidence suggest a strong association between PMDD and major depression. Both conditions share several key psychologic symptoms, although those of premenstrual dysphoria are distinct in that they occur exclusively during the luteal phase of the menstrual cycle. Women with PMDD have an increased history of mood disorder,11,12 are more likely to experience postpartum depression, and are at greater risk of experiencing major depressive disorders in later life.13,14 In addition, family and twin studies indicate that there is a significant genetic contribution to the cause of PMDD.15,16 Polymorphisms of genes that regulate the synthesis (tryptophan hydroxylase [TPH1]), membrane reuptake (serotonin [5-hydroxytryptamine] transporter [5-HTT]), and catabolism (monoamine oxidase A [MAOA]) of serotonin have been studied in numerous psychiatric conditions.17-21 Thus, it seemed a logical progression to investigate patients with PMDD. In this study, we genotyped a series of common polymorphisms in the TPH1, 5-HTT, and MAOA genes in 2 populations of regularly ovulating female subjects: a case group with clinically established PMDD and a healthy control group. The allelic and genotype frequencies were compared in both cohorts. Our aim was to determine whether specific polymorphic genotypes of 5-HTT, TPH1, and MAOA (individually or in combination with others) are associated with PMDD.

1255

Material and methods The study was approved by the University Hospital of North Staffordshire Ethics Committee, and informed written consent was obtained from each participant. Women were recruited from a variety of sources: from advertisement on the hospital Intranet system, from general gynecology clinics, and from a specialized PMS clinic. One hundred five European white women between the ages of 18 and 48 years were enlisted and categorized into 2 groups: women with PMDD and control subjects. All of the women reported regular menstrual cycles (28 G 4 days), and none of the women was receiving oral contraceptives, hormone replacement therapy, or psychotropic drugs. Any woman with a known existing or previous psychiatric disorder was excluded. In accordance with DSM-IV criteria, subjects were categorized clinically by prospective symptom rating with the use of the Daily Record of Severity of Problems scale, based on self-assessment reports that spanned 2 consecutive menstrual cycles.22 Day 1 of each cycle was identified by the first day of menses. Symptom ratings of days 6 to12 and the 7 days immediately before the next menstrual period were used to calculate the mean follicular and mean luteal scores, respectively. Women were diagnosed with PMDD if there was a R200% increase in severity of R1 or a R100% increase of R2 PMDD-defining symptoms during the luteal phase compared with the follicular phase in both menstrual cycles. The control group comprised women who reported no significant premenstrual symptoms and who did not meet the aforementioned criteria. Eight polymorphic sites were chosen for genetic analysis. Three sites were located in the 5-HTT gene: LPR (a 44-nucleotide insertion/deletion in the promoter region giving rise to long [L] or short [S] alleles),23 VNTR-2 (comprising 9, 10, or 12 copies of a 17–base pair repeat element in intron 2),24 and 30 UTR G/T (a single nucleotide polymorphism that is located in the 30 untranslated region).19 Three single nucleotide polymorphisms were studied in the TPH1 gene: the promoter polymorphisms G-6526A and G-5806T25 and the A218C marker in intron 7.26 Two polymorphisms were targeted in the MAOA gene: the VNTR-1 promoter comprising 3, 3.5, 4, or 5 copies of a 30–base pair repetitive sequence located 1.2 kilobases upstream of exon 127 and the Fnu 4HI polymorphism in exon 8.28 DNA was extracted from leukocytes in ethylenediaminetetraacetic acid–anticoagulated blood with the use of standard techniques. Genomic regions that encompassed each of the 8 polymorphic sites were amplified in duplicate by polymerase chain reaction (PCR) for 35 cycles. With the exception of the 5-HTT LPR polymorphism, each 25 mL reaction mixture contained approximately 100 ng DNA, 1X Taq DNA polymerase buffer that contained 1.5 mmol/L MgCl2, 12.5 pmol of each

1256 Table I

Magnay et al Oligonucleotide primer sequences and amplification conditions for PCR of 5-HTT, TPH1, and MAOA polymorphisms

Polymorphism

Oligonucleotide primer sequences

PCR cycling parameters

Restriction enzyme

5-HTT LPR

Forward: 50 -GGCGTTGCCGCTCTGAATGC-30 Reverse: 50 -GAGGGACTGAGCTGGACAACCAC-30 Forward: 50 GTCAGTATCACAGGCTGCGAG-30 Reverse: 50 -TGTTCCTAGTCTTACGCCAGT-30 Forward: 50 -CCGCTTGAATGCTGTGTAACACAC-30 Reverse: 50 -GTACCCTTCCAATAATAACCTCC-30 Forward: 50 -ATGGTACTTACTAGCCTGTG-3 Reverse: 50 -CTGTCTCCACAGTTTTGCC-3 Forward: 50 -CTTCGTTATGTGTACAGTCC-3 Reverse: 50 -TAGGACTGCAGTGCTTCTC-3 Forward: 50 -TTCAGATCCCTTCTATACCCCAGA-3 Reverse: 50 -GGACATGACCTAAGAGTTCATGGC-3 Forward: 50 -ACAGCCTGACCGTGGAGAAG-30 Reverse: 50 -GAACGGACGCTCCATTCGGA-30 Forward: 50 -GATCCCTCCGACCTTGACT-30 Reverse: 50 -CTTCTTCTTCCAGAAGGCC-30

Touchdown PCR

d

58 C (30 sec), 72 C (30 sec), 94 C (30 sec)

d

5-HTT VNTR-2 5-HTT 30 UTR G/T TPH1 G-6526A TPH1 G-5806T TPH1 A218C MAOA VNTR-1 MAOA Fnu 4H1

56 C (30 sec), 72 C (45 sec), 94 C (30 sec) 56 C (45 sec), 72 C (45 sec), 94 C (45 sec) 58 C (30 sec), 72 C (30 sec), 94 C (30 sec) 60 C (30 sec), 72 C (30 sec), 94 C (30 sec)

Mse I (T) Mae III (G) Mbo 1 (A) Eco 471 (G) Msl 1 (T)

62 C (45 sec), 72 C (45 sec), 94 C (45 sec)

Nhe 1 (C) HpH 1 (A) d

55 C (30 sec), 72 C (30 sec), 94 C (30 sec)

Fnu 4H1 (G)

Restriction endonucleases for those polymorphisms that require restriction digests are shown. The cleaved variant is identified in parentheses.

primer, 5 nmol of deoxyribonucleoside triphosphate, and 0.5 units Taq DNA polymerase. Oligonucleotide primer sequences and PCR cycling parameters are listed in Table I. The 5-HTT LPR polymorphism was amplified with the use of the Qiagen HotStar PCR kit. Each 25 mL reaction mixture contained approximately 100 ng DNA, 1X Qiagen buffer, 1X Q-solution, 12.5 pmol of each primer, 5 nmol of deoxyribonucleoside triphosphate, and 0.5 units HotStar Taq DNA polymerase. PCR consisted of a touchdown protocol with an initial denaturation of 15 minutes at 95 C, followed sequentially by 1 cycle of annealing at 66 C (30 seconds), 64 C (30 seconds), and 62 C (30 seconds), then 32 cycles of 61 C (30 seconds), each with extension at 72 C (30 seconds), and denaturation at 94 C (30 seconds). 5-HTT LPR and VNTR-2 alleles were resolved on 2.5% agarose gels that contained 0.5 mg/mL ethidium bromide, and the bands were viewed under ultraviolet light. MAOA VNTR-1 alleles were identified on 8% polyacrylamide gels that were stained with silver nitrate. The remaining polymorphisms required digestion of PCR products with restriction endonucleases to identify the genotype. All restriction digests were incubated in duplicate for 2 hours in a total reaction volume of 20 mL that contained 3 to 5 mL of PCR product, 1X reaction buffer, and 1 or 2 units of the respective restriction endonuclease that is shown in Table I. Wherever possible, pairs of complementary enzymes were used in separate digest reactions, each of which targeted a different allele. Digest products were electrophoresed on 2% or 2.5% agarose gels. All genotypes were determined independently by 2 researchers, without prior knowledge of clinical group status. Chi-square tests were performed to determine conformation to the Hardy-Weinberg equilibrium and any association between each genotype/allelic frequency and

clinical category. Exact probability tests were used to compare genotype and allelic distribution between the PMDD and control group with the use of Stat Xact-4 software (Cytel Corp, Cambridge, MA). Clinical significance was considered at exact probability values of !.05. In addition, all possible combinations of TPH1, 5-HTT, and MAOA genotypes were assessed with a stepwise logistic regression analysis to determine whether any specific polymorphic profiles predicted clinical category.

Results One hundred five European white women were categorized into 2 groups: women with PMDD (n = 53; age range, 27-46 years; mean, 37.7 years) and control subjects (n = 52; age range, 22-48 years; mean, 36.2 years). For each marker, the genotype distribution and allelic frequencies in the PMDD and control groups are shown in Tables II and III. All genotype distributions conformed to the Hardy-Weinberg equilibrium, except for MAOA Fnu 4H1 in the PMDD cohort (chi-square test, 5.5981; degree of freedom, 1). This deviation was attributed to the complete absence of the G/G genotype in women who were diagnosed with premenstrual dysphoria. Chi-square test and stepwise logistic regression analysis of all genotypes showed no significant association between any genotype and clinical category, and there were no significant allelic distribution profiles in either group.

Comment To our knowledge, this study represents the first genotypic analysis of any TPH1 and MAOA polymorphisms in premenstrual dysphoria. However, a PMDD case-control

Magnay et al

1257

Table II Distribution of genotypes, chi-square test, and exact probability values for polymorphisms in the 5-HTT, TPH1, and MAOA genes in control (n = 52) and PMDD (n = 53) subjects

Genotype 5-HTT LPR L/L L/S S/S 5-HTT VNTR-2 9/10 10/10 10/12 12/12 5-HTT 30 UTR G/G G/T T/T TPH1 A-6526G G/G G/A A/A TPH1 G-5806T T/T T/G G/G TPH1 A218C A/A A/C C/C MAO-A VNTR-1 3/3 3/4 3.5/3.5 3.5/4 4/4 5/4 MAOA Fnu 4HI G/G T/G T/T

Control subjects (n)

PMDD subjects (n)

2.66

2

.29

1.81

3

.67

0 1 (2%) 8 (15%) 11 (21%) 17 (33%) 18 (34%) 27 (52%) 23 (43%) 2.89

2

.27

14 (27%) 11 (21%) 27 (52%) 23 (43%) 11 (21%) 19 (36%) 0.043

2

.98

9 (17%) 10 (19%) 22 (42%) 22 (42%) 21 (41%) 21 (39%) 0.09

2

.96

9 (18%) 10 (19%) 22 (42%) 21 (40%) 21 (40%) 22 (41%) 0.12

2

.94

5.00

5

.43

12 (23%) 11 (21%) 22 (42%) 24 (45%) 18 (35%) 18 (34%) (11%) 5 (29%) 23 1 (2%) 1 (58%) 22 1

Allelic frequency (n)

ChiDegrees Exact square of P test freedom value

18 (35%) 21 (40%) 26 (50%) 29 (55%) 8 (15%) 3 (5%)

6 15 0 1 30 0

Table III Distribution of alleles, chi-square test, and exact probability values for polymorphisms in the 5-HTT, TPH1, and MAOA genes in control subjects (n = 52) and PMDD subjects (n = 53)

(10%) (43%) (2%) (2%) (41%) (2%) 5.30

2

.055

3 (6%) 0 (0%) 17 (33%) 26 (49%) 32 (62%) 27 (51%)

comparison of the 3 5-HTT markers that were studied here was reported by Melke et al,29 who also found no significant association between 5-HTT genotype and PMDD. There may be several explanations for our negative findings. First, clinical categorization of patients with PMDD can present a formidable challenge because of the subjective nature of symptom interpretation. However, we used a robust established protocol, which was based on standard DSM-IV criteria, to characterize subjects.22 Second, the possibility of population stratification should be considered. In studies that comprise subjects

Allele 5-HTTLPR L S 5-HTT VNTR-2 9 10 12 5-HTT 30 UTR G T TPH1 A-6526G G A TPH1 G-5806T T G TPH1 A218C A C MAOA VNTR-1 3 3.5 4 5 MAOA Fnu 4H1 G T

Control subjects

PMDD subjects

ChiDegrees Exact square of P test freedom value 1.23

1

.32

2.21

2

.28

2.29

1

.17

0.03

1

.89

0.001

1

1

0.015

1

1

2.92

3

.36

0.17

1

.75

62 (60%) 71 (67%) 42 (40%) 35 (33%) 0 1 (1%) 33 (32%) 41 (39%) 71 (68%) 64 (60%) 55 (53%) 45 (42%) 49 (47%) 61 (58%) 40 (38%) 42 (40%) 64 (62%) 64 (60%) 40 (38%) 41 (39%) 64 (62%) 65 (61%) 46 (44%) 46 (43%) 58 (56%) 60 (57%) 27 (26%) 33 (31%) 1 (1%) 3 (3%) 76 (73%) 69 (65%) 0 1 (1%) 23 (22%) 26 (25%) 81 (78%) 80 (75%)

who were taken primarily from a local community, it is important to include healthy control subjects to gauge typical genotype and allelic frequencies, although this may yield results that are not representative of the wider population. Furthermore, significant genetic variations may be encountered if different ethnic groups are included. We attempted to minimize this latter variable by using ethnically matched European white patients and control subjects who were drawn from the local female population who lived in Stoke-on-Trent, UK. The allelic frequencies in our control group concurred with frequencies in other European white studies17-20,25-27,30,31 for all polymorphisms except 5-HTT VNTR-2; the 10- and 12-repeat variants (32% and 68%, respectively) differed considerably from other reports (typically, 42%-49% and 51%-55%).18,19,32 Hence, these findings may represent a local genotypic anomaly for the VNTR-2 marker. In addition, although the frequency of MAOA Fnu 4H1 G and T alleles was similar in both cases and control subjects, we did not detect any G/G genotypes

1258 in our PMDD population. The frequency of this genotype in white female control subjects has been reported to be low (7%),30 which concurs with our control group (6%). The fact that no G/G genotypes were detected in the PMDD cohort may either reflect this low population incidence or indicate an important finding. It certainly merits further investigation in a larger group of subjects. Third, the lack of association between genotype and PMDD may be due to sample size. The detection of a small single genetic effect may require an analysis of large numbers of patients. However, it is probably unrealistic to expect a single polymorphism to be the sole factor that is responsible for triggering premenstrual dysphoria. A more likely scenario might be combined genotypes from functionally related genes acting in a synergistic, additive, or antagonistic manner that confers an overall risk for PMDD. We adopted a polygenic approach and focused on key polymorphisms in genes that are involved with serotonin production, catabolism, and reuptake. We were unable to identify either a single genetic marker or a combined polymorphic profile for susceptibility to PMDD. Cautious interpretation of the present study is warranted, both by the preliminary nature of these findings and by their basis in simple association analysis. Nevertheless, within the limits that are imposed by the sample size, the polymorphisms that were studied here do not represent major risk factors for PMDD.

Magnay et al

8.

9.

10.

11. 12.

13.

14. 15.

16.

17.

18.

Acknowledgment 19.

We thank Dr Paul Hoban for his expert advice and for proofreading of the manuscript.

References 1. Angst J, Sellaro R, Merikangas KR, Endicott J. The epidemiology of perimenstrual psychological symptoms. Acta Psychiatr Scand 2001;104:110-6. 2. American Psychiatric Association. Diagnostic and statistical manual of mental disorders. 4th ed. Washington (DC): American Psychiatric Association; 1994. 3. Ashby CR Jr, Carr LA, Cook CL, Steptoe MM, Franks DD. Alteration of platelet serotonergic mechanisms and monoamine oxidase activity in premenstrual syndrome. Biol Psychiatry 1988; 24:225-33. 4. Halbreich U, Tworek H. Altered serotonergic activity in women with dysphoric premenstrual syndrome. Int J Psychiatry Med 1993;23:1-27. 5. Parry BL. The role of central serotonergic dysfunction in the aetiology of premenstrual dysphoric disorder: therapeutic implications. CNS Drugs 2001;15:277-85. 6. Ho H, Olsson M, Westberg L, Melke J, Erikkson E. The serotonin reuptake inhibitor fluoxetine reduces sex steroid-related aggression in female rats: an animal model of premenstrual irritability? Neuropsychopharmacology 2001;24:502-10. 7. Erikkson E, Alling C, Andersch B, Andersson K, Berggren U. Cerebrospinal fluid levels of monoamine metabolites: a preliminary study of their relationship to menstrual cycle phase, sex steroids

20.

21.

22.

23.

24.

25.

26.

and pituitary hormones in healthy women and in women with PMS. Neuropsychopharmacology 1994;11:210-3. Fitzgerald M, Malone KM, Harrison WM, McBride PA, Endicott J, Cooper T, et al. Blunted serotonin response to fenfluramine challenge in premenstrual dysphoric disorder. Am J Psychiatry 1997;154:556-8. Steege JF, Stout AL, Knight DL, Nemeroff CB. Reduced platelet tritium-labeled imipramine binding sites in women with premenstrual syndrome. Am J Gynecol 1992;167:168-72. Dimmock PW, Wyatt KM, Jones PW, O’Brien PM. Efficacy of selective serotonin-reuptake inhibitors in premenstrual syndrome: a systematic review. Lancet 2000;356:1131-6. Halbreich U, Endicott J. The relationship of dysphoric premenstrual changes to depressive disorders. Acta Psychiatr Scand 1985;71:331-8. Critchlow DG, Bond AJ, Wingrove J. Mood disorder and personality assessment in premenstrual dysphoric disorder. J Clin Psychiatry 2001;62:688-93. Pearlstein TB, Frank E, Rivera-Tovar A, Thoft JS, Jacobs E, Miecqowski TA. Prevalence of axis I and axis II disorders in women with late luteal phase dysphoric disorder. J Affect Disord 1990;20:129-34. Graze KK, Nee J, Endicott J. Premenstrual tension predicts future major depressive disorder. Am J Psychiatry 1990;81:210-2. Kendler KS, Silberg MC, Neale RC, Kessler, Heath AC, Eaves LJ. Genetic and environmental factors in the aetiology of menstrual, premenstrual and neurotic symptoms: a population based twin study. Psychol Med 1992;22:85-100. Wilson CA, Turner CW, Keye WR Jr. Firstborn adolescent daughters and mothers with and without premenstrual syndrome: a comparison. J Adolesc Health 1991;12:130-7. Melke J, Landen M, Baghei F, Rosmond R, Holm G, Bjorntorp P, et al. Serotonin transporter gene polymorphisms are associated with anxiety-related personality traits in women. Am J Med Genet 2001;105:458-63. Coyle N, Jones I, Robertson E, Lendon C, Craddock N. Variation at the serotonin transporter gene influences susceptibility to bipolar affective puerperal psychosis. Lancet 2000;356:490-1. Battersby S, Ogilvie AD, Blackwood DHR, Sehn S, Muqit MMK, Muir WJ, et al. Presence of multiple functional polyadenylation signals and a single nucleotide polymorphism in the 30 untranslated region of the human serotonin transporter gene. J Neurochem 1999;72:1384-8. Turecki G, Zhu Z, Tzenova J, Lesage A, Seguin M, Tousignant M, et al. TPH and suicidal behavior: a study in suicide completers. Mol Psychiatry 2001;6:98-102. Rubinsztein DC, Leggo J, Goodburn S, Walsh C, Jain S, Paykel ES. Genetic association between monoamine oxidase A microsatellite and RFLP alleles in bipolar affective disorder: analysis and meta-analysis. Hum Mol Genet 1996;5:779-882. Endicott J, Harrison W. Daily record of severity of problems form. New York: Department of Research Assessment and Training, New York State Psychiatric Institute; 1990. Heils A, Teufel A, Petri S, Stober G, Reiderer P, Bengel D, et al. Allelic variation of the human serotonin transporter gene expression. J Neurochem 1996;66:1-4. Lesch KP, Balling U, Gross J, Strauss K, Wolozin BL, Murphy DL, et al. Organization of the human serotonin transporter gene. J Neural Transm 1994;95:157-62. Rotondo A, Mazzanti CM, Nielson DA, Schuebel KE, Lezza A, Gemignani A, et al. Lack of association of promoter region variation in tryptophan hydroxylase and serotonin transporter with obsessive compulsive disorder and eating disorders [Abstract]. Neuropsychiatric Genet 1997;21:621. Nielsen DA, Jenkins GL, Stefanisko KM, Jefferson KK, Goldman D. Sequence, splice site and population frequency distribution analyses of the polymorphic human tryptophan hydroxylase intron 7. Mol Brain Res 1997;45:145-58.

Magnay et al 27. Sabol SZ, Hu S, Hamer D. A functional polymorphism in the monoamine oxidase A gene promoter. Hum Genet 1998;103:273-9. 28. Hotamisligil GS, Breakefield XO. Human monoamine oxidase A gene expression determines level of enzyme activity. Am J Hum Genet 1991;49:383-92. 29. Melke J, Westberg L, Landen M, Sundblad C, Eriksson O, Baghei F, et al. Serotonin transporter gene polymorphisms and platelet [3H] paroxetine binding in premenstrual dysphoria. Psychoneuroendocrinol 2003;28:446-58.

1259 30. Marshall SE, Bird TG, Hart K, Welsh KI. Unified approach to the analysis of genetic variation in serotonergic pathways. Am J Med Genet (Neuropsychiatr Genet) 1999;88:621-7. 31. Rees M, Norton N, Jones I, McCandless F, Scourfield J, Holmans P, et al. Association studies of bipolar disorder at the human serotonin transporter gene (hSERT; 5HTT). Mol Psychiatry 1997;2:398-402. 32. Collier DA, Arranz MJ, Sham P, Battersby S, Vallada H, Gill P, et al. The serotonin transporter is a potential susceptibility factor for bipolar affective disorder. Neuroreport 1996;7:1675-9.