International Journal of Neuropsychopharmacology (2007), 10, 401–404. Copyright f 2006 CINP doi:10.1017/S1461145706007073
Association between a polymorphism in the promoter region of the TPH2 gene and the personality trait of harm avoidance
B R I EF R E P O R T
CINP
Martin Reuter, Yvonne Kuepper and Juergen Hennig Center of Psychobiology and Behavioral Medicine, Department of Psychology, University of Giessen, Germany
Abstract In a genetic association study the role of the x703 G/T (rs4570625) polymorphism, located in the promoter region of the tryptophan hydroxylase 2 gene (TPH2), in personality traits was investigated in a sample of 404 healthy Caucasian subjects. A significant association between harm avoidance (HA), a trait related to anxiety, and the x703 G/T polymorphism was detected supporting the findings by Gutknecht and colleagues. Received 28 February 2006; Reviewed 3 April 2006; Revised 18 May 2006; Accepted 29 May 2006; First published online 19 December 2006 Key words : Harm avoidance, personality, TPH2 gene.
Introduction The serotonergic (5-HT) system plays an important role in a number of psychopathological states including suicidal behaviour, anxiety disorders and depression and seems also to be related to personality traits of negative emotionality, e.g. anxiety, impulsivity and aggressiveness. Molecular genetics research has produced a number of association studies supporting these findings. Especially genes expressing receptors, transporters and enzymes involved in 5-HT metabolism have been investigated. The tryptophan hydroxylase 1 gene (TPH1) which is the rate-limiting enzyme of 5-HT synthesis is one of those candidate genes that have been reported to be associated with aggression and suicidal behaviour in numerous independent studies (e.g. Hennig et al., 2005; Nielsen et al., 1998). However, these positive findings have been criticized because the TPH1 gene is primarily expressed in the periphery and SNPs of the TPH1 gene that showed positive associations to the phenotypes under investigation were located in intron 7, and no effect on splicing resulting in exon skipping was demonstrated (Shaltiel et al., 2005). Therefore, positive association studies of TPH1 seem to result from Address for correspondence : PD Dr. M. Reuter, Justus-Liebig-University of Giessen, Department of Psychology, Otto-Behaghel-Str. 10F, D-35394 Giessen, Germany. Tel. : ++49-641-9926154 Fax : ++49-641-9926159 E-mail :
[email protected]
linkage disequilibrium of the intronic SNP with other functional as yet unidentified gene variations. Recently, Walther et al. (2003) identified a second TPH isoform – referred to as TPH2 – in mice, that is predominantly expressed in the brain stem, while the classical TPH gene – now labelled TPH1 – is expressed in the gut, pineal gland, spleen and thymus. The authors also identified a human TPH2 ortholog on human chromosome 12p21.1. SNPs in the TPH2 gene have now been detected and first studies showed associations between TPH2 gene variation and depression, ADHD, autism and suicide (Coon et al., 2005; Walitza et al., 2005; Zhou et al., 2005; Zill et al., 2004). Moreover, functional studies underscore the impact of the TPH2 x703 G/T SNP on amygdala responses to emotional stimuli (Canli et al., 2005). The present study is an independent replication study of another study by Gutknecht and colleagues who reported an association between the functional TPH2 x703 G/T SNP and the personality traits of Harm Avoidance (HA) and Persistence in a sample of healthy Caucasian volunteers (Gutknecht et al., 2006). In this first association study on the relationship between gene variants of the TPH2 gene and personality traits there was a significant negative association between the number of T alleles and the scores in the temperaments HA and Persistence. Based on these findings our hypothesis was that the prevalence of the T allele of the x703 G/T allele is associated with lower scores in HA and Persistence.
402
M. Reuter et al.
Method In order to investigate the role of TPH2 gene variation in anxiety and negative emotionality we tested the functional x703 G/T SNP for associations with personality traits measured by Cloninger’s Temperament and Character Inventory (TCI; Cloninger et al., 1993) in a sample of 404 healthy Caucasian subjects of German origin (113 men and 291 women; age : mean= 23.69 yr, S.D.=5.38 yr). All participants were members of the Giessen Gene Brain Behaviour Project (GGBBP) gene data bank and were recruited by e-mail advertisement or by telephone. The members of the gene data bank were free of any psychiatric diagnosis. The personality traits of HA, Novelty Seeking, Persistence and Reward Dependence are the basic temperaments in Cloninger’s biosocial theory of personality (Cloninger et al., 1993). HA is characterized by anticipatory worry, fear of uncertainty, shyness, and fatiguability and Persistence is related to ambition and eagerness. DNA was extracted from buccal cells to avoid a selective exclusion of subjects with blood and injection phobia. Purification of genomic DNA was performed with a standard commercial extraction kit (High Pure PCR Template Preparation kit ; Roche Diagnostics, Mannheim, Germany). Genotyping of the TPH2 x703 G/T polymorphism was performed by real-time PCR using fluorescence melting-curve detection analysis by means of the Light Cycler System (Roche Diagnostics). The primers and hybridization probes used (TIB MOLBIOL, Berlin, Germany) and the PCR protocols were as follows – forward primer : 5k-TCCATATAACTCTGCATAGAGGCA-3k; reverse primer : 5k-GATATCCATTGCCTCAAGCA-3k ; anchor hybridization probe: 5k-LCRed640-CATGCAAATGTGTGAGTGTATATATGTGTAATG-phosphate-3k; sensor [G] hybridization probe : 5k-TCTGACTTGACATATTCTAATTTTG -fluorescein-3k. The PCR run comprised 55 denaturation cycles (95 xC, 0 s, ramp rate 20 xC/s), annealing (61 xC, 10 s, ramp rate 20 xC/s), acquisition of the fluorescence signal (40 xC, 1 s, ramp rate 20 xC/s) and extension (72 xC, 10 s, ramp rate 20 xC/s) which followed an incubation period of 10 min (95 xC) to activate the FastStart Taq DNA Polymerase of the reaction mix (Light Cycler FastStart DNA Master Hybridization Probes ; Roche Diagnostics). After amplification a melting curve was generated by keeping the reaction time at 40 xC for 2 min and then heating slowly to 75 xC with a ramp rate of 0.2 xC/s. The fluorescence signal was plotted against temperature to yield the respective melting points (Tm) of the two alleles. Tm for the T allele was 51.6 xC and for the G allele 58.8 xC.
Table 1. Genotype frequencies dependent on personality scores TT
GT
GG
n
Harm Avoidance Low High
15 (7.5 %) 65 (32.5 %) 120 (60.0 %) 200 4 (2.0 %) 88 (43.1 %) 112 (54.9 %) 204
Persistence Low High
7 (4.1 %) 70 (35.7 %) 119 (60.7 %) 196 12 (5.7 %) 83 (39.9 %) 113 (54.3 %) 208
Novelty Seeking Low High
8 (3.9 %) 83 (41.3 %) 110 (54.7 %) 201 11 (5.4 %) 70 (34.5 %) 122 (60.1) 203
Reward Dependence Low 7 (3.5 %) 84 (42.2 %) 108 (54.3 %) 199 High 12 (5.9 %) 69 (33.6 %) 124 (60.5) 205 Total
19 (4.7 %) 153 (37.9)
232 (57.4 %) 404
Test for Hardy–Weinberg Equilibrium: x2=0.971, d.f.=1, p>0.05. Association between TPH2 x703 G/T and Harm Avoidance: x2=10.06, d.f.=2, p=0.007; Monte-Carlo simulation based on 10 000 samples: p=0.005, 99 % CI=0.004–0.007. Association between TPH2 x703 G/T and Persistence: x2=2.22, d.f.=2, p=0.329. Association between TPH2 x703 G/T and Novelty Seeking: x2=2.19, d.f.=2, p=0.335. Association between TPH2 x703 G/T and Reward Dependence: x2=3.80, d.f.=2, p=0.149.
The study was carried out in accordance with the ethical principals of the Declaration of Helsinki of the World Medical Association (WMA) and was approved by the Ethics Committee of the German Psychologist Association. Data were analysed by x2 tests after gender- and age-adjusted personality scores had been median dichotomized. We controlled the personality scores for the influence of age and gender by entering these two variables as predictors in a regression analysis with personality trait as the dependent variable. Standardized residuals of the regression analysis were the basis of the median dichotomization. This strategy is in agreement with the method applied by Gutknecht et al. (2006). Additional ANCOVA analyses with age and gender as covariates were calculated to demonstrate the stability of the results across different statistical methods. Results Our results show a significant association between the TPH2 x703 G/T SNP and HA (x2=10.06, d.f.=2, p=0.007). The genotype distribution in Table 1
The TPH2 gene and harm avoidance
403
Table 2. Results of the ANCOVA analyses with the covariates sex and age [values are mean (S.D.)]
Harm Avoidance Persistence Novelty Seeking Reward Dependence
TT
GT
GG
F(2, 399) (p)
12.62 (6.93) 5.07 (2.07) 21.46 (5.80) 17.63 (3.36)
16.81 (6.95) 4.57 (2.08) 21.64 (5.81) 16.85 (3.34)
15.93 (6.95) 4.46 (2.07) 22.07 (5.80) 17.26 (3.35)
3.27 (0.039) 0.81 (0.445) 0.31 (0.731) 0.93 (0.394)
indicates that the prevalence of the TT genotype is y4-fold lower in subjects scoring high on HA. Due to the fact that our findings were based on the rare TT genotype (n=19) we merged our data with the those of Gutknecht et al. which resulted in a total number of 28 TT genotypes (in a total sample size of 740 subjects) and a greater significance regarding the association with HA (x2=16.45, d.f.=2, p=0.0003). The three other temperaments of the TCI, Persistence, Novelty Seeking, and Reward Dependence were not related to the x703 G/T polymorphism. In order to control the robustness of the results independently of the statistical methods applied we also analysed the data by means of a parametric approach. The ANCOVA results confirmed the results of the x2 tests (see Table 2). The association between the x703 G/T SNP and HA could be demonstrated. The three other temperaments were not related to the promoter polymorphism of TPH2.
negative emotionality (Canli et al., 2005). Also the two other temperaments, Novelty Seeking and Reward Dependence, of Cloninger’s TCI were not related to the x703 G/T. The association between HA and the x703 G/T that is even significant after Bonferroni correction is caused by the rare TT genotype. To consolidate our replication study we have pooled our data with the original data of Gutknecht et al. This resulted in an increase of carriers of the TT genotype from 19 to 28 and a further increase of the effect. Parametric analyses corroborated the results of the non-parametric x2 tests. HA was the only temperament that was associated with the promoter polymorphism of TPH2. Biochemical and expression profiling studies are required to confirm the functional relevance of TPH2 x703 G/T SNP, i.e. whether the TT genotype is related to an altered rate of 5-HT synthesis. Acknowledgements
Discussion Our findings corroborate the results reported by Gutknecht and colleagues (2006) who found a significant single marker- and haplotype-based association between the functional TPH2 x703 G/T SNP and the personality traits of HA in a sample of healthy volunteers. However, we could not replicate the association between the x703 G/T SNP and the personality trait of Persistence reported by Gutknecht et al. Due to the larger sample size of the present study in comparison to the Gutknecht et al. study, the possibility that the non-replication of the association with Persistence is caused by a lack of power, seems implausible. The Gutknecht et al. sample showed similar effects for Persistence and for HA in a smaller sample than ours. Therefore, the Persistence effect seems to be less robust than the effect reported for HA. Given the fact that many association studies fail to replicate, confirmation of an effect of the x703 G/T SNP on HA by our own independent study emphasizes the role of TPH2 gene variation in anxiety-related personality traits and
We thank Professor Lesch (University of Wu¨rzburg) and Dr Alexander Strobel (University of Dresden) for their stimulating intellectual input and for making the independent control sample available to us. Statement of Interest None. References Canli T, Congdon E, Gutknecht L, Constable RT, Lesch KP (2005). Amygdala responsiveness is modulated by tryptophan hydroxylase-2 gene variation. Journal of Neural Transmission 112, 1479–1485. Cloninger CR, Svrakic DM, Przybeck TR (1993). A psychobiological model of temperament and character. Archives of General Psychiatry 50, 975–990. Coon H, Dunn D, Lainhart J, Miller J, Hamil C, Battaglia A, Tancredi R, Leppert MF, Weiss R, McMahon W (2005). Possible association between autism and variants in the brain-expressed tryptophan hydroxylase gene (TPH2).
404
M. Reuter et al.
American Journal of Medical Genetics, B Neuropsychiatric Genetics 135, 42–46. Gutknecht L, Jacob C, Strobel A, Kriegenbaum C, Mu¨ller J, Zeng Y, Markert C, Escher A, Wendland J, Reif A, et al. (2006). Tryptophan hydroxylase-2 gene variation influences personality traits and disorders related to emotional dysregulation. International Journal of Neuropsychopharmacology. doi:10.1017/S1461145706007437. Hennig J, Reuter M, Netter P, Burk C, Landt O (2005). Two types of aggression are differentially related to serotonergic activity and the A779C TPH polymorphism. Behavioral Neuroscience 119, 16–25. Nielsen DA, Virkkunen M, Lappalainen J, Eggert M, Brown GL, Long JC, Goldman D, Linnoila M (1998). A tryptophan hydroxylase gene marker for suicidality and alcoholism. Archives of General Psychiatry 55, 593–602. Shaltiel G, Shamir A, Agam G, Belmaker RH (2005). Only tryptophan hydroxylase (TPH)-2 is relevant to the CNS. American Journal of Medical Genetics, B Neuropsychiatric Genetics 136, 106.
Walitza S, Renner TJ, Dempfle A, Konrad K, Wewetzer C, Halbach A, Herpertz-Dahlmann B, Remschmidt H, Smidt J, Linder M, et al. (2005). Transmission disequilibrium of polymorphic variants in the tryptophan hydroxylase-2 gene in attention-deficit/hyperactivity disorder. Molecular Psychiatry 10, 1126–1132. Walther DJ, Peter JU, Bashammakh S, Hortnagl H, Voits M, Fink H, Bader M (2003). Synthesis of serotonin by a second tryptophan hydroxylase isoform. Science 299, 76. Zhou Z, Roy A, Lipsky R, Kuchipudi K, Zhu G, Taubman J, Enoch MA, Virkkunen M, Goldman D (2005). Haplotype-based linkage of tryptophan hydroxylase 2 to suicide attempt, major depression, and cerebrospinal fluid 5-hydroxyindoleacetic acid in 4 populations. Archives of General Psychiatry 62, 1109–1118. Zill P, Baghai TC, Zwanzger P, Schule C, Kser D, Rupprecht R, Moller HJ, Bondy B, Ackenheil M (2004). SNP and haplotype analysis of a novel tryptophan hydroxylase isoform (TPH2) gene provide evidence for association with major depression. Molecular Psychiatry 9, 1030–1036.
