Molecular Psychiatry (1997) 2, 224–226  1997 Stockton Press All rights reserved 1359–4184/97 $12.00 IMMEDIATE COMMUNICATION a Finnish cohort no relationship was observed between Novelty Seeking11 and the long repeat alleles. (NS) is one of four distinct dimen- No association between sions of that are measured by Cloninger’s tridimensional personality questionnaire (TPQ).12–15 the We found no association between the D4DR repeat gene regulatory region polymorphism and the other three dimensions of per- sonality: (HA), polymorphism and the (RD134), and Persistence (RD2). In order to further examine the role of common genetic polymorphisms Tridimensional Personality in the determination of personality traits this cohort was genotyped for two additional coding region poly- Questionnaire (TPQ) morphisms,10 a glycine to serine substitution in the temperament of harm D3 receptor (D3DR) and a cysteine to serine substitution in the 5-HT2C serotonin receptor (HTR2C). Our results showed that RD134 and RD2 were signifi- avoidance cantly reduced by the presence of the less common 5- 1 1 1 HT polymorphism. There was also a significant RP Ebstein , I Gritsenko , L Nemanov , 2Cser A Frisch2, Y Osher3 and RH Belmaker3 interaction between the two dopamine receptor poly- morphisms and the serotonin polymorphism on 1Research Laboratory, S Herzog Memorial Hospital, PO RD134. 16 Box 35300, Jerusalem 91351; 2Felsenstein Medical Lesch and his colleagues recently reported an Research Center, Sackler School of Medicine, Tel-Aviv association between anxiety-related traits with a University; 3Beersheva Mental Health Center, Faculty of newly-described functional polymorphism in the sero- Health Sciences, Ben-Gurion University of the Negev, PO tonin transporter transcriptional control region (5- Box 4600, Beersheva, Israel HTTLPR). Anxiety-related traits were analyzed by related personality instruments.12–15 Although the sub- jects in this study did not complete the TPQ, the Keywords: serotonin transporter; promoter; polymorphism; authors used weighted regression equations to obtain harm avoidance; TPQ; tridimensional personality question- estimated TPQ scores. The authors predicted that the naire; anxiety; personality 5-HTTLPR genotype would be related to HA which A functional polymorphism in the upstream regulatory incorporates many aspects of anxiety and was corre- region of the serotonin transporter gene has been lated with NEO-PI-R . The 5-HTTLPR was, recently reported to be associated with anxiety-related indeed, found to be associated with estimated scores traits assessed by the NEO-PI-R. Individuals both for HA. In order to replicate the findings of Lesch and hetero- and homozygous for the short form of a highly his colleagues,16 and further elucidate the interactions repetitive regulatory element in this gene have signifi- between common genetic polymorphisms in determin- cantly higher neuroticism scores. We have attempted to ing personality dimensions, we examined the 5- replicate these findings in a normal cohort of 120 indi- HTTLPR polymorphism in our original cohort for an viduals, whom we have previously examined for associ- association to the TPQ personality dimension of HA. ation between personality dimensions and other sero- In the current study, no relationship was observed tonergic and dopaminergic receptor polymorphisms. The Tridimensional Personality Questionnaire (TPQ) between the 5-HTTLPR polymorphism and any of the was used to assess personality dimensions in this four TPQ personality dimensions. cohort. No association was observed in the present The 5-HTTLPR polymorphism was analyzed for 120 study between individuals grouped by the long and normal adult male and female volunteers. The fre- short form of the transporter gene and any of the per- quencies in the population of the long allele (l) and sonality dimensions measured by the TPQ including short alleles (s) were 0.47 and 0.53 respectively and Harm Avoidance, which incorporates many aspects of genotypes were distributed according to Hardy–Wein- anxiety and is correlated with NEO-PI-R Neuroticism. berg equilibrium (l/l = 0.20, l/s = 0.55 and s/s = 0.25). Normal personality traits, as measured by dimen- As shown in Table 1, no significant differences in mean sional scales of personality assessment, are partially TPQ scores for each of the four personality dimensions inherited and between 30–60% of the observed vari- (HA, RD134, RD2 and NS) were observed when the ance can be accounted for by genes.1–4 Only recently, subjects were grouped according to 5-HTTLPR geno- however, have several candidate gene polymorphisms type (one-way Anova; HA by genotype, l/l, s/l, s/s: been connected with particular human temperament F = 0.366, P = 0.694). Nor was any difference observed traits.5–11. We reported an association between a human when the l/l vs l/s and s/s genotypes were compared personality trait, Novelty Seeking, and the long repeat (HA by genotype: F = 0.100, P = 0.752). No significant allele of the D4 dopamine receptor exon III polymor- difference in genotype frequency was observed phism.5 Although our results were confirmed by between the Ashkenazi and non-Ashkenazi cohort another laboratory7 employing a different personality (␹2 = 0.52, P = 0.77, Pearson). Nor was any significant questionnaire in an ethnically distinct population, in difference observed between TPQ scores and 5- No association between 5-HTTLPR polymorphism and HA RP Ebstein et al 225 Table 1 TPQ personality scores in subject groups sorted by Chronic psychiatric disorders that are associated with 5-HTTLPR polymorphism secondary depressions, such as , are also associated with inflated Harm Avoidance scores rela- Genotype Novelty Reward Persistence Harm tive to that observed before illness. State effects are seeking dependence (RD2) avoidance quite large in some individuals depending on the (NS) (RD134) (HA) severity of the mood change. Depressed mood accounts for 10% of the variability in HA, inflating HA scores l/l (n = 23) 16.52 ± 1.11 13.91 ± 0.75 4.91 ± 0.41 12.86 ± 1.22 from 13 to 15, and could mask or skew associations = ± ± ± ± l/s (n 66) 16.25 0.60 13.49 0.40 5.04 0.25 12.15 0.76 with genetic factors such as those described by Lesch = ± ± ± ± s/s (n 32) 15.40 0.90 14.37 0.57 5.40 0.33 13.15 0.81 and his colleagues,16 which account for only a small percentage of the overall variance. ± TPQ results are reported as mean raw scores s.e.m. The spate of recent studies,5–11,16 which were spurred by the reported association between Novelty HTTLPR genotype when the population was grouped Seeking and the D4DR exon III repeat polymorphism,10 into Ashkenazi and non-Ashkenazi cohorts now appear to be subject to the uncertainty associated (Ashkenazi: F = 0.74, P = 0.48 and non-Ashkenazi: with psychiatric genetics in the past decade. These F = 0.27, P = 0.76) or male (F = 0.22, P = 0.80) and issues may be related to the growing consensus that female (F = 0.22, P = 0.80) cohorts. Finally, no signifi- psychiatric diseases are likely both polygenic and gen- cant effect of 5-HTTPLR genotype on TPQ scores was etically heterogeneous18 and it now appears that a simi- observed when second-order factors of the HA dimen- lar complex genetic architecture may underlie normal sion were analyzed by comparing the s/s and s/l vs the personality. l/l genotypes (HA1, worry and pessimism: F = 0.05, P = 0.81; HA2, fear of uncertainty: F = 0.22, P = 0.63; HA3, shyness: F = 0.54, P = 0.46; HA4, fatigability: Materials and methods F = 0.007, P = 0.93). We have now genotyped this cohort for four coding Subjects region genetic polymorphisms (D4DR exon III repeat, Normal volunteers were recruited from students and HTR2Ccys→ser, D3DRgly→ser and 5-HTTLPR) allowing an staff at the Ben-Gurion University Department of analysis by four-way Anova of possible interactions Behavioral Sciences and the Beersheva Mental Health between these genotypes and TPQ personality Center as previously described.5 Briefly, volunteers domains. Although we have previously observed an gave informed consent and the protocol was approved interaction between three genotypes and RD134 and by the Ben-Gurion University Helsinki Committee. 10 RD2 in this cohort, in the present study no main There were 67 males and 54 females and the average effects or interactions were observed when HA was age was 29.7 ± 7.9 (mean ± s.d.) years. The ethnic com- analyzed by four-way Anova which included genotype position was 89 Ashkenazi Jews and 31 non-Ashkenazi information for these four common polymorphisms subjects (Sephardic Jews, Arab, Druze). The volunteers (HA by D3DR, D4DR, 5-HT2c and 5-HTTPLR: filled out a Hebrew version of the TPQ, which consists = = F5-HTTPLR 0.12, P 0.88). of 100 questions with yes-no answers, and donated 16 The study by Lesch and his colleagues demon- 20 cc of blood by venipuncture. strated that the serotonin regulatory region polymor- phism contributes a modest 3–4% of the total variance and 7–9% of the genetic variance to anxiety. Since gen- Genotyping etic factors account for 40–60% of the total variance in DNA was extracted using a Qiamp kit (Qiagen, Hilden, this trait, Lesch et al16 suggested that an additional 10– Germany). PCR amplification was carried out by a lab- 15 genes of similar size effects may be involved in oratory technician blind to subject TPQ score using Pfu determining this trait. Since any single gene in a mul- exo-minus polymerase (Stratagene, La Jolla, CA, USA) tiple gene system is neither necessary nor sufficient to and a high denaturing temperature (98°C). The primers determine the trait, it is not unlikely that the determi- employed were SIL 1 5Ј ggC gTT gCC gCT CTg AAT nation of a complex trait across both individuals and gC 3Ј and SIL 2 5Ј gAg ggA CTg AgC Tgg ACA ACC population groups reflects partially overlapping sets of AC 3Ј. The reaction mixture contained the following shared as well as sets of non-shared genes. Although components: 1 × Pfu buffer (Stratagene), 0.5 ␮M pri- lack of association between the 5-HTTPLR genotype mers, 100 ng DNA, 200 ␮M dATP, dTTP, dCTP, and HA in the present study is disappointing, it is not 100 ␮M dGTP and 100 ␮M deazo-dGTP, 1.2 U Pfu, and an unexpected result of repeated investigations 12% DMSO in a total volume of 15 ␮l. After an initial designed to confirm initial findings across diverse cul- denaturation step of 98°C for 2.5 min, amplification tural and ethnic categories. was carried out for 40 cycles (98°C for 45 s, 55°C for Another possible explanation for our failure to con- 45 s, and 72°C for 90 s) in a Perkin-Elmer Cetus 9600 firm a role of the 5-HTTPLR genotype is the occasional thermal cycler. A 5-min final extension at 72°C was state dependence of this particular trait. Empirical employed. The reaction mixture was electrophoresed studies show that Harm Avoidance tends to covary on a 2% Metaphor gel (FMC, Rockland, ME, USA) with weakly with current and anxiety states.17 ethidium bromide to screen for genotypes. No association between 5-HTTLPR polymorphism and HA RP Ebstein et al 226 Statistical analysis 9 Jonsson E, Nothen MM, Gustavsson JP, Neidt H, Brene S, Associations between TPQ test scores and 5-HTTLPR Tylec A, Propping P, Sedvall G. DRD4 polymorphisms and genotype were assessed by Anova using SPSS for Win- personality traits in a Swedish sample of healthy volun- dows. teers. Psychiatric Gen 1996; 6: 165. 10 Ebstein RP, Segman R, Benjamin J, Osher Y, Nemanov L, Belmaker RH. 5-HT2c (HTR2C) serotonin receptor gene Acknowledgements polymorphism associated with the human personality trait of reward dependence: interaction with dopamine D4 Dr L Nemanov is the recipient of a grant from the Isra- receptor (D4DR) and dopamine D3 (D3DR) polymor- eli Ministry of Absorption (Scientific Absorption). Part phisms. Am J Med Genet (Neuropsychiatric Genetics) of this work was supported by a grant from the Ger- 1997; 74: 65–72. man–Israeli Binational Foundation for Research and 11 Malhotra AK, Virkunnen M, Rooney W, Eggert M, Linno- Development (RPE and BL) and the Israeli Institute ila M, Goldman D. The association between the dopamine for Psychobiology. D4 receptor (D4DR) 16 amino acid repeat polymorphism and Novelty Seeking. Mol Psychiatry 1996; 1: 388–391. 12 Cloninger CR. 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