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Molecular Psychiatry (2003) 8, 118–122 & 2003 Nature Publishing Group All rights reserved 1359-4184/03 $25.00 www.nature.com/mp

ORIGINAL RESEARCH ARTICLE

Association between a dinucleotide repeat polymorphism of the estrogen receptor alpha gene and personality traits in women L Westberg1, J Melke1, M Lande´n2, S Nilsson3, F Baghaei4, R Rosmond4, M Jansson1, G Holm4, P Bjo¨rntorp4 and E Eriksson1 1

Department of Pharmacology, Go¨teborg University, Go¨teborg, Sweden; 2Institute of Clinical Neuroscience, Section of Psychiatry, Go¨teborg University, Go¨teborg, Sweden; 3Department of Mathematical Statistics, Chalmers University of Technology, and Department of Clinical Genetics, Go¨teborg University, Go¨teborg, Sweden; 4Institute of Heart and Lung Disease, Go¨teborg University, Go¨teborg, Sweden

Keywords: steroid hormone receptor; irritability; aggression; anxiety; neuroticism; Karolinska Scales of Personality

Estrogens are known to play a key role in the regulation of various aspects of behavior. In order to study the potential contribution of genetic variation in the estrogen receptor (ER) alpha to specific personality traits, we investigated a repeat polymorphism in the ER alpha gene in 172 42-year-old women who had been assessed using the Karolinska Scales of Personality (KSP). Based on the hypothesis that there is a relationship between the length of a repeat polymorphism and gene function,1 the alleles were divided into two groups: short and long. In order to elucidate the possible influence of the ER alpha gene on the different aspects of personality measured by means of the KSP, the possible association between this gene and four different factors (‘neuroticism’, ‘psychoticism’, ‘non-conformity’, and ‘extraversion’) was analysed. ‘Neuroticism’, ‘psychoticism’, and ‘non-conformity’ all appeared to be associated with the ER alpha gene. After correction for multiple comparisons by means of permutation analysis, the associations with the factor ‘non-conformity’— including the subscales ‘indirect aggression’ and ‘irritability’—and the factor ‘psychoticism’—including the subscale ‘suspicion’—remained significant. The results suggest that the studied dinucleotide repeat polymorphism of the ER alpha gene may contribute to specific components of personality. Molecular Psychiatry (2003) 8, 118–122. doi:10.1038/ sj.mp.4001192

Many studies have shown that inter-individual variations in measures of personality are to a great extent, heritable.2 Linkage between anxiety traits and several loci on different chromosomes has been suggested,3 and association studies have provided support for the involvement of a number of different candidate genes in this context.4 Transcription factors are one particular group of genes that have recently been attributed importance for behavior and personality.5 In this study, the possible association between one well-established ligand-activated transcription

factorFthe estrogen receptor (ER) alphaFand personality traits is the subject of investigation. Estrogen is known to influence neonatal brain development as well as neurogenesis and transmitter function in the adult organism.6 The influence of estrogen on various aspects of mental functionFincluding cognition, mood, libido, and aggressionFis also well established.7–10 The actions of estrogen are mediated by ERs of two subtypesFalpha and betaFthat are both widely distributed throughout the human brain. The expression of the ER alpha subtype appears to dominate in the hypothalamus and amygdala, suggesting that this subtype is an important mediator of effects of estrogen on emotional and reproductive behavior.11 This notion also gains support from recent studies showing ER alpha knock-out mice to differ from wild-type mice, not only with respect to aggression and sexual activity, but also with respect to learning and memory.12–14 The ER alpha gene is located on chromosome 6q25.1. Its promoter region contains a polymorphic TA repeat that has been associated with coronary heart disease15 in men, and bone mineral density16 and endometriosis17 in women. As yet, studies on a putative association between this ER alpha polymorphism and behavior have been sparse. Comings et al,18 however, have reported an association between the length of this repeat sequence and anxiety in men; moreover, in two subsequent studies, using multivariate analysis of a large number of genes, the same research group reported an association between the ER alpha polymorphism and personality traits (measured by means of the temperament and character inventory)19 as well as with conduct disorder.20 Support for the influence of the ER alpha gene on anxiety-related personality traits was gained also by a recent longitudinal study of children and adolescents.21 To further examine the possible importance of the ER alpha repeat polymorphism for behavior, we studied 172 women recruited from the normal

Estrogen receptor alpha gene and personality traits L Westberg et al

Figure 1 Allele frequency of the dinucleotide repeat polymorphism of the ER alpha gene in 172 women.

population and assessed with the Karolinska Scales of Personality (KSP). In all, 17 different alleles of the ER alpha gene were identified, comprising 183–215 bp (Figure 1). The allele distribution was similar to that reported by others. The cutoff limit applied to achieve three groups of similar size was 191 (r191 ¼ short ¼ S; 4191 ¼ long ¼ L); the same cutoff was also used in previous studies by Comings et al.18 A principal component analysis comprising all KSP subscales revealed that 40% of the variation in the different subscales in our cohort of 42-year-old women could be attributed to the first principal component. The influence of the studied gene on the personality traits assessed by KSP gains support from the finding that subjects displaying the SS genotype differed from the rest of the subjects (SS+SL) (dominant model) with respect to the first principal component (P-value corrected for multiple comparisons: P ¼ 0.008). When the four different KSP factors (see Materials and methods) were separately analysed, comparison of the different genotypes using the dominant model (SS vs SL/LL) revealed no association between genotype and ‘extraversion’ (comprising the subscales ‘impulsiveness’ and ‘monotony avoidance’). In contrast, all other three factorsF‘neuroticism’, ‘psychoticism’, and ‘non-conformity’Fappeared to be associated with the studied ER alpha polymorphism (Table 1). After the P-values had been corrected for multiple comparisons using a step-down permutation procedure22,23 (see Materials and methods), the associations between genotype and ‘non-conformity’ and ‘psychoticism’, respectively, remained significant. A subsequent analysis of the four subscales

included in the ‘non-conformity’ factorF‘social desirability’, ‘indirect aggression’, ‘verbal aggression’, and ‘irritability’Frevealed ‘indirect aggression’ and ‘irritability’ to be significantly associated with genotype. Within the ‘psychoticism’ factor, the ‘suspicion’ subscale remained significant after correction for multiple comparisons by means of the step-down permutation procedure. Had P-values instead been adjusted for multiple comparisons using a crude Bonferroni correction, one factorF‘non-conformity’ Fand one subscale within this factorF‘irritability’F would have remained significant (Table 1). When the recessive model was applied (LL vs SL/ SS), the groups did not differ significantly with respect to the first principal component or with respect to the four different factors. Our data therefore support the notion that long alleles are dominant with respect to the aspects of the phenotype examined in this study. Both sex and age may influence the outcome of questionnaire-based personality assessment. A strength of this study is that all subjects were not only of the same sex, women, but also born in the same year. The apparent association between the ER alpha gene and behavioral traits associated with aggression and irritability is intriguing, given the well-established influence of estrogen, both neonatally and in the adult animal, on aggressive behavior in adult rats and mice.24 In humans, the administration of estrogen has been reported to both increase and decrease aggression, depending on the age, sex, and underlying condition of the subjects studied.25–27 The observed association between short alleles and high anxiety scores is opposite to what has previously been reported by Comings in men.18 The notion that the ER alpha may influence the same kind of behavior in women and in men, but in the opposite direction, gains some support from recent animal studies, suggesting that male ER alpha knock-out mice display less aggressive behavior,13 and female ER alpha knock-out mice display more aggressive behavior,12 than wild-type mice. The analysis of data, dividing the subjects into those with long and short microsatellites, respectively, is based on the assumption that there may be a relationship between the length of a repeat polymorphism in regulating sequences of a gene and promoter function.1 That this indeed may be a common feature of many repeat polymorphisms gains support from recent studies.28 Direct support for such a role of the studied polymorphism in the ER alpha promoter is as yet not available. The notion that it is of functional importance, however, does gain support from several previous reports suggesting this polymorphism to be associated with various aspects of phenotype, including bone mineral density and endometriosis (see above). The ER alpha gene is transcribed from several alternative promoters that appear to be regulated in a

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Table 1 The KSP scoresa in a normal population of women displaying LL, SL, or SS alleles of the TA-repeat polymorphism of the ER alpha gene KSP

LL (n=47)

SL (n=70)

SS (n=55)

Pb

Pc

Pd

Pe

Factor: neuroticism Somatic anxiety Psychic anxiety Muscular tension Psychasthenia Inhibition of aggression Guilt Socialization

46.977.4 46.279.5 43.5710.5 47.5711.4 46.4713.1 45.879.0 47.475.7 48.5711.1

48.277.6 46.7710.2 44.6710.5 50.778.9 46.9712.2 48.179.1 48.778.6 48.1710.8

50.076.9 50.1710.2 46.8710.3 51.379.5 48.1710.4 48.977.7 48.477.6 43.5712.4

0.089 0.25 0.24 0.037 0.60 0.070 0.36 0.22

0.053 0.028 0.12 0.25 0.47 0.22 0.90 0.010

0.42

0.17

Factor: psychoticism Detachment Suspicion

46.778.8 43.3710.2 50.0710.5

46.878.8 44.2711.0 49.4710.3

50.8710.3 47.6710.5 54.1712.1

0.25 0.21 0.46

0.0079 0.033 0.014

0.063

0.043 0.062 0.049

Factor: non-conformity Social desirability Indirect aggression Verbal aggression Irritability

49.476.3 46.877.7 51.379.8 47.6710.0 45.577.3

49.476.8 48.779.6 53.5711.5 49.078.9 43.879.1

53.477.0 45.276.6 57.6711.2 51.779.1 49.678.9

0.14 0.78 0.035 0.10 0.59

0.00034 0.047 0.0066 0.029 0.00040

0.0027 0.38 0.053 0.23 0.0033

0.0024 0.11 0.036 0.11 0.0030

Factor: extraversion Impulsiveness Monotony avoidance

51.079.3 48.9710.9 53.1711.2

52.477.8 51.179.0 53.779.1

51.576.4 50.378.1 52.778.7

0.42 0.23 0.89

0.81 0.94 0.64

1

0.71

a The personality test scores have been standardized using normative data to have a mean7SD of 50710 (=T-scores). Pb=significance levels of unpaired comparisons of recessive model; LL vs SS+SL (t-test). Pc=significance levels of unpaired comparisons of dominant model; SS vs LL+SL (t-test). Pd=Bonferroni corrected significance levels of comparisons of dominant model. Pe=corrected significance levels of comparisons of both dominant and recessive models with a step-down permutation procedure using 100 000 permutations.

cell- and tissue-specific manner. At least two of these promoters, A and B, seem to be important for the expression of the ER alpha gene in the brain; notably, the two mRNA isoforms derived from these two promoters seem to exhibit different patterns of expression in different brain regions.11 Since the TA-repeat polymorphism investigated in this study is located between the A and B promoters, it is tempting to speculate that the length of this polymorphism may influence promoter usage and hence receptor expression in the brain in a region-specific manner. Of interest in this context is also the finding that a sequence shown to act as a strong enhancer element in the regulation of ER alpha expression is located in the vicinity of the studied repeat polymorphism.16 To conclude, our results provide support for the notion that the ER alpha gene may influence various aspects of personality in women. Such an influence may be due either to an involvement of this receptor in neonatal brain development, or to an acute influence of this receptor on the activity of brain circuits involved in the regulation of behavior. A detailed discussion of how the studied polymorphism may influence brain function must await further clarification of the relationship between repeat length and receptor activity. Molecular Psychiatry

Materials and methods Subjects All women born on uneven days in the year of 1956 and living in Go¨teborg, Sweden, constituted the primary cohort (n ¼ 1137) (for details, see Rosmond and Bjo¨rntorp29). Of these subjects, 80% responded to questionnaires and reported self-measurements of body weight, height, and circumferences over the waist and hips. The waist/hip circumference ratio (WHR) was then used for a selection of 450 women in total with low, median, or high WHRs, respectively. Of these women, 270 (60%) volunteered to provide blood samples for hormone analyses and genotyping. In the population studied, WHRs appeared normally distributed. Women with one or two parents probably or certainly being non-Caucasian (n ¼ 17) were excluded from further analyses. Also excluded were subjects that had not responded to all questions in the inventory, and for which data regarding all personality traits were hence not available. In total, the present study comprised 172 women who had responded to all questions within the KSP inventory and had been genotyped with respect to the ER alpha polymorphism. The study protocol was approved by the ethical committee of Go¨teborg University.


Personality assessment The participants completed the KSP,30 which has been widely used in previous studies examining biological correlates of personality traits.5,30,31 The KSP consists of 135 items forming 15 subscales and measuring relatively stable personality traits. These subscales are often classified into four factors covering different dimensions of temperament: ‘neuroticism’, ‘psychoticism’, non-conformity’, and ‘extraversion’.31–33 Inter-individual variations in several of the KSP scales have been shown to be partly heritable.2 All KSP personality test scores were standardized using normative data to have an expected mean7SD of 50710 ( ¼ T-scores). Genotyping Venous blood was collected from each subject, and genomic DNA was isolated using the QIAamp DNA Blood Mini Kit (Qiagen). The TA repeat of the ER alpha gene was amplified by polymerase chain reaction (PCR) in a total volume of 15 ml containing 50 ng DNA, 1.5 mM MgCl2, 1 U HotstarTaq polymerase (Qiagen), and 0.3 mM each of the primers described by Comings et al.18 The forward primer 50 -AGA CGC ATG ATATAC TTC ACC-30 was labeled with 6-FAM and used together with the reverse primer 50 GTT CAC TTG GGC TAG GATAT-30 . The temperature profile was 951C for 15 min followed by 35 cycles of 951C for 30 s, 601C for 30 s, 721C for 30 s, and with a final incubation at 721C in 7 min. The fluorescently labeled DNA fragments were analysed by size with automated capillary electrophoresis using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). Data analysis Prompted by the tentative assumption that there is a relationship between the length of microsatellites and gene function,1 it was decided before any results were available that the alleles should be divided into two groups of approximately equal size with the median as cutoff. Alleles containing r191 bp were defined as ‘short’ (S), whereas those with 4191 bp were defined as ‘long’ (L). This resulted in three equally sized subgroups, comprising subjects with two short alleles (SS), subjects with two long alleles (LL), and subjects with one short and one long allele (SL). No preassumption was made regarding which of the variants are recessive and dominant, respectively; hence, all analyses were undertaken in accordance both with the assumption that L may be dominant (dominant model: SS vs SL/LL) and with the alternative possibility that L may be recessive (recessive model: LL vs SS/SL). Many of the 15 KSP subscales display a considerable positive or negative correlation. To explore the relationship between the different scales in our cohort of 42-year-old women, a principal component analysis was undertaken. The possible association between the first component and the ER alpha polymorphism was analysed using t-tests according to a dominant (SS vs SL/LL) and a recessive (LL vs

SS/SL) model, respectively. The larger of these two absolute t-values was used as statistic and the P-value assessed by means of permutation. In order to further elucidate the possible relationship between specific aspects of personality and the studied gene, the 15 KSP subscales were grouped into four factors, each comprising subscales displaying a considerable degree of correlation, and arguably reflecting related aspects of the personality: ‘neuroticism’, ‘psychoticism, ‘non-conformity’, and ‘extraversion’ (see Table 1). The grouping of subscales was based on previous studies.32,33 With respect to the placing of the ‘irritability’ and ‘inhibition of aggression’ subscales, previous reports, however, have not been unanimous; in some studies, these subscales have been included in the ‘neuroticism’ factor,33 whereas in others they have been included in the ‘non-conformity’ factor.32 On the basis of the correlation structure of the scores for the different subscales displayed by the cohort studied in this paper, ‘irritability’ was included in the ‘non-conformity’ factor, whereas ‘inhibition of aggression’ was included in the ‘neuroticism’ factor. For analyses of the possible relationship between the four factors and the ER alpha gene, summation scales were obtained for the four factors by calculating the mean of all items within one factor, using 100X for those scalesF‘socialization’ and ‘social desirability’Fdisplaying negative loading in relation to the other scales within that factor. For comparison of the different genotypes with respect to these summation scales, t-test was used. In the case where a factor displayed a significant association with genotype (after correction of P-values for multiple comparisons), the possible relationships between genotype and the different subscales within that factor were analyzed. The levels of significance for all t-tests undertaken were corrected for multiple comparisons by means of a step-down permutation procedure (comprising 100 000 permutations),22,23 automatically taking into account the correlation between the scales as well as the correlation between a dominant and a recessive t-test. Combined with the step-down principle, this is a more powerful procedure than a crude Bonferroni correction. For the purpose of comparison, P-values adjusted by means of Bonferroni correction are, however, presented as well.

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Acknowledgements This study was supported by grants from the Swedish Medical Research Council (Grants No. 8668 and 251), Lundberg’s Foundation, Wallenberg’s Foundation, Thuring’s Foundation, and Lundbeck’s Foundation. We thank Dr Lisa Ekselius and J Petter Gustavsson for valuable comments regarding the Karolinska Scales of Personality, and Inger Oscarsson, Gunilla Bourghardt, and Anna Nilsson for skillful technical assistance. Molecular Psychiatry


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Correspondence: L Westberg, Department of Pharmacology, Go¨teborg University, PO Box. 431, SE 405 30 Go¨teborg, Sweden. E-mail: [email protected] Received 11 December 2001; revised 6 May 2002; accepted 9 May 2002