Do the Dimensions of the Temperament and Character Inventory Map a Simple Genetic Architecture? Evidence From Molecular Genetics and Factor Analysis

The participants included 946 men and women in the National Institute on Aging’s Baltimore Longitudinal Study of Aging (27). The participants in this study are generally healthy, well-educated, community-dwelling volunteers who visit the Gerontology Research Center every 2 years for biomedical and psychosocial testing. After complete description of the study to the participants, written informed consent was obtained. The subjects who consented to participate consisted of 478 men aged 21 to 94 years (mean=62.1, SD=15.5) and 468 women aged 20 to 93 years (mean=55.4, SD=15.5). The ethnic composition of the total sample was 83.5% white (N=790), 13.1% African American (N=124), and 3.4% other (including Asian Americans, Native Americans, and Hispanics, N=32). The mean education level of the sample was 16.8 years (SD=2.4). DNA was available for only 587 participants (321 men and 266 women). All 587 were genotyped for the D4DR polymorphism. DNA and funds for 5-HTTLPR genotyping were available for 425 participants (217 men and 208 women).

The Temperament and Character Inventory is a 240-item, true-false, self-report questionnaire that measures seven dimensions of personality, including the original three Tridimensional Personality Questionnaire scales (or temperaments) of novelty seeking, harm avoidance, and reward dependence (4, 5). The Temperament and Character Inventory also includes a fourth temperament dimension, persistence, which was originally subsumed under reward dependence, and three character dimensions: self-directedness, cooperativeness, and self-transcendence. The Temperament and Character Inventory also measures 25 more specific traits that define the temperament and character dimensions. Details regarding the development, reliability, and factor structure of the Temperament and Character Inventory are provided elsewhere (4). Coefficient alpha, an index of internal consistency, reflects the degree to which items in a scale are tapping similar content. For the present sample, the coefficient alphas were 0.76, 0.86, 0.70, 0.60, 0.83, 0.80, and 0.87 for novelty seeking, harm avoidance, reward dependence, persistence, self-directedness, cooperativeness, and self-transcendence, respectively.

Genomic DNA was extracted from whole blood by standard techniques. The yield of the genomic DNA was assessed by electrophoresis: 2 ml of DNA and a HindIII-digested l standard DNA and genomic DNA concentration standards were subjected to electrophoresis on a 1% agarose gel containing 0.5 mg/ml of ethidium bromide, with subsequent visualization by ultraviolet transillumination. Oligonucleotide primers used in the primary amplification reaction were synthesized by means of standard methods. The positive control included a DNA sample of the most common allele, D4DR 4,4, in parallel with every set of test samples. The amplification reaction contained the following in a final volume of 60 ml: 25–250 ng of DNA, 50 pmol of each primer (as already described), 10% DMSO and buffer, nucleotides (200 mM each of dATP, dTTP, and dCTP and 100 mM each of dGTP and 7-deaza-dGTP), and Taq polymerase and buffer. Initial denaturation of the genomic DNA template was accomplished by heating at 95˚C for 5 minutes. Each of 30 cycles of amplification included annealing for 40 seconds at 54˚C, extension of the primers at 72˚C for 1 minute, and denaturation at 95˚C for 45 seconds. The amplified samples (15–20 ml) were loaded onto a 1.5% agarose gel and electrophoresed at 100 V for 2 to 3 hours. Each set of gel samples included a DNA amplification size marker, the D4DR 4,4 amplification control, a D₄ amplification ladder, a DNA negative control, and the test samples. The DNA fragments were visualized on an ultraviolet transilluminator and recorded by photographing the gel.

We used several approaches to defining the short versus long D4DR groupings reported in the literature (7, 8). The first approach defined the short polymorphism by the absence of the exon III seven-repeat allele and the long polymorphism by the presence of any seven-repeat allele. Because four- and seven-repeat alleles account for the majority of alleles in the population, a second approach compared the 4,4 genotype to the 4,7 genotype. A third approach defined the short polymorphism as two copies of two to five exon III repeats (s/s genotype) and the long polymorphism as one or two copies of six to eight exon III repeats (s/l and l/l genotypes). A fourth approach contrasted the s/s, s/l, and l/l genotypes. We also attempted to replicate the finding of Ekelund et al. (21) that the two- and five-repeat alleles were associated with novelty seeking.

Genomic DNA was extracted from whole blood by standard techniques. Polymerase chain reaction primers and conditions were as described by Lesch et al. (9). Briefly, primers flanking 5-HTTLPR and corresponding to the nucleotide positions –1416 to –1397 and –910 to –888 of the serotonin transporter gene 5¢-flanking regulatory region were used to generate 484- or 528-base-pair fragments. This polymerase chain reaction amplification was performed in a final volume of 30 ml consisting of 50 ng of genomic DNA, 2.5 mM deoxyribonucleotides, 0.1 mg 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 seconds, extension at 72˚C for 1 minute, and denaturation at 95˚C for 30 seconds for 35 cycles. The fragments were separated by agarose gel electrophoresis using 4% gels, and the genotypes were screened by ethidium bromide staining. DNA from 60 randomly selected subjects was sent to an independent laboratory to confirm the veridicality of our 5-HTTLPR assays. Perfect (100%) interlaboratory agreement was obtained.

We used two approaches defining the short and long 5-HTTLPR groupings reported in the literature (9, 23). In the first approach, participants with either one or two copies of the short variant of 5-HTTLPR were grouped together (s/s and s/l genotypes) and were contrasted to participants homozygous for the long variant of 5-HTTLPR (l/l genotype). The second method contrasted the s/s, s/l, and l/l genotype groups.

All statistical tests were carried out by using SAS software, release 6.12 (SAS, Cary, N.C.). Associations between genotype group and scores on the Temperament and Character Inventory scale were assessed by one-way analysis of variance (ANOVA). Because concerns about ethnic differences have been raised in prior studies (25), we repeated the analyses while controlling for the demographic variables of race (white versus nonwhite), age, and gender. All analyses were repeated within the subsample of white participants.

For D4DR, subjects with the absence (short) versus presence (long) of any seven-repeat alleles were compared in terms of scores on the overall Temperament and Character Inventory scale for novelty seeking and on the four subscales within novelty seeking. Additional D4DR genotype comparisons included 1) 4,4 (short) versus 4,7(long); 2) short (s/s) versus long (l/l plus s/l); 3) all three groups (l/l, s/l, and s/s); and 4) presence versus absence of two- and five-repeat alleles. For 5-HTTLPR, the short (s/s plus s/l) and long (l/l) 5-HTTLPR genotype groups were compared on the overall harm avoidance scale and the four harm avoidance subscales. The harm avoidance scores of the three 5-HTTLPR genotype groups (l/l, s/l, and s/s) were also compared.

Following the method Cloninger et al. (4), we evaluated the phenotypic structure of the Temperament and Character Inventory by conducting a principal components factor analysis of the 25 subscales. Exploratory factor analyses using a variety of indicators (Horn’s parallel analysis, Scree test, and eigenvalue greater than 1.0) suggested that fewer than seven factors should be extracted (Costa et al., manuscript in preparation). However, because the temperament-character model posits seven factors, seven factors were extracted. Varimax and promax rotations were examined.

The frequencies of the D4DR polymorphism alleles in the total sample were as follows: allele 2, 8.9%; allele 3, 3.7%; allele 4, 64.3%; allele 5, 1.1%; allele 6, 0.3%; allele 7, 20.4%; and allele 8, 1.1%. One participant had 10 repeats on the second D4DR allele. These distributions are similar to those reported previously (7, 8). The hypothesized difference in scores on the overall scale for novelty seeking between individuals with no seven-repeat allele (short) and those with any seven-repeat allele (long) was not found (Table 1). An analysis comparing the scores for novelty seeking of the 4,4 and 4,7 genotype groups was also not significant. Comparison of the groups with long (l/l plus s/l) and short (s/s) polymorphisms also resulted in no significant differences. An analysis of the three D4DR genotype groups (l/l, s/l, and s/s) also resulted in no significant differences in novelty seeking. When participants with the presence of any two- or five-repeat alleles were compared to those without these alleles, no significant effects involving the genotype were obtained. No significant D4DR group differences were found when the analyses were performed on the four subscales of novelty seeking: exploratory excitability, impulsiveness, extravagance, and disorderliness. When the analyses were statistically controlled for the effects of race, age, and gender and when the analyses were repeated within the subsample of white participants, no significant D4DR group differences emerged for novelty seeking or its subscales.

The frequencies of 5-HTTLPR alleles in the total sample were 58% for the long allele (l) and 42% for the short allele (s). The three genotype frequencies were distributed according to Hardy-Weinberg equilibrium (l/l=0.35, s/l=0.47, and s/s=0.18) and are similar to those reported previously (28, 29). Contrary to findings in a prior report (9), when the long (l/l) and short (s/l plus s/s) genotype groups were compared on the harm avoidance dimension of the Temperament and Character Inventory, no significant effect for genotype group was obtained (Table 2). No significant 5-HTTLPR genotype group differences were found for the four subscales for harm avoidance: anticipatory worry (F=0.47, df=1, 423, p=0.49), fear of uncertainty (F=1.96, df=1, 423, p=0.16), shyness with strangers (F=1.06, df=1, 423, p=0.30), and fatigability (F=1.09, df=1, 423, p=0.30). An analysis of the three 5-HTTLPR genotype groups (l/l, s/l, and s/s) also indicated no significant differences in scores for harm avoidance. Analyses that statistically controlled for the effects of race, age, and gender and analyses within the subsample of white participants also resulted in no significant genotype differences for harm avoidance or its subscales.

The results of the varimax and promax rotations were similar. For comparison to the table in the Temperament and Character Inventory Manual (5), Table 3 presents the factor structure loadings from the promax-rotated principal components analysis of scores on the 25 subscales of the Temperament and Character Inventory for the participants in our study. The factor structure loadings can be interpreted as the correlation between each subscale and the underlying factor. The observed factor structure of the Temperament and Character Inventory instrument does not correspond to the hypothesized seven-factor structure of the temperament-character model. In particular, Table 3 reveals two recurring problems with the Temperament and Character Inventory. First, according to the temperament-character model, all the subscales of each temperament or character factor should load on the same factor. Yet, several subscales have split or joint loadings. For example, the subscales for novelty seeking define two separate factors (IV and V), and the subscales for reward dependence have their chief loadings on three different factors (II, III, and IV). Second, several of the factors are defined by an admixture of subscales from the temperament and character domains. For example, factor I is clearly defined by the four harm avoidance subscales but also has significant inverse loadings from three of the five self-directedness subscales. Factors II and III combine the temperament scales of reward dependence with the character scales of self-transcendence and cooperativeness, respectively. The conceptual distinction between temperament and character is not empirically supported in these analyses of the Temperament and Character Inventory.