Genetic Variants of Dopamine Receptor D4 and [PDF]

Genetic Variants of Dopamine Receptor D4 and Psychopathology by ALessandro Serretti, Fabio Macciardi, Marco Catalano, Laura Bettodi, and Enrico Smeraldi

are derived from operational criteria (American Psychiatric Association 1994), and their main achievement pertains to the reliability of the diagnostic process, not to its validity. In addition, family studies do not univocally validate diagnoses (Maier et al. 1992, 1993; Taylor 1992; Heun and Maier 1993; Taylor et al. 1993; Winokur et al. 1993, 1995). Existing diagnoses guarantee partial clinical similarity, not biological homogeneity. In fact, no symptom or biological marker has ever revealed sufficient discriminating power among psychiatric diagnoses (Elliott 1992; Koyama and Yamashita 1992; Cannon and Marco 1994; Claridge 1994; Csernansky and Newcomer 1994), and even drug response shows a strong degree of variation within the same diagnosis. Thus, psychiatric diagnoses do not guarantee any biological homogeneity; moreover, the use of psychiatric clinical diagnoses has been considered to be reductive of the amount of information available from the whole symptomatologic presentation of patients (Tsuang and Faraone 1990; Cloninger 1994; Ginsburg et al. 1996; Vollenweider et al. 1997). This is a troublesome issue for genetic research, where biologically homogeneous samples are needed. The problem is usually addressed using multiple diagnostic levels from narrow to broad, but that solution can frequently and spuriously increase result significance (Cloninger 1994). Our working approach is to use psychopathology independently of clinical diagnoses. Thus, our search for candidate genes may be directed toward intermediate psychopathological structures that do not overlap with psychiatric diagnoses, but that are more closely related to the underlying biologic and genetic background. Because genetic liability may be expressed as psychopathological traits shared by several psychiatric disorders (Cloninger 1994; Serretti et al. 1996), we have

Abstract There is much evidence to indicate that the dopamine receptor D4 (DRD4) gene is involved in psychiatric disorders. We investigated the correlation between DRD4 gene polymorphism and the psychopathology of major psychoses, independently of diagnoses. Some 461 inpatients affected by major psychoses were assessed by the Operational Criteria checklist for psychotic illness and typed for DRD4 variants. The four symptomatologic factors—mania, depression, delusion, and disorganization—were used as phenotype definitions. DRD4 Exon 3 long allele variants were associated with high delusional scores, with the most significant difference between alleles 2 and 7 (p 0.004). DRD4 variants may, therefore, constitute a liability factor for development of delusional symptomatology in patients with major psychoses. Key words: Psychopathology, DRD4, psychiatric genetics, mood disorders, schizophrenia. Schizophrenia Bulletin, 25(3):609-618,1999. Major psychiatric disorders tend to aggregate in families. Clinical practice supports this observation to the extent that ill relatives constitute the only accepted risk factor. Familial aggregation suggests that genetic factors play a role in the etiopathogenesis of major psychoses. But despite the fact that twin and family studies have confirmed the existence of genetic factors, no exact model of transmission has yet been established (Risch 1990). Beyond formal genetics, recent molecular genetic approaches permit investigation of specific genetic susceptibilities through both candidate genes and genome scans. Those approaches have produced some positive results, but they are still far from producing ultimate findings (Risch and Botstein 1996). A possible explanation for the lack of conclusive results could lie in the definition of the phenotype, which at present is mainly based on psychiatric diagnoses. These

Reprint requests should be sent to Dr. Enrico Smeraldi, Department of Neuroscience, Istituto Scientifico H. San Raffaele, University of Milano School of Medicine, Via Luigi Prinetti 29, 20127 Milano, Italy.

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developed a phenotypic definition based on the observed symptomatology independently of the clinical diagnosis (Serretti et al. 1996). A few other studies have adopted this strategy with promising results in both Alzheimer's disease and psychiatric disorders (Weisgraber et al. 1994; Ginsburg et al. 1996; Bellivier et al. 1997; Gottesman 1997;Yaffeetal. 1997). Dopamine pathways are involved in the pathophysiology of schizophrenia and mood disorders (Kahn and Davis 1995; Willner 1995). Since the dopamine D4 receptor gene (DRD4) was cloned (Van Tol et al. 1991), it has received considerable interest because of its many interesting properties. The DRD4 mRNA distribution profile differs from other dopamine receptors in having elevated values in limbic areas, such as the frontal cortex and amygdala, involved in the pathophysiology of major psychoses (Van Tol et al. 1991; Matsumoto et al. 1995; Murray et al. 1995). Postmortem studies of schizophrenia patients detected a sixfold elevation of D4 sites (Seeman et al. 1993; Sumiyoshi et al. 1995). The DRD4 gene is extremely polymorphic. It has one polymorphism located in the third exon coding for the third cytoplasmatic loop of the receptor (Van Tol et al. 1991, 1992) and consisting of a variable number of copies of a 48 base pair (bp) sequence, from 2 to 10. A second polymorphism is characterized by a polymorphic 12 bp repeat in exon 1 and codes for a sequence of four amino acids in the extracellular N-terminal part of the receptor (Catalano et al. 1993). Other known DRD4 gene polymorphisms include a T -* G substitution in nucleotide position + 581 (Seeman et al. 1994), and the presence of a nonsense 13 bp deletion in exon 1 (Nothen et al. 1994). Furthermore, two novel polymorphisms and a rare deletion variant were recently reported (Cichon et al. 1995). The 48 bp variation exhibited different pharmacological activities. Dopamine stimulation of the DRD4 receptors decreases cAMP formation. The potency of dopamine's inhibition of cAMP formation was reduced about twofold for the 7 allele compared with the short allele variants (Asghari et al. 1995). DRD4 also shows a high affinity for clozapine, an antipsychotic used for schizophrenia and manic states. Moreover, a different dissociation constant for clozapine in the absence of sodium was demonstrated between short and long alleles of the 48 bp polymorphism (Van Tol et al. 1992; Calabrese et al. 1996; Marder 1996). In conclusion, variants of the DRD4 gene may produce functionally different receptor proteins with a potential influence on the psychopathology of major psychoses. The aim of this study was to investigate the association of exon 1 and exon 3 polymorphisms of the DRD4 gene with major psychoses symptomatology, within basic psychopathological areas.

Methods Sample. Some 461 psychiatric inpatients (females = 244, males = 217; mean age = 42.93 years, standard deviation [SD] = 14.41; mean age at onset = 29.48 years, SD — 13.25) consecutively admitted to the Department of Neuropsychiatry at the Institute H. San Raffaele were included in this study. All the patients were evaluated using the Operational Criteria (OPCRIT) checklist for psychotic illness (McGuffin et al. 1991). Lifetime diagnoses were assigned by two independent psychiatrists on the basis of interviews and medical records, according to DSM-III—R criteria (American Psychiatric Association 1987). We included all subjects affected by major psychoses. However, the presence of concomitant diagnoses of mental retardation and drug dependence, together with somatic or neurological illnesses that impaired psychiatric evaluation (e.g., hypothyroidism mimicking a depressive state) represented exclusion criteria. The subjects included were affected by schizophrenia, including all subtypes (n - 162), major depressive disorder (n = 83, including 26 obsessive-compulsive comorbid subjects), bipolar disorder (n = 152), delusional disorder (n = 56), and psychotic disorder not otherwise specified (NOS) (n = 8). Informed consent was obtained from all probands, who were unrelated and of Italian descent with antecedents from all parts of Italy. DNA Analysis. Genomic DNA was extracted from anticoagulated thawed blood according to the method of Lahiri and Nurnberger (1991). For the D4 exon 3 typing, a polymerase chain reaction (PCR) was carried out with primers and conditions as described elsewhere (Macciardi et al. 1994). This PCR polymorphism is particularly complex and detects a 7 allele system (A2 = 0.1, A3 = 0.03, A4 = 0.7, A5 = 0.01, A6 = 0.01, A7 = 0.15, A8 = 0.01). For exon 1, PCR conditions were also as described elsewhere (Catalano et al. 1993) and 446 subjects were typed. Statistical Analysis. In a previous article we described the factoring process of the OPCRIT checklist in a sample of patients affected by major psychoses (Serretti et al. 1996). We identified four factors: 1. Excitement: excessive activity, reduced need for sleep, pressured speech, elevated mood, thoughts racing, increased sociability, increased self esteem, irritable mood, distractibility, agitated activity, dysphoria, grandiose delusions, reckless activity 2. Depression: loss of pleasure, loss of energy/tiredness, diminished libido, excessive self reproach, slowed activity, poor appetite, poor concentration, suicidal ideation, weight loss, diurnal variation, early morning waking, delusions of guilt

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3. Delusion: persecutory delusions, well-organized delusions, delusions of influence, widespread delusions, primary delusional perception, delusions and hallucinations lasting for 1 week, persecutory/jealous delusions and hallucinations, thought insertion, thought withdrawal, thought broadcast, third-person auditory hallucinations 4. Disorganization: speech difficult to understand, incoherent, positive formal thought disorder, inappropriate affect, bizarre behavior, bizarre delusions, blunted affect, negative formal thought disorder, deterioration from premorbid level of function These factors explain 50 percent of the variance of the selected OPCRIT items and were confirmed in an independent sample. The factors were present, in various degrees, in all the clinical diagnoses considered. Standardized factor scores have been derived from these factors and were considered to be dependent variables when investigating their distribution across DRD4 alleles and genotypes. Of the original 461 subjects, factor scores could be calculated only for 446 subjects due to missing items. For all calculations we excluded variants 3, 5, 6, and 8 because in our sample they were poorly represented (their cumulative prevalence was only 0.06). Differences were assessed using t test or one-way analysis of variance (ANOVA), with Newman-Keul (N-K) test to evaluate post hoc comparisons; frequencie were compared by chi-square test. The power of our sam pie was tested considering an alpha value of 5 percent two tailed. Our sample featured mixed mood and schizc phrenia spectrum disorders, which could introduce a bia in the analysis of the genetic effect. The diagnostic com position of our sample was not established a priori, and i must be considered as randomly extracted from all possi ble sets of samples. To control for this bias, we repeate the analysis using a mixed effect two-factor ANOVA i which D4 typing was considered the fixed effect and diag nostic status a random effect. For this calculation we use BMDP 3V program with restricted maximum likelihoo method (Dixon 1990).

Results The association between DRD4 variants and excitement, depressive, disorganized, and delusional symptomatology was estimated using one-way ANOVA (table 1). The ANOVA on factor scores showed a significant difference on delusion factor (F = 5.52, df = 2,840, p = 0.004; mixed model F = 5.37, df = 2,840, p = 0.005), with a post hoc significant difference between the 2 and the 7 bp repeats O = 0.0006; N-K p < 0.01) (see figure 1). The 7 bp repeats groups had the highest scores on this factor while the 4 bp repeats group had intermediate scores between the 7 and the 2 repeats. The power of detection for the observed difference in the delusional factor was 0.83 in our sample. Subgroups considering sex and diagnoses did not show any significant deviation from the observed results. The genotype analysis is summarized in table 2. Overall ANOVA did not show significant differences, but

Figure 1. Distribution of Delusion score according to alleles at DRD4 exon 3

DRD4 Exon 3 Alleles

Table 1. Factor Scores and DRD4 exon 3 aileles DRD4 exon 3 alleles 2 repeat 4 repeat 7 repeat Total

Excitement Mean 0.050 -0.051 0.108 -0.013

Depression

SD

Mean

SD

1.044 1.011 1.052 1.023

0.195 0.152 0.053 0.146

1.121 1.146 1.003 1.052

Delusion Mean -0.049 0.105 0.3821 • 0.117

Disorganization SD

Mean

SD

n

1.037 1.057 0.934 1.043

-0.081 0.006 -0.091 -0.083

0.962 1.051 1.030 1.015

138 591 114 843

Note.—Rare alleles are not displayed. SD = standard deviation. High delusional scores are significantly associated with long exon 3 alleles (F = 5.52, df = 2,840, p = 0.004).

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Table 2.

A. Serretti et al.

Factor scores and DRD4 exon 3 genotypes

DRD4 exon 3 genotypes 2/2 2/4 2/7 4/4 4/7 7/7 Total

Depression

Excitement Mean 0.259 0.084 -0.567 -0.103 0.25 0.109 0.005

SD 1.093 1.072 0.528 0.998 1.070 1.165 1.032

Disorganization

Delusion Mean

Mean

SD

-0.091 0.311 0.176 0.180 0.043 0.064 0.165

1.017 1.150 1.076 1.031 1.012 1.032 1.052

-O.043 -0.077 0.174 0.066 0.400 0.373 0.107

SD

Mean

SD

n

1.025 1.044 1.209 1.087 0.892 0.887 1.048

0.314 -0.267 -0.118 0.040 0.054 -0.728 -0.031

0.917 0.866 1.200 1.076 1.020 0.609 1.026

18 81 16 200 77 9 401

Note.—Rare genotypes are not displayed. SD = standard deviation.

a significant post hoc comparison on the delusion factor was detected between the 2/4 and the 4/7 genotypes (p = 0.004; N-K p < 0.05). Variants of DRD4 gene showed a pharmacological activity related to the number of repeats; in our sample we observed a linear increase in delusional scores with the number of 48 bp repeats on the DRD4 genotype (figure 2). We then performed a linear regression of delusional score on genotypes considered as a

variable ranging from 1 (corresponding to genotype 2/2) to 6 (corresponding to genotype 7/7). We observed a significant linear regression for the number of 48 bp variants on delusional scores (r = 0.111, F = 5.53, df = 1,444, p = 0.019). DRD4 exon 1 variants were not related to any factor (table 3), even when subjects were stratified for sex and diagnoses. A possible interaction between exon 1 and exon 3 was hypothesized and an haplotype analysis was performed stratifying exon 3 on exon 1 variants, but no significant interaction was detected. Diagnostic group could constitute a stratification bias but the mixed model ANOVA formally excluded this issue. Table 4 reports the delusion factor scores distribution on DRD4 exon 3 stratified for diagnostic group. The DRD4 exon 3 variants difference remained significant only for the bipolar group, but delusional values were also higher for the 7 allele for schizophrenia, major depressive, and NOS psychoses.

Figure 2. Distribution of Delusion score according to genotypes at DRD4 exon 3

Discussion In our sample, the intensity of delusional symptomatology increased linearly from short to long alleles of DRD4 exon 3, producing a significant linear regression on delusional scores. Because delusional scores are the weighted sum of OPCRIT items, we also cross-tabulated exon 3 variants with any of the 11 items that constitute the delu-

DRD4 Exon 3 Genotypes

Table 3.

Factor Scores and DRD4 exon 1 alleles

DRD4 exon 1 alleles 1 2 Total

Excitement

Depression

Delusion

Disorganization

Mean

SD

Mean

SD

Mean

SD

0.010 -0.289 -0.012

1.031 0.780 1.018

0.129 -0.002 0.119

1.050 0.974 1.045

0.078 0.117 0.081

1.008 1.192 1.022

Mean -0.033 0.018 -0.030

Note.—Scores are not associated with DRD4 exon 1 alleles and genotypes (data not shown); SD = standard deviation.

612

SD

n

1.037 1.021 1.035

828 64 892

I—*

0.739 0.083 -0.361 0.219 0.245 0.875 0.234

0.316 0.213 0.305 0.237

Mean

0.240 (3) 0.667 (8) 0.000(1) 0.738 (30) 0.561 (8) 0.000(1) 0.677(51)

0.616(15) 0.691 (80) 0.577(11) 0.665(106)

SD

Delusional

0.705 (49) 0.720(190) 0.713(44) 0.730 (283)

SD -0.338 -0.469 -0.189 -0.407

Mean

-0.697 -0.406 0.233 -0.519 -0.112 0.215 -0.374 0.643 (7) 0.735 (33) 0.000(1) 0.699 (60) 0.677 (32) 0.914(5) 0.727(138)

-1.209 -0.289 -0.188 -0.594 -0.150 -0.289 -0.426

0.000(1) 1.070(13) 1.115(5) 0.931 (22) 1.035(5) 0.000(1) 0.971 (47)

0.988 (22) 0.937 (66) 0.919(12) 0.941 (100)

SD

Major Depressive

(F=6.16 , df = 2,280, p = 0.0024)

-0.467 -0.423 -0.022 -0.369

Mean

Bipolar

— -1.185 -1.210 -0.957 — — -1.046

-1.192 -1.012 -1.210 -1.055

Mean

— 0.056 (8) 0.015(2) 0.684(16) — — 0.543 (26)

0.049(11) 0.594 (42) 0.015(2) 0.524 (55)

SD

Major Depressive + doc

-0.483 0.119 0.033 0.016

Mean

— 0.000(1) 0.000(1) 0.544 (2) 0.593 (3) — 0.747 (7)

1.085 (2) 0.505 (9) 0.983 (4) 0.692(15)

SD

Psychotic NOS

— 0.285 -1.250 -0.183 0.461 — 0.007

Note.—The number of subjects is displayed in parentheses. SD = standard deviation; NOS = not otherwise specified; doc = obsessive compulsive disorder.

Total

in

genotypes 1.114(7) 0.442 1.082 0.926(18) 1.254 0.795 (6) 0.954 (70) 0.949 0.729 (29) 1.096 0.849 1.301 (2) 0.985 0.906(132)

DRD4 exon 3 2/2 2/4 2/7 4/4 4/7

1.001 (39) 0.912(204) 0.748(41) 0.903 (284)

SD

alleles 0.845 0.969 1.116 0.973

Mean

Schizophrenia

Delusion factor scores and DRD4 exon 3 alleles and genotypes stratified for diagnoses

DRD4 exon 3 2 4 7 Total

Table 4.

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Schizophrenia Bulletin, Vol. 25, No. 3, 1999

findings may suggest a sampling effect: High frequencies of subjects with delusional symptoms may increase the allele 7 frequency. When using psychiatric diagnoses as the phenotype definition, it would also be useful to include a symptomatologic description of subjects. However, the extent of variance in symptoms explained was low. The DRD4 48 bp polymorphism only explained about 2 percent (effect size 0.1) of the variation in delusional symptomatology, an observation that is in agreement with a supposedly minor effect of the gene coding for the D4 receptor in the development of delusional symptomatology. While the variance explained is statistically informative, we were also interested in the epidemiological influence of DRD4 on delusional symptomatology. To accomplish this task we split the delusional factor into the original 11 OPCRIT items. The increase in risk of having at least one delusional item, given the presence of the DRD4 7 allele, was 2.24 (odds ratio [OR],/? = 0.016). When using the items most associated with DRD4 long alleles (persecutory delusions, delusions of influence, widespread delusions, persecutory/jealous delusions and hallucinations), the risk was significantly higher (OR = 2.9l,p = 0.0004). In the latter case, we calculated the attributable fraction, that is, the proportion of subjects that would not present those delusional symptoms in the absence of the risk factor. In other words, the attributable fraction reveals the extent to which the risk factor contributes to the delusional symptoms. The attributable fraction was 3.4 percent; that means that in the absence of allele 7 we would observe 3.4 percent fewer subjects with delusional symptoms.

sion factor. The allele 7 variant of DRD4 was significantly associated with four delusion factor items: persecutory delusions, delusions of influence, widespread delusions, and persecutory/jealous delusions and hallucinations. For the remaining items, the association was present but not significant. In the present study, we used a mixed sample of patients affected by mood, schizophrenia, and delusional disorders. This heterogeneity might indicate that the observed differences on the delusional factor are simply the result of a spurious correlation (e.g., an excess of delusional subjects with allele 7). However, delusional disorder was not associated with DRD4 exon 3 variants (Serretti et al., in press); moreover, the same association of delusional symptomatology with allele 7 was observed for all the diagnoses. A formal demonstration of this last point was achieved by considering diagnoses through a mixed model ANOVA and observing the substantial stability of the results. Thus, the reported association could be considered an extended effect of long alleles on delusional symptoms among psychotic patients. DRD4 exon 1 variants did not show any association with symptom factors. We hypothesized that exon 1 might interact with exon 3 on symptoms, but, among subjects with exon 1 deletion, exon 3 variants did not differ significantly in their symptom scores. Recently, an association of DRD4 with the novelty seeking personality trait has been demonstrated in normal subjects (Benjamin et al. 1996; Ebstein et al. 1996; Ebstein and Belmaker 1997), although no univocal confirmations followed (Jonsson et al. 1997; Ono et al. 1997; Sander et al. 1997; Vandenbergh et al. 1997). Dopamine transmission is involved both in the individual variations in novelty seeking (Cloninger et al. 1996) and in the development of delusion (Kahn and Davis 1995). It might be hypothesized that there is a broad effect of the altered dopamine receptor D4 on the dopaminergic system, producing an increase of the novelty seeking personality trait and, among psychiatric patients, an increase in delusional symptoms independently of diagnosis. The relevance of long alleles for psychopathology is also indicated by a recent article (LaHoste et al. 1996) describing an association of long DRD4 alleles with attention deficit hyperactivity disorder. DRD4 gene has been investigated in major psychiatric disorders and associations have been reported (Catalano et al. 1993; Lim et al. 1993; Manki et al. 1996). Previous findings reported an excess of 7 allele in persons with bipolar disorder (Lim et al. 1993) that subsequently was not confirmed (Lim et al. 1994) and reported no results in persons with schizophrenia (Shaikh et al. 1993, 1994; Petronis et al. 1995). A recent article reported an association between DRD4 gene (allele 2) and unipolar disorder in a Japanese population (Manki et al. 1996). Our

To our knowledge, our sample is the largest in any published study, and the small effect observed would not have been detectable in smaller samples. We scored subjects with a lifetime prospect of ever-present symptoms; that should partially circumvent any bias derived from the cross-sectional nature of the study. This sample does not include control subjects, because our objective was to test the effect of liability factors on psychopathological traits among affected individuals, not on the development of disease. Thus, a mixed psychiatric sample constitutes the best means to verify this hypothesis. However, it might be of interest to compare DRD4 exon 3 allele frequencies in subjects with high delusional scores with those of controls. To this end, ill subjects are compared to a sample of control subjects currently analyzed for a companion paper (Serretti et al., in press). DRD4 alleles 7 and 2 for subjects with high delusional scores differ from those for subjects with low delusional scores, but not from controls (see table 5). Ethnic origin is frequently a cause of stratification bias; however, our sample is composed of subjects with Italian antecedents back to grandparents and no other origin was allowed. Most studies, including the present one,

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Table 5.

Schizophrenia Bulletin, Vol. 25, No. 3,1999

DRD4 exon 3 alleles and genotype frequencies among patients and controls Patients Controls

DRD4 alleles 2 3 4 5 6 7 8 Total

Very high delusion

High delusion

Low delusion

All

n

%

n

%

n

%

n

%

n

%

98 26 655 8 7 143 5 942

0.10 0.03 0.70 0.01 0.01 0.15 0.01

56 14 294 7 3 75 1 450

, 0.12 0.03 0.65 0.02 0.01 0.17 0.01

43 7 213 5 3 53 0 324

0.13 0.02 0.66 0.02 0.01 0.16 0.00

82 15 297 4 3 39 2 442

0.191 0.03 0.67 0.01 0.01 0.091 0.01

138 29 591 11 6 114 3 892

0.16 0.3 0.66 0.01 0.01 0.13 0.01

Genotypes 12 2/2 0.03 8 0.04 7 0.05 10 0.05 18 0.05 57 0.12 2/4 31 0.16 23 0.16 50 0.251 81 0.20 14 0.04 0.04 0.04 16 2/7 0.03 8 6 8 0.04 94 0.47 0.53 200 4/4 229 0.49 70 0.48 106 0.50 102 54 77 4/7 0.22 0.27 37 0.25 23 0.11 1 0.19 12 4 0.02 9 7/7 0.03 5 0.03 4 0.03 0.02 426 200 147 401 Total 201 Note.—High delusional subjects (delusional factor scores > 0.0) did not show higher frequencies of 7 allele compared to controls. Subjects with extreme delusion scores (> 0.5) were not different from controls. Subjects presenting low delusion scores were significantly different from both controls and the remaining subjects. 1 p < 0.001 versus controls and high delusion score subjects.

use multiple statistical testing. Therefore, significance levels would be unlikely to survive correction in many cases. However, analyses of candidate genes have high a priori probabilities of association, and multiple correction could not demonstrate the best choice (Carey 1994; Levinson 1997). Replication using independent samples or familybased association designs should be considered more suitable strategies. In conclusion, DRD4 variants were related to psychopathology: long DRD4 exon 3 alleles may constitute a liability factor for delusional symptoms in major psychoses. The use of a symptomatological definition of phenotype complements the usual genetic research strategies.

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The Authors Alessandro Serretti, M.D., is Researcher; Fabio Macciardi, M.D., Ph.D., and Marco Catalano, M.D., are Lecturer of Psychiatry and Vice Psychiatrist-in-Chief; Laura Bellodi, M.D., is Associate Professor of Psychiatry and Psychiatrist-in-Chief; Enrico Smeraldi, M.D., is Professor of Psychiatry and Head of the Department of Psychiatry, Istituto Scientifico H. San Raffaele, University of Milano School of Medicine, Milano, Italy.

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