Molecular Psychiatry (2002) 7, 695–705  2002 Nature Publishing Group All rights reserved 1359-4184/02 $25.00 www.nature.com/mp ORIGINAL RESEARCH ARTICLE Relationship between adverse effects of treatment and D2 receptor polymorphisms in patients with schizophrenia R Kaiser1,2, P-B Tremblay1, F Klufmo¨ller1, I Roots1 and J Brockmo¨ller1,2

1Institute of Clinical Pharmacology, Universita¨tsklinikum Charite´, Humboldt Universita¨t zu Berlin, Berlin, Germany; 2Institute of Clinical Pharmacology, Universita¨tsklinikum der Georg-August-Universita¨t Go¨ttingen, Go¨ttingen, Germany

Extrapyramidal adverse symptoms (EPS) represent a major type of adverse events in treat-

ment with which share high affinity to the dopamine D2 receptor (DRD2). Genetic variants of this receptor may modulate the therapeutic response and the severity of adverse symptoms of . We analyzed nine known polymorphisms of the DRD2 in 665 schizophrenic patients with European Caucasian ethnic background and compared the intensity of acute dystonia, extrapyramidal symptoms, akathisia, and tardive dyskinesia between carriers of different DRD2 genotypes. In a subgroup of 40 patients with most severe extrapyramidal symptoms we sequenced the coding region including the exon-

intron junctions of the DRD2 gene. Functionally relevant DRD2 amino acid variants (Ser310, Cys311) were rare or were not found at all (Ala96). Complete sequence analysis of sufferers from the most severe adverse effects revealed two new intronic polymorphisms and a silent polymorphism in exon 7, but no new amino acid variants beyond those which are already known. We found no significant association between these polymorphisms and the intensity of the different types of adverse neurologic effects of the antipsychotics. These results were obtained by correlating adverse events with each of the nine single nucleotide polymorphisms and by correlation with the estimated haplotypes. In conclusion, genetic variations in the DRD2 gene were no major predictors of the individually variable adverse effects from antipsy- chotic treatment in Caucasian schizophrenic patients. Molecular Psychiatry (2002) 7, 695–705. doi:10.1038/sj.mp.4001054

Keywords: dopamine D2 receptor; DRD2; polymorphism; schizophrenia; adverse effects; BARS; AIMS; EPS; dyskinesia; akathisia

Introduction striatal dopamine resulting in an increased oxidative cellular stress.6,7 Administration of typical antipsychotics in the treat- Akathisia is a frequent adverse effect of antipsy- ment of schizophrenia is associated with a high rate of chotic treatment and is characterized by periodic undesirable neurologic effects such as acute dystonia, movements of the legs and inner restlessness. A dopa- pseudoparkinsonism (EPS), akathisia and tardive dys- minergic misbalance may be the underlying mech- kinesia. These may be observed in up to 50–75% of anism since the occurrence of akathisia was related to patients and may require discontinuation of treatment D and D receptor occupancy.8–10 in up to 10% of patients.1,2 1 2 Tardive dyskinesia is a later and most pronounced Antidopaminergic drugs produce acute dystonia and adverse event appearing as abnormal, involuntary, pseudo-parkinsonian symptoms and in a subgroup of irregular, repetitive movements of the orofacial muscu- long-term or high-dose treated patients these symp- lature and limbs found in approximately 25% of toms may persist after discontinuation of the medi- patients. Alteration in the dopamine D or D receptor cation in the form of tardive dyskinesia.3,4 It has been 1 2 mediated striatonigral or striatopallidal pathways and hypothesized that neuroleptics irreversibly desensitize a loss of striatal neurons might be involved in the and depopulate receptors5 or that they pathogenesis indicating a neurotoxic effect of long- increase the neuronal firing rates and release of nigro- term antipsychotic treatment.11,12

The dopamine D2 receptor (DRD2) is the primary binding target of all typical antipsychotics. It belongs Correspondence: Prof Dr J Brockmo¨ller, Abteilung fu¨ r Klinische to the family of receptors coupled to heterotrimeric Pharmakologie, Universita¨tsklinikum der Georg-August-Universi- cyclic guanine nucleotide binding regulatory proteins ta¨tGo¨ttingen, Robert Koch Str 40, D-37075 Go¨ttingen, Germany. E-mail: jurgen.brockmollerȰmed.uni-goettingen.de (G-proteins). The DRD2 activates intracellular signaling Received 3 July 2001; revised 31 July 2001; accepted 30 Nov- by inhibition of cAMP synthesis through interaction ember 2001 with Gi-like proteins. As shown in Figure 1, the DRD2 Dopamine D2 receptor polymorphisms and adverse events R Kaiser et al 696

Figure 1 The genomic structure and the examined polymorphisms of the DRD2 receptor gene. Boxes represent the exons. Shadowed boxes represent the coding sequences within the exons. Transmembrane domains (I–VII) were characterized by black boxes. All polymorphisms are indicated with their respective localization according to the published sequence.13 Polymor- phisms indicated by * have been found by sequencing in the subgroup of 40 individuals with the severest EPS.

gene has eight exons and spans at least 270 kb. The kum Benjamin Franklin) and from three community presumed promoter sequence is separated by the large psychiatric hospitals (Wilhelm-Griesinger Kranken- (greater than 250 kb) first intron from the protein- haus, Kliniken im Theodor Wenzel Werk, Urban- coding sequence.13,14 Several variants of the DRD2 gene Krankenhaus). Only subjects of German Caucasian

are known: one substitution (Val96Ala) within the descent were included. second transmembrane domain and two substitutions Patients with organic or -induced psychosis as

(Pro310Ser, Ser311Cys) within the third transmembrane revealed by clinical history, magnetic resonance tom- domain and a number of silent polymorphisms.15 Pre- ography, computerized tomography, or toxicological vious studies using positron emission tomography laboratory findings and patients younger than 18 or (PET) have shown that a threshold DRD2 occupancy of older than 70 years were excluded. Not all patients about 65–75% during antipsychotic treatment is neces- could be evaluated by the rating scales given below sary to obtain a therapeutic effect.16 Individuals with because they were either not able to participate in the the TaqIA1 and TaqIB1 allele were shown to have a neurological tests for adverse effects or were dismissed decreased striatal DRD2 density and showed a better prior to the respective test dates from the hospitals clinical response to a DRD2 antagonist.17–20 In contrast, within a few days after admission to the hospital. In individuals with the −141C insertion allele of the pro- addition, we excluded all patients with repeated moter showed an increased DRD2 density.17 Moreover, admission to the hospital during the study period. Of

in vitro the Ala96 variant had a 50% reduced binding the 665 patients initially recruited, we could analyze affinity for , and dopamine the different scores for the adverse effects from 584

whereas the Cys311 and Ser310 variants were markedly patients at observation point day 2–4, from up to 518 less effective in inhibiting cAMP synthesis than the patients at day 12–16 and from up to 390 patients at 21,22 wild-type or the Ala96 variant. day 26–30. To explore the bias possibly caused by Therefore, these variations in the DRD2 receptor those who could not be explored for adverse events, gene might explain individual differences in the sus- we compared the genotype frequencies in both groups ceptibility to develop adverse drug effects in schizo- (those who completed the evaluation and those who phrenic patients. We tested this hypothesis in a multi- did not complete the evaluation), but no significant dif- center prospective trial in patients receiving typical ferences were found by use of the chi-square test. This antipsychotic drugs. result excludes at least a major selection bias. Informed consent was obtained from all subjects. The study was approved by the Ethics Committee of the Universitats- Materials and methods ¨ klinikum Charite´ (Humboldt-Universita¨t zu Berlin) and Subjects the Universita¨tsklinikum Benjamin Franklin (Freie A hospital-based, multicenter, naturalistic, prospec- Universita¨t Berlin). tive, non-interventional study was performed with All 665 patients were acute hospitalized patients unrelated schizophrenic patients from Berlin, Ger- with schizophrenia according to DSM-IV criteria, many. We recruited 665 schizophrenic patients (53.6% including 368 schizo-paranoid (DSM-IV 295.3), 134 male, 46.4% female) with a mean age of 38.3 years schizo-affective (295.7), 45 residual (295.6), 41 cata- (range 18–70 years, SD = 12.1) and a median age of tonic (295.2), 23 disorganized (295.1), 11 schizophrenic onset of 28.7 years (range 14–66 years, SD = 9.1) from form (295.4), six undifferentiated (295.9), and 37 other two university psychiatric hospitals in Berlin and unclassified subtypes. Diagnosis was assigned on (Universita¨tsklinikum Charite´ and Universita¨tsklini- the basis of interviews and medical records according

Molecular Psychiatry Dopamine D2 receptor polymorphisms and adverse events R Kaiser et al 697 to DSM-IV criteria. All psychiatrists involved in the for 1 min 50 s; TaqIB: 94°C for 1 min, 60°C for 1 min, study received detailed instructions on how to apply 72°C for 1 min 50 s; TaqID: 94°C for 1 min, 60°C for 1 ° ° ° the DSM-IV criteria prior to the study. Age at first epi- min, 72 C for 1 min 20 s; Val96Ala: 94 C for 30 s, 58 C ° ° ° sode, duration, familial predisposition, number of for 1 min, 72 C for 45 s; Pro310Ser: 94 C for 1 min, 60 C ° ° recurrent exacerbations, smoking behavior and current for 1 min, 72 C for 1 min 30 s; Ser311Cys: 94 C for 1 therapeutic response (using the positive and negative min, 60°C for 1 min, 72°C for 1 min 30 s; and for the Ͼ ° symptoms scale), and were documented. Leu141Leu (423G A) polymorphism: 94 C for 30 s, Incident cases were 131 patients and 534 patients suf- 54°C for 30 s, 72°C for 1 min. For the −241AϾG and fering from recurrent exacerbation. −141C insertion/deletion polymorphism: two cycles of Adverse effects of antipsychotics were evaluated for 94°C for 30 s, 68°C for 30 s, 72°C for 45 s, followed by the extent of acute dystonia using an adverse effect 35 cycles of 94°C for 30 s, 66°C for 30 s, 72°C for 45 s. reporting form. The extrapyramidal symptoms All PCR-products were digested with restriction (pseudoparkinsonism) were recorded by the Extrapyr- enzymes according to the manufacturer’s protocol, sep- amidal Symptom Scale (EPS) published by Simpson arated by 3.0% agarose gel electrophoresis and stained and Angus.23 Akathisia was described by the Barnes with ethidium bromide for UV visualization. Akathisia Rating Scale (BARS).24 Tardive dyskinesia was measured by use of the Abnormal Involuntary Sequence analysis Movement Scale (AIMS).25 Besides the formal criterion For sequencing we first amplified exons 3–5 (10× buffer for tardive dyskinesia,26 the raw AIMS score was used 1, initial denaturation at 94°C for 2 min, followed by without data reduction. Interviews took place on day 35 cycles of 96°C for 10 s, 61°C for 20 s, 68°C for 3 min 3(2–4), day 14 (12–16) and day 28 (26–30) after admis- and a final extension period of 72°C for 7 min) and sion to the hospital. Inter-rater reliability was assured exons 6–8 (10× buffer 2, initial denaturation at 94°C for by regular monthly meetings of the clinical investi- 2 min, followed by 35 cycles of 96°C for 10 s, 62°C for gators. 20 s, 68°C for 5 min and a final extension period of Of the typical antipsychotics, , flupen- 72°C for 7 min) using the Expand Long Template PCR thixol, clopenthixol, , and fluphenazine were system (Roche, Germany) and the respective primers administered most frequently. Applied dosages at the (exon 3–5: primer pair 3.1, 5.2; exon 6–8: primer pair day of examination were calculated as chlorpromazine 6.1, 8.2, Table 1). Exon 2 was amplified using the adjusted dosage: therefore dosages of haloperidol were primer pair 2.1, 2.2 and the following PCR cycle con- multiplied by 50, flupenthixol by 30, clopenthixol by ditions: initial denaturation at 94°C for 2 min, followed 6, perazine by 1.2 and fluphenazine by 50. The by 35 cycles of 95°C for 20 s, 60°C for 20 s, 72°C for 1 attending psychiatrists were blinded with respect to min and a final extension period of 72°C for 7 min. the patients genotype. The following sequencing reaction was carried out with exon specific primers as shown in Table 1: the Methods PCR cycle sequencing program consisted of an initial High-molecular-weight genomic DNA was prepared denaturation at 94°C for 2 min, followed by 25 cycles from venous blood using the standard phenol chloro- with an initial denaturation at 96°C for 15 s, annealing form extraction. Laboratory staff was blind to the psy- at 57°C for 15 s, extension at 60°C for 4 min and a final chiatric observations. Analysis for the −241AϾG and extension period of 72°C for 7 min using a GeneAmp −141C insertion/deletion polymorphism of the DRD2 9600 Perkin-Elmer Cetus PCR machine. Analysis was gene was modified from Arinami et al27 using buffer 1 performed using a Big-Dye Primer cycle sequencing kit of the Expand Long PCR Kit (Roche, Germany). Analy- on a ABI 310 automated capillary DNA sequencer sis of the TaqI-polymorphisms have been modified (Perkin Elmer, USA) and compared with the published according to Grandy et al28 (TaqIA), to Castiglione et sequence (GenBank accession number AF050737). al29 (TaqIB) and to Kidd et al30 (TaqID). PCR-analysis of the Pro310Ser polymorphism was modified according Statistical methods 31 to Higuchi et al and of the Ser311Cys polymorphism All statistical analyses were performed by the SPSS, according to Arinami et al.32 All amplification reac- Version 8.01 software. Significance of frequency differ- tions were performed in a total volume of 25 ␮l, con- ences of the different genotypes was assessed by Pear- taining 100 ng DNA, 140 ␮M dNTP, 1 ␮M of each son’s ␹2 test or, if any cell count was less than 5, by primer, 10× buffer (Perkin Elmer, Roche, USA) and 1 Fisher’s exact test. The limit of significance was set to unit of AmpliTaq polymerase (Perkin Elmer, Roche, 0.05. Correlation analysis to search for relationships USA). between the intensity of the EPS-, AIMS- and BARS- Applied primer sequences, restriction enzymes and scores and the potential clinical predictors or polymor- Mg2+ concentrations are shown in Table 1. The PCR phisms of the DRD2 was conducted using the Spear- program of all these reactions consisted of 35 cycles, man rank test. Effects of the different DRD2 genotypes an initial denaturation at 94°C for 2 min, and a final on drug effects measured by the EPS-, AIMS- and extension period of 72°C using a GeneAmp 9600 Per- BARS-scores were analyzed using the Kruskal–Wallis kin-Elmer Cetus PCR machine. The conditions of the test, the Mann–Whitney U-test (two sided) and the cyclic PCR reactions were as follows: For the TaqIA- Jonckhere–Terpstra test (two sided). Logistic regression polymorphism: 94°C for 1 min, 53°C for 1 min, 72°C analysis was performed to elucidate the association

Molecular Psychiatry Dopamine D2 receptor polymorphisms and adverse events R Kaiser et al 698 Table 1 PCR primer sequences, enzymes and Mg2+-concentrations for the analysis of DRD2 polymorphisms and for DNA sequence analysis of the DRD2 coding region

Polymorphism Primer Enzyme Mg2+ (mM)

−241AϾG fd: GAC GGT GAG GAC CCA GCC TGC MaeIII 2.3 −141Cins/del rs: CGG TTC GGC ACT GAA GCT GGA C BstNI 2.3 TaqIB fd: GAT ACC CAC TTC AGG AAG TC TaqI 2.5 rs: GAT GTG TAG GAA TTA GCC AGG TaqID fd: CCC AGC AGG GAG AGG GAG TA TaqI 0.8 rs: GAC AAG TAC TTG GTA AGC ATG

Val96Ala fd: TGG ATC CCC ACA AGA CTT GCA GCT G AciI 2.0 rs: GGC TCA CCT GTC GAT GCT GAT

Leu141Leu fd: TGT GCA TAT TGT GGA GTG Bsp1407I 1.5 rs: CAG GCA AAC AAA AGC AGA

Pro310Ser fd: ACC ACC AGC TGA CTC TCC CCG GC HaeIII 1.0 rs: GGA AGG ACA TGG CAG GGA ATG GGA C

Ser311Cys fd: ACC ACC AGC TGA CTC TCC CCG ACC GGT Sau96I 1.3 rs: GGA AGG ACA TGG CAG GGA ATG GGA CCT TaqIA fd: CCG TCG ACG GCT GGC CAA GTT GTC TA TaqI 1.0 rs: CCG TCG ACC CTT CCT GAG TGT CAT CA

Sequencing primers Exon code 2 fd: GGC CCT CTC ACT GAC ACC TTG T 2.1 rs: AGC CAG TGG GGA GCT AGA TCC 2.2 3 fd: ATC GGC TCC AGG AGT ACC 3.1 rs: GCA CAG CAT CAC AGA CAC AG 3.2 4 fd: TGT GCA TAT TGT GGA GTG 4.1 rs: CAG GCA AAC AAA AGC AGA 4.2 5 fd: ATG GGC TCT TGT GGA ATT 5.1 rs: CCC CCA CCT GAG CAT AAG ATG A 5.2 6 fd: CCC CCA CCC TTC TGA TCT CT 6.1 rs: CCC TAG GGG CAG AAA GAC CT 6.2 7 fd: ATG CCT GGG AAC TTG TCC 7.1 rs: GGC AGG GAA TGG GAC CTT TCA 7.2 8 fd: GGC GCA CTG GGT GTG GGT GTT 8.1 rs: TGG AGC CAA GCG AAC ACT 8.2

fd, Forward primer; rs, reverse primer; del, deletion; ins, insertion.

between scores of EPS, AIMS and BARS and the DRD2 morphisms, and the C−141 insertion/deletion polymor- polymorphisms taking age, sex, chlorpromazine- phism. In contrast, the two variants with an amino acid

adjusted dose, dose of anticholinergic drugs, number substitution, Pro310Ser and Ser311Cys were less frequent of recurrent exacerbations and smoking simultaneously and were not found in any subject in homozygous com-

into account. For this purpose, patients were grouped bination. The Val96Ala variant was not detected in any into subgroups with low and high scores of the rating subject. Comparison of all patients with schizophrenia scales. Linkage disequilibrium and estimated haplo- or in the major subgroups (DSM-IV 295.3, 295.4, 295.7, types were assessed using the linkage utility program 295.6, 295.2) disclosed no significant differences for Equilibrium Haplotype (EH).33 the frequencies of the tested genotypes (Table 2). We did not find any relationship between the DRD2 recep- tor genotypes with family history, number of recurrent Results exacerbations or age at the first manifestation. As shown in Figure 1 and Table 2, there was extensive Sequencing of the protein coding DRD2 exons 2–8 genetic variation in the DRD2 gene in schizophrenic including the exon–intron junctions from genomic patients. Particularly frequent were the TaqI restriction DNA of the 40 patients with the most severe EPS out site variants known as TaqIA, TaqIB, and TaqID poly- of 584 patients with data for the Simpson Angus scale

Molecular Psychiatry Dopamine D2 receptor polymorphisms and adverse events R Kaiser et al 699 Table 2 Frequencies of the DRD2 genotypes in schizophrenic patients (n = 665)

Polymorphism All schizophrenic Schizo-paranoid Schizo-affective Residual

1/1 1/2 2/2 1/1 1/2 2/2 1/1 1/2 2/2 1/1 1/2 2/2 (% of n: 665) (% of n: 368) (% of n: 134) (% of n: 45)

−241AϾG (1: A, 2: G) 89.3 10.4 0.3 89.4 10.3 0.3 86.6 12.7 0.7 86.7 13.3 0.0 −141Cins (1: del, 2: ins) 1.2 15.8 83.0 0.8 15.2 84.0 2.2 15.7 82.1 0.0 20.0 80.0 TaqIB (2: cleavable) 2.3 22.3 75.5 2.7 23.4 73.9 1.5 21.6 76.9 0.0 17.8 82.2 TaqID (2: cleavable) 32.8 49.9 17.3 29.6 53.0 17.4 32.1 49.3 18.7 40.0 53.3 6.7

Val96Ala 100.0 0.0 0.0 100.0 0.0 0.0 100.0 0.0 0.0 100.0 0.0 0.0 Leu141Leu (1: G 2: A) 95.5 4.5 0.0 95.1 4.9 0.0 94.8 5.2 0.0 100.0 0.0 0.0 Pro310Ser (1: P, 2: S) 99.4 0.6 0.0 98.9 1.1 0.0 100.0 0.0 0.0 100.0 0.0 0.0 Ser311Cys (1: S, 2: C) 95.3 4.7 0.0 95.1 4.9 0.0 95.5 4.5 0.0 97.8 2.2 0.0 TaqIA (2: cleavable) 2.7 28.4 68.9 3.0 26.9 70.1 3.0 29.9 67.2 2.2 26.7 71.7

Del, deletion; ins, insertion.

Table 3 Polymorphisms found in the DRD2 gene in patients with the severest EPS (n = 40)

Exon/Intron Exchange Wild type Variant Position Change Frequency of variant allele

Exon 4 GϾA CTGTA CTATA 141 Leu/Leu 0.025 Exon 7 CϾG GTCCC GTGCC 311 Ser/Cys 0.013 Exon 7 CϾT CACGG CATGG 313 His/His 0.33 Exon 7 CϾT CCCGA CCTGA 319 Pro/Pro 0.54 Intron 3 AϾC AGAAG AGCAG +74 (upstream exon 4) 0.038 Intron 8 GϾA CCGGC CCAGC +55 (upstream exon 8) 0.73

revealed no new amino acid variants, but a number of sex, number of recurrent exacerbations or smoking silent polymorphisms, as shown in Table 3. behavior. As shown in Table 4 there was a significant corre- For the AIMS-score, there was a significant corre- lation between chlorpromazine adjusted dose taken lation with age (P Ͻ 0.001 for every date of within 24 h before scoring and the intensity of pseudo- examination) and number of recurrent exacerbations (P parkinsonian symptoms. Intensity of the EPS-score was Ͻ 0.001 for every date of examination). Intensity of the significantly correlated with the chlorpromazine BARS-score was significantly correlated with the num- adjusted dosage and dose of (P Ͻ 0.001 for ber of recurrent exacerbations (P Ͻ 0.001 at day 2–4, P every date of examination); biperiden was the almost Ͻ 0.05 at day 12–16) and with chlorpromazine exclusively administered anticholinergic agent. We did adjusted dosage (P Ͻ 0.06 at day 2–4, P Ͻ 0.005 at day not find an association between the EPS-score and age, 12–16, P = 0.01 at day 26–28).

Table 4 Correlation of adverse effects with clinical predictors

Pseudoparkinsonism Tardive dyskinesia Akathisia (EPS-score) (AIMS-score) (BARS-score)

Study day 2–4 12–16 26–30 2–4 12–16 26–30 2–4 12–16 26–30

Age ns ns ns 0.3 0.2 0.2 ns ns ns Chlorpromazine 0.2 0.3 0.2 ns ns ns 0.1 0.1 0.1 adjusted dosage Dose of biperiden 0.2 0.2 0.1 ns ns ns ns ns ns Sex ns ns ns 0.1 ns ns ns ns ns smoking ns ns ns ns ns ns ns ns ns Recurrent exacerbation ns ns ns 0.3 0.2 0.2 0.1 0.1 ns

Spearman’s rank correlation coefficients are given if the respective correlation was statistically significant.

Molecular Psychiatry Dopamine D2 receptor polymorphisms and adverse events R Kaiser et al 700 Analysis of the severity of the extrapyramidal symp- trend to decreased mean values of the AIMS-score

toms in relation to the DRD2 genotypes did not show any compared with individuals homozygous for the Pro310 statistically significant correlation (Table 5). But, patients allele at day 2–4 (mean value of AIMS score: 0.0 vs 1.7) = heterozygous for the Ser310 allele (n 3) had a trend to at day 14 (0.0 vs 1.5) and at day 28 (0.0 vs 1.4). lower mean value of EPS-score than patients With respect to akathisia, patients heterozygous for

homozygous for the Pro310 allele. Mean of the EPS-score the Ser310 allele had a tendency to lower mean values at day 2–4 after start of therapy was 2.3 in heterozygous of the BARS-score than patients homozygous for the

carriers of the Ser310 variant compared with 4.6 in those Pro310 allele. This could be observed at day 2–4 (mean homozygous for the Pro310 allele. The relationship was value of the BARS-score: 1.3 vs 2.2), at day 14 (0.3 vs similar at the two subsequent examinations with a mean 2.4) and at day 28 (1.0 vs 2.0). For the Ser311Cys poly- EPS score of 2.7 vs 4.6 at day 14 and 1.0 vs 4.5 at day 28. morphism we could not observe a significant difference The individual cumulative doses of anticholinergic in the intensities of the EPS-score stratified for the drugs were used as an additional parameter reflecting respective genotypes. acute dystonia and pseudoparkinsonism. Biperiden As shown in Table 5 patients heterozygous for the

was almost the only anticholinergic drug used in the Cys311 allele had decreased mean values of the AIMS- participating hospitals and the correlation of the cumu- score compared with individuals homozygous for the

lative dose of biperiden with the eight DRD2 polymor- Ser311 allele at day 2–4 (mean value: 0.8 vs 1.8) and phisms did not reveal any significant association. day 28 (mean value: 0.8 vs 1.5). The mean value of the There was no significant association of any DRD2 applied chlorpromazine adjusted dosages between genotype with the severity of AIMS (Table 5). But these patients stratified for the genotypes did not dif-

patients heterozygous for the Ser310 allele showed a fer significantly.

Table 5 Intensity of adverse effects as function of the DRD2 polymorphism at three observation points. Given are the mean value and standard deviation (SD)

EPS AIMS BARS

Day 2–412–16 26–30 2–412–16 26–30 2–412–16 26–30 n = 584 512 384 584 518 384 583 514 390

−241AϾG (1: A, 2: G) 1/1 4.6 (4.7) 4.6 (4.5) 4.5 (4.5) 1.7 (3.7) 1.5 (3.4) 1.4 (3.4) 2.2 (2.5) 3.0 (2.7) 2.1 (2.5) 1/2 4.2 (3.9) 3.9 (3.8) 3.8 (3.5) 1.5 (3.2) 1.5 (3.2) 1.5 (3.1) 2.5 (3.0) 2.6 (3.0) 1.0 (1.7) −141Cins (1: del, 2: ins) 1/1 2.7 (3.0) 4.4 (5.3) 3.4 (3.3) 2.2 (3.6) 1.9 (3.2) 0.8 (1.3) 3.0 (2.2) 3.0 (3.3) 4.3 (5.9) 1/2 4.9 (4.6) 4.8 (4.4) 4.3 (4.5) 1.7 (3.0) 1.6 (2.9) 1.5 (2.7) 2.2 (2.4) 2.4 (2.7) 1.8 (2.3) 2/2 4.5 (4.6) 4.5 (4.4) 4.5 (4.4) 1.7 (3.7) 1.5 (3.5) 1.4 (3.4) 2.2 (2.6) 2.3 (2.7) 2.0 (2.5) TaqIB (2: cleavable) 1/1 3.4 (4.2) 3.9 (3.6) 4.2 (2.8) 1.5 (2.4) 1.9 (3.2) 1.5 (2.4) 2.9 (2.6) 3.2 (2.7) 2.5 (2.3) 1/2 4.1 (4.3) 4.4 (4.5) 4.1 (4.0) 1.7 (3.9) 2.0 (4.1) 1.6 (3.7) 1.9 (2.1) 2.2 (2.4) 1.9 (2.2) 2/2 4.7 (4.7) 4.6 (4.5) 4.6 (4.6) 1.7 (3.6) 1.4 (3.1) 1.4 (3.3) 2.3 (2.7) 2.3 (2.8) 1.9 (2.6) TaqID (2: cleavable) 1/1 4.8 (4.8) 4.6 (4.7) 4.9 (5.3) 1.5 (3.3) 1.3 (3.1) 1.4 (3.7) 2.3 (2.6) 2.2 (2.8) 2.0 (2.8) 1/2 4.7 (4.6) 4.6 (4.4) 4.3 (4.0) 1.9 (3.9) 1.6 (3.7) 1.4 (3.2) 2.2 (2.5) 2.3 (2.7) 1.9 (2.3) 2/2 4.0 (4.2) 4.5 (4.5) 4.1 (4.1) 1.6 (3.4) 1.8 (3.0) 1.5 (2.9) 2.3 (2.6) 2.4 (2.8) 2.1 (2.4)

Leu141Leu (1: G 2: A) 1/1 4.6 (4.7) 4.6 (4.5) 4.5 (4.5) 1.7 (3.6) 1.6 (3.4) 1.4 (3.7) 2.2 (2.5) 2.3 (2.7) 1.9 (2.5) 1/2 4.4 (3.6) 3.9 (3.7) 4.1 (3.0) 1.5 (3.0) 1.2 (2.4) 1.2 (2.1) 2.5 (3.2) 2.4 (3.1) 2.6 (2.6) 2/2 5.0 (0.0) 0.0 (0.0) 7.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 6.0 (0.0)

Pro310Ser (1: P, 2: S) 1/1 4.6 (4.6) 4.6 (4.5) 4.5 (4.4) 1.7 (3.6) 1.5 (3.4) 1.4 (3.3) 2.2 (2.6) 2.4 (2.7) 2.0 (2.5) 1/2 2.3 (2.3) 2.7 (1.2) 1.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 1.3 (2.6) 0.3 (0.6) 1.0 (0.0)

Ser311Cys (1: S, 2: C) 1/1 4.6 (4.6) 4.5 (4.5) 4.4 (4.4) 1.8 (3.7) 1.5 (3.4) 1.5 (3.4) 2.3 (2.6) 2.3 (2.8) 2.0 (2.5) 1/2 4.4 (5.1) 4.9 (4.5) 4.9 (4.6) 0.8 (1.7) 1.4 (3.0) 0.8 (2.2) 1.3 (1.4) 2.0 (2.1) 1.7 (2.3) TaqIA (2: cleavable) 1/1 3.4 (4.2) 3.6 (3.4) 3.3 (3.0) 2.1 (2.9) 2.3 (3.2) 2.0 (2.7) 2.8 (2.5) 3.3 (2.5) 1.9 (2.3) 1/2 4.6 (4.5) 4.8 (4.5) 4.4 (4.2) 1.6 (3.7) 1.7 (3.9) 1.7 (3.7) 2.1 (2.3) 2.2 (2.5) 2.0 (2.2) 2/2 4.6 (4.7) 4.5 (4.5) 4.5 (4.6) 1.8 (3.6) 1.4 (3.2) 1.3 (3.2) 2.3 (2.7) 2.4 (2.8) 1.9 (2.6)

Del, deletion; ins, insertion.

Molecular Psychiatry Dopamine D2 receptor polymorphisms and adverse events R Kaiser et al 701 Patients homozygous for the TaqIA1 allele (the allele patients with higher rating scores of adverse effects reported to be associated with decreased receptor compared with patients with lower scores. The analy- density) showed a trend to lower EPS-scores than sis presented in Table 6 refers to the measurements at patients heterozygous or homozygous for the TaqIA2 day 2–4. The corresponding analysis was performed at allele, especially in the subgroup of males: mean value the two other observation points (days 12–16 and 26– of the EPS score at day 2–4 was 2.4 in patients homo- 30) but again, no differences were found. zygous for the TaqIA1 allele compared with 4.9 in indi- As shown in Table 7, all TaqI-polymorphisms viduals homo- or heterozygous for the TaqIA2 allele (TaqIA, B, D) were in strong linkage disequilibrium (statistically not significant). The two subsequent with each other and linked with the −141CIns/Del examinations showed the same tendency with a polymorphism. Moreover the TaqID polymorphism − Ͼ median of the EPS score of 3.4 vs 4.5 at day 14 and 3.3 was linked with the 241A G variant and the Ser311- vs 4.7 at day 28. The same tendency could be observed Cys polymorphism. About 512 possible estimated two for the TaqIB polymorphism. The mean value of the locus haplotypes could be found for these nine loci for applied chlorpromazine adjusted dosages between the entire study population of 665 patients (␹2 = patients stratified for the different genotypes of these 1597.47, P Ͻ 0.001, df = 511) by using the linkage util- polymorphisms differed not significantly. ity program.33 Six haplotypes accounted for over 80% In contrast to EPS-score, patients homozygous or het- of the haplotypes (Table 6). erozygous for allele 2 of the TaqIA polymorphism showed a trend to lower mean value of the BARS score compared with patients homozygous for the allele 1, Discussion especially in the subgroup of males (day 2–4: median of the BARS score: 2.0 vs 2.0, at day 14: 1.0 vs 5.0, at We wanted to explore whether genetic variants in the day 28: 0.0 vs 2.8). The same tendency could be DRD2 gene may predict adverse effects of drug treat- observed for the linked TaqIB polymorphism. ment with DRD2 antagonists. However, we failed to In addition using logistic regression analysis we detect a statistically significant association with nine found no significant association between scores of EPS, single nucleotide polymorphisms or with the haplo- AIMS and BARS as dependent variable and the differ- types. In the subgroup of patients suffering from the ent DRD2 polymorphisms taking age, gender, chlorpro- most severe adverse events we did not detect any new mazine adjusted dose, dose of anticholinergic agents, amino acid variants or other variants for which func- number of recurrent exacerbations and smoking simul- tional relevance might be deduced from genotype. taneously into account. As expected, significant correlations could be The eight polymorphic loci within or near the DRD2 observed between the chlorpromazine adjusted dosage gene are partially linked. The estimation of the and extrapyramidal symptoms or akathisia. The num- chromosomal organization showed that three haplo- ber of recurrent exacerbations was significantly corre- types accounted for over 80% of all haplotypes. As lated with intensity of involuntary movements shown in Table 6, there was no significant difference (reflecting tardive dyskinesia) or with akathisia. More- in the frequency of the estimated haplotypes between over, age was correlated with the intensity of involun-

Table 6 Haplotypes of the DRD2 receptor variants in relation to the intensity of adverse effects at study day 2–4

Estimated haplotypes Acute EPS BARS AIMS Dystonia

−241 −141 TaqID TaqIB L141L P310S S311C TaqIA All yes no Ͻ6 Ͼ5 =0 Ͼ0 Ͻ3 Ն3 n = 665 91 574 386 184 241 327 459 109

A Ins 2 1 G P S 2 50.7 55.6 50.2 49.1 52.9 51.2 49.8 50.2 48.2 A Ins 2 2 G P S 2 21.6 23.6 21.2 22.8 20.9 22.5 22.0 20.5 28.6 A Del 1 2 GPS1 11.5 7.2 12.1 11.5 11.0 11.0 12.2 12.2 11.1 A Ins 2 1 G P S 1 3.0 3.5 2.8 2.0 4.0 3.3 2.0 2.5 4.1 G Ins 2 2 G P S 2 2.9 1.6 3.1 2.4 2.7 2.4 2.5 2.9 2.0 G Ins 2 1 G P S 2 1.8 2.3 1.6 2.2 1.1 1.4 2.3 2.1 1.5 A Ins 2 2 GPC 2 1.8 1.6 1.8 1.8 2.2 2.3 1.5 2.2 0.8 A Ins 2 2 GPS1 1.8 1.1 2.0 2.0 1.7 0.7 3.0 2.3 0.0 A Ins 2 1 A P S 2 1.5 0.0 1.7 1.6 0.5 0.0 1.7 1.7 0.0 A Del 1 2 G P S 2 1.1 0.6 1.1 1.3 0.5 1.8 0.4 1.4 0.0 Miscellaneous others 2.3 2.9 2.4 3.3 2.5 4.1 5.6 4.3 3.7

The frequency of the estimated haplotypes assuming association and the respective allelic organization in % within the respect- ive group of adverse effects are presented. Del, deletion; ins, insertion; EPS, rating scale for pseudoparkinsonism; BARS, rating scale for akathisia; AIMS, rating scale for tardive dyskinesia.

Molecular Psychiatry Dopamine D2 receptor polymorphisms and adverse events R Kaiser et al 702 Table 7 Linkage analysis of the DRD2 receptor variants. Presented is the P-value of the chi-square test for expected vs found genotypes

− Ͼ − 241A G 141Cins TaqIB TaqID Leu141Leu Pro310Ser Ser311Cys TaqIA

Allele frequency, %a 5.4 9.2 13.5 42.4 2.2 0.3 2.3 17.2 −241AϾG 0.006 −141Cins 0.05 Ͻ0.001 0.01 TaqIB Ͻ0.001 Ͻ0.001 TaqID 0.045 0.001 Ͻ0.001

Leu141Leu Pro310Ser Ser311Cys 0.04 TaqIA

aFrequency of the more rare allele. Del, deletion; ins, insertion.

tary movements. These clinical predictors of adverse accordance with this, the results of DRD2 density stud- events (Table 2) have been described earlier.34–36 ies in humans are contradictory.17,44 In our sample the Since typical antipsychotics mediate their adverse frequency of this deletion variant was similar com- events via the dopamine D2 receptor, altered signal pared to other Caucasian samples40–43 and we could transduction by DRD2 variants may result in alter- not detect any association between this polymorphism ations of the intensities of these adverse events. For and the clinical intensity of adverse events. In contrast most of the polymorphisms studied here there is some to Ohara et al,45 we could not find a difference in the evidence about functional and medical impact. For the age of first manifestation of the disease stratified for TaqIA polymorphism, individuals either homo- or het- the different genotypes. Reports in Caucasians showed erozygous for the TaqIA1 allele appear to have lower either the opposite41 or no association.42,43 We could 17–19 37 DRD2 density, but this finding is controversial. not find any individual with the Ala96 variant, as con- Moreover, these patients showed a better therapeutic firmed by two different PCR-assays. Moreover, no indi- response.20 However, in agreement with Mihara et al38 vidual in the group analyzed by DNA sequencing we could not find a significant association between showed this mutation. Gejman et al46 described this adverse events measured by the EPS-score and the variant in a single Caucasian woman with

TaqIA 1 allele. dependence and drug abuse and the Ala96 variant Chen et al39 showed that females homozygous for the showed an up to 50% reduced binding affinity for cloz- TaqIA2 allele had a higher risk for tardive dyskinesia apine, chlorpromazine and dopamine in vitro21,22 but compared to females carrying the TaqIA1 allele. We this variant is apparently extremely rare in Caucasians.

could not find such an association, either in the sub- The Pro310Ser variant resides in the cytoplasmatic group of females with and without dyskinesia or third loop of the receptor protein. In vitro data showed between incident patients and patients with recurrent a less effective inhibition of the cAMP synthesis for the

exacerbations of the disease; females homozygous for Ser310 variant upon exposure to various antipsychotics the TaqIA2 allele showed lower intensities of the AIMS in comparison with the wild type.22 In our study only

score. Nevertheless it remains questionable, whether three individuals were heterozygous for the Ser310 vari- and how much the TaqIA polymorphism modulates ant. Two other authors have investigated this variant the expression of the DRD2 receptor, since this poly- in patients with alcoholism and found a similarly low morphism is located 10 kb upstream of the 3Ј non frequency as in our population.31,46 The heterozygous coding region of the gene. To explain the positive find- individuals of our study did not show any special ings it was argued that this polymorphism may be phenotype. All three were characterized as schizo- linked with an unknown functional mutation of the paranoid and only one patient had a family history of promotor region which might influence the expression schizophrenia. The mean age of first manifestation was of the gene. However, the only discussed functional 26.7 years (SD: 2.5) and was not different from those

promotor variant of the DRD2 which is linked with the homozygous for the Pro310 allele (mean age 28.7 years, TaqIA polymorphism is the −141C insertion variant SD: 9.1). With respect to side effects, we observed a

(Table 7). consistent trend: patients heterozygous for the Ser310 The deletion variant of the of the −141C allele showed lower EPS-, AIMS- and BARS-scores

insertion/deletion polymorphism of the promoter may than patients homozygous for the Pro310 allele. 27 reduce the protein expression in vitro. It was hypo- The Ser311Cys polymorphism is also located in the thesized that the increased DRD2 density in the brain G-protein coupling region. The Cys311 variant was of schizophrenic patients may be explained in part by markedly less effective in inhibiting cAMP synthesis 21,22 this promoter variant. Some authors reported an associ- than the wild type and the Ser310 variant. In the ation of this variant with schizophrenia, however, current study the frequency of Cys311 was 4.7% in het- others could not confirm these observations.27,40–43 In erozygous individuals. In previous reports the fre-

Molecular Psychiatry Dopamine D2 receptor polymorphisms and adverse events R Kaiser et al 703 quency of schizophrenic individuals heterozygous for pate or who left the hospitals within 7 days after admis-

Cys311 varied between 3.6% up to 8.5% in Asian popu- sion. But we could not find a significant difference of lations32,47–51 and was less frequent (1.8–5.0%) in Cau- the genotype frequencies between patients who could casians.46,52–58 Arinami et al32 firstly reported a signifi- not be sufficiently characterized clinically and those cantly increased allele frequency of the cysteine allele who completed the evaluation. The study included in schizophrenic Japanese patients (5.4%) compared to patients receiving variable doses of different antipsy- controls (1.8%). Furthermore, the age of onset was chotics, a factor which could be considered by multi- lower in patients with the cysteine allele and most of variate statistical analysis, but of course, power would the allele carriers had a positive family history. But in have been greater if only one drug had been used. all other association studies in Caucasians, except for Moreover, the DRD2 polymorphisms may modify the one observation,54 no associations between this variant response to antipsychotics treatment in their interac- and schizophrenia was found46,52,53,55–59 and all further tions with other genes such as those coding for drug studies in Asians also did not confirm the previous metabolizing enzymes or other receptors. It is typical findings.47,49,51,60,61 In contrast to Arinami et al,32 in our of many genetic polymorphisms, that their medical sample most carriers of the cysteine allele had no fam- impact alone is mostly only moderate, but may become ily history (75.9%) compared to those with such a fam- important in specific gene–gene or gene–environment ily history (24.1%). interactions. Thus, in spite of the negative findings in We observed a non significant trend of an increased this study, it is justified still to consider DRD2 variants age of onset between carriers of the Cys311 variant in future pharmacogenomic studies. (mean age: 31.8 years, SD: 10.3) compared with the One direction of further studies will be to consider others (mean age 28.5 years, SD: 9.0). We did not find multiple candidate genes and their interactions. Some any homozygous individual, but this is compatible authors reported an association or a tendency between 62–66 with the low allele frequency of 0.023. dyskinesia and the dopamine D3 receptor gene but In conclusion, analysis of genetic variants in the others could not replicate this finding.67,68 Of course, dopamine D2 receptor as predictors of neurological candidate genes may also be found in other gene adverse events from antipsychotics drug treatment was groups such as those coding for or gluta- a rational candidate gene approach. However, our rela- mate receptors or in genes coding free radical detox- tively large sample did not reveal a significant impact ification, drug transport, or drug biotransformation. and, as shown above, data published by others are inconsistent. Moreover the frequency of the functional amino acid variants was too rare in Caucasians to give Acknowledgements significant results. The finding that the small number This study was supported by the German Ministry for of individuals heterozygous for the Ser310 allele had a Education and Research (BMBF), grants 01EC9408 and trend to lower EPS-, AIMS- and BARS-scores may be 01ZZ9511. We thank Prof Dr R Uebelhack, Prof Dr B 22 inconsistent with in vitro data of Cravchik et al, since Nickel and Dr A Mackert for constant support of this we would expect increased adverse effects due to the study in their departments. 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Molecular Psychiatry