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1Department of Adult Psychiatry, University of Medical Sciences, Poznan, ... School of Medicine, Bydgoszcz, Poland; 3Laboratory of Psychiatric Genetics,.
Molecular Psychiatry (2001) 6, 718–724  2001 Nature Publishing Group All rights reserved 1359-4184/01 $15.00 www.nature.com/mp

ORIGINAL RESEARCH ARTICLE

Dopamine D3 receptor (DRD3) gene polymorphism is associated with the intensity of eye movement disturbances in schizophrenic patients and healthy subjects JK Rybakowski1, A Borkowska2, PM Czerski3 and J Hauser1,3 1

Department of Adult Psychiatry, University of Medical Sciences, Poznan, Poland; 2Neuropsychology Laboratory, Department of Psychiatry, University School of Medicine, Bydgoszcz, Poland; 3Laboratory of Psychiatric Genetics, Department of Psychiatry, University of Medical Sciences, Poznan, Poland

Keywords: schizophrenia; dopamine; eye movement disturbances; DRD3 gene polymorphism Altered dopamine neurotransmission and eye movement disturbances have been implicated in the pathogenic process of schizophrenia. So far, molecular genetic studies have shown little association between schizophrenia and polymorphism of any dopamine receptor or transporter genes except for some findings concerning D3 receptor (DRD3) gene. Eye movement disturbances occur in a majority of patients with schizophrenia and in a proportion of their first-degree relatives and they have been suggested as a phenotypic marker in genetic studies of this illness. Here we report an association between the Ser9Gly polymorphism of the DRD3 gene and the intensity of eye movement disturbances (fixation and smooth pursuit) observed in 119 schizophrenic patients and in 94 unrelated healthy control subjects. In schizophrenic patients, the mean intensity of both kinds of eye movement disturbances was highest in individuals with the Ser-Ser genotype, significantly lower in Ser-Gly and lowest in the Gly-Gly genotype. The Ser-Ser genotype was more prevalent in patients with a higher intensity of both fixation (58.1 vs 23.9% P ⬍ 0.001) and smooth pursuit disturbances (52.3 vs 25.8%, P ⬍ 0.02) and the Ser-Gly genotype frequency was lower in patients with higher fixation disturbances (37.0 vs 60.9%, P ⬍ 0.02). In control subjects, the genotype frequency Ser-Ser was higher in subjects with any degree of eye movement disturbances compared to subjects without such disturbances both for fixation and smooth pursuit performance (81.0 vs 50.7%, P ⬍ 0.05 and 79.2 vs 50.0%, P ⬍ 0.05, respectively). In control subjects the frequency of Ser-Gly was lower in the first group, for either fixation or smooth pursuit, compared to normal performers (9.5 vs 43.8%, P ⬍ 0.01 and 8.3 vs 45.7, P ⬍ 0.005, respectively). We suggest that the DRD3 Ser9Gly polymorphism may be a contributing factor to the performance of eye movements used as a phenotypic marker of schizophrenia. Molecular Psychiatry (2001) 6, 718–724.

The hypothesis of altered dopamine neurotransmission in schizophrenia proposed on the basis of the mechanism of antipsychotic drugs action gained some support from recent neuroimaging data.1 However, molecular genetic studies performed in recent decades did

not show a strong association between schizophrenia and any gene coding either dopamine receptor or transporter except for dopamine receptor D3 (DRD3) gene polymorphism, where the relationship was found with some aspects of this illness. DRD3 is localized in limbic and cortical brain areas and probably affects locomotion as well as emotional and cognitive processes.2 DRD3 knock-out mice show abnormalities in spontaneous level of locomotor activity.3 The DRD3 gene was mapped to chromosome 3q13.3 and its polymorphic site in exon 1 with a serine to glycine substitution has been postulated as a useful investigating tool in psychiatric disorder.4 Association studies of this polymorphism with schizophrenia have been mostly negative, including the most recent work of Joober et al.5 However, in two recent meta-analyses, a possible association with the Ser-Ser genotype was obtained: one in the general population6 and another in a Caucasian population of schizophrenics.7 Also, Jo¨nsson et al8 in Swedish patients with schizophrenia suggested a linkage to a DNA marker in close proximity to the DRD3 gene on chromosome 3q. Recently, a genotypic association between the DRD3 receptor polymorphism and a susceptibility to neuroleptic-induced motor disturbances such as tardive dyskinesia and akathisia in schizophrenic patients was obtained in a number of studies9–12 except for one.13 Furthermore, the association between the DRD3 polymorphism and a schizoaffective variant of schizophrenia was reported.14 The identification of vulnerability genes for schizophrenia is compounded by the wide range of phenotypic variability postulated in this illness and it has recently been suggested that in genetic studies some clinical or biochemical markers should be used to diminish this variability.15 Among those, neurophysiological markers such as eye movement disturbances have achieved a wide acceptance. Some degree of eye movement disturbances may be found in about 40–80% of patients with schizophrenia, in about 25–40% of their healthy first-degree relatives and in less than 10% of healthy control subjects.16 The most frequent eye movement disturbances occurring in schizophrenia include reduction of smooth pursuit gain, an increased

DRD3 gene polymorphism and eye movement disturbances JK Rybakowski et al

number of intrusive saccades during smooth pursuit movement and abnormalities of antisaccadic task, eg antisaccade latency and antisaccade errors rate.17,18 They also include abnormalities of visual fixation such as a decreased number of fixation points, a longer fixation time and an increased number of intrusive saccades occurring during fixation both in horizontal and vertical components.18,19 To date, only one study showed possible mapping of eye movement disturbances to a locus on chromosome 6, using a genomic scan for the specific region of 6q21–23.20 Further testing of possible linkage of eye movement disturbances to specific markers on chromosomes 8, 9, 20 and 22, performed by these authors yielded negative results, although they confirmed their previous findings with chromosome 6.21 Since the intensity of eye movement disturbances is rather a continuous than a discreet phenomenon, the definition and quantification of these is necessary to make any meaningful comparison or correlation in genetic studies. In this respect we have chosen the measurements of eye tracking performance during two tasks: (1) point fixation on a central position on the computer screen; and (2) smooth pursuit at the moving point on Lisajou curves. The pattern of eye tracking performance and its disturbances was identified for both fixation and smooth pursuit task on a 0–3 scale where 0 = no disturbances, 1 = minimal, 2 = moderate, and 3 = severe disturbances. The examples of each pattern are shown in Figures 1 and 2. Both schizophrenic patients and healthy control subjects were genotyped for the Ser9Gly polymorphism of the DRD3 gene. There were no deviations from Hardy– Weinberg equilibrium in the groups studied. Allele frequencies and the distribution of three kinds of genotypes (Ser-Ser, Ser-Gly and Gly-Gly) were not significantly different in schizophrenic patients and control subjects (Table 1). Genotype distributions of DRD3 were similar either for total (␹2 = 3.06, df = 2, NS) or for male or female subjects (␹2 = 3.24, df = 2, NS, and ␹2 = 2.58, df = 2, NS, respectively). The numbers and percentages of subjects showing a given degree of eye tracking disturbances in both experimental groups are shown in Table 2. The vast majority (⬎90%) of schizophrenic patients had some degree of eye movement disturbances. On the other hand, 3/4 of control subjects had normal eye tracking performance. There was a significant correlation between the performance on two eye movement tasks both in schizophrenic patients and in control subjects as calculated by the Spearman test (r = 0.55, P ⬍ 0.001 and r = 0.63, P ⬍ 0.001, respectively). When allele frequencies and genotype distribution were compared in schizophrenic patients broken down according to their performance as none-minimal and moderate-severe eye movement disturbances, there were significant differences between these groups (Table 3). The frequency of allele 1 (serine) was significantly higher in patients with more eye tracking dysfunction occurring both during fixation and smooth pursuing task (␹2 = 12.9, df = 1, P ⬍ 0.001 and ␹2 = 23.0,

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Figure 1 The examples of disturbances of eye tracking recording during fixation task, according to a 0–3 scale.

df = 1, P ⬍ 0.001, respectively). The strength of the association between allele 1 frequency and a higher degree of eye movement disturbances, as expressed by the odds ratio (OR) was 2.88 for fixation and 3.94 for smooth pursuit. Distribution of DRD3 genotypes was also different in patients with varying intensity of disturbances, both for fixation (␹2 = 15.2, df = 2, P ⬍ 0.001) and for smooth pursuit (␹2 = 10.2, df = 2, P = 0.006). The relative frequency of the Ser-Ser genotype was higher in patients with a higher degree of eye movement disturbances compared to lower ones for both Molecular Psychiatry

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Figure 2 The examples of disturbances of eye tracking recording during smooth pursuit task, according to a 0–3 scale.

fixation (58.1 vs 23.9%, ␹2 = 12.6, df = 1, P ⬍ 0.001) and for smooth pursuit movement (52.3 vs 25.8, ␹2 = 5.45, df = 1, P ⬍ 0.02). On the other hand, the relative frequency of the Ser-Gly phenotype was lower in patients with a higher intensity of fixation disturbances (37.0 vs 60.9%, ␹2 = 5.55, df = 1, P ⬍ 0.02). In Table 4, the mean intensity of eye movement disturbances in schizophrenic patients with various DRD3 Molecular Psychiatry

genotypes was shown. In both fixation and smooth pursuit performance, the highest intensity of disturbances is seen with the Ser-Ser genotype, lower with Ser-Gly and lowest with the Gly-Gly phenotype. Significant differences between groups were obtained by ANOVA analysis both for fixation (P ⬍ 0.001) and smooth pursuit disturbances (P ⬍ 0.001). Post hoc analysis by Tukey test for unequal numbers for the fixation test revealed a significant difference between Ser-Ser and Ser-Gly (P = 0.01) as well as between the Ser-Ser and Gly-Gly phenotype (P = 0.01). The same pattern of difference was obtained for smooth pursuit disturbances (Ser-Ser vs Ser-Gly, P ⬍ 0.001; Ser-Ser vs Gly-Gly, P = 0.01). A similar analysis of relationship between the intensity of eye movement disturbances and allele frequencies and DRD3 genotype distribution was attempted in healthy subjects. However, due to a low number of subjects which fell into the moderate-severe category we decided to compare those showing no disturbance at all (category 0) with subjects displaying the degrees of disturbances assessed as 1, 2 and 3 combined (Table 5). Although not significant by chi-square test, the strength of the association between allele 1 frequency and any degree of eye movement disturbances, as expressed by the OR was 2.26 for fixation and 1.86 for smooth pursuit. There were also significant differences between the two groups as to the distribution of DRD3 genotypes both for fixation (␹2 = 8.33, df = 2, P = 0.016) and smooth pursuit performance (␹2 = 11.44, df = 2, P = 0.003). The relative frequency of the Ser-Ser genotype was higher in subjects with any degree of eye movement disturbances compared to subjects showing no disturbances at all, for both fixation (81.0 vs 50.7%, ␹2 = 4.94, df = 1, P ⬍ 0.05), and for smooth pursuit movement (79.2 vs 50.0, ␹2 = 5.08, df = 1, P ⬍ 0.05). On the other hand, the relative frequency of the Ser-Gly phenotype, compared to normal performers, was lower both in subjects with fixation disturbances (9.5 vs 43.8%, ␹2 = 6.90, df = 1, P ⬍ 0.01) as well as for those with smooth pursuit disturbances (8.3 vs 45.7%, ␹2 = 9.26, df = 1, P ⬍ 0.005). The findings of our study suggest a contribution of the DRD3 gene to the quality of eye tracking performance both in schizophrenic patients and healthy subjects. In contrast to the results of studies with neuroleptic-induced tardive dyskinesia or akathisia in schizophrenic patients where the allele 2 (glycine) of DRD3 gene was connected with higher susceptibility to these motor disorders,9–12 the highest intensity of eye movement disturbances in schizophrenia was found in patients homozygous for the Ser-Ser genotype. Both in schizophrenic patients and in healthy control subjects, the Ser-Ser genotype was significantly more prevalent and the Ser-Gly genotype less prevalent in subjects with a higher degree of eye movement disturbances. It seems that allele 1 (serine) of the DRD3 receptor may be mostly associated with a higher intensity of eye movement disturbances. This may also show that vulnerabilities to neuroleptic-induced motor disorders

DRD3 gene polymorphism and eye movement disturbances JK Rybakowski et al

Table 1

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Allele frequencies and genotype distributions of DRD3 in schizophrenic patients and control subjects

DRD3 genotype

Allele frequency

Schizophrenic – total n = 119 Control – total n = 94 Schizophrenic – male n = 74 Control – male n = 40 Schizophrenic – female n = 45 Control – female n = 54

Ser (1) n (%)

Gly (2) n (%)

Ser-Ser n (%)

Ser-Gly n (%)

Gly-Gly n (%)

163 (68.5%) 142 (75.5%) 103 (69.6%) 60 (75.0%) 60 (66.7%) 82 (75.9%)

75 (31.5%) 46 (24.5%) 45 (30.4%) 20 (25.0%) 30 (33.2%) 26 (24.1%)

54 (45.4%) 54 (57.4%) 34 (45.9%) 24 (60.0%) 20 (44.4%) 30 (55.6%)

55 (46.2%) 34 (36.2%) 35 (47.3%) 12 (30.0%) 20 (44.4%) 22 (40.7%)

10 (8.4%) 6 (6.4%) 5 (6.8%) 4 (10.0%) 5 (11.1%) 2 (3.7%)

Table 2 The degree of eye movement disturbances in schizophrenic patients and control subjects Schizophrenic patients n = 119

0 – none 1 – minimal 2 – moderate 3 – severe

Genotype distribution

Control subjects n = 94

Fixation

Smooth pursuit

Fixation

Smooth pursuit

9 (7.6%) 37 (31.1%) 42 (35.3%) 31 (26.0%)

7 (5.9%) 24 (20.2%) 39 (32.8%) 49 (41.1%)

73 (77.6%) 15 (16.0%) 5 (5.3%) 1 (1.1%)

70 (74.5%) 13 (13.8%) 9 (9.6%) 2 (2.1%)

and for oculomotor (eye movement) disturbances are different pathological processes. The neurophysiological processes underlying eye movement performance are associated with the coordi-

nation of a number of both cortical and subcortical brain structures. These may overlap with neural circuits believed to display defective functioning in schizophrenia22 as well as with the areas of localization of the D3 receptor.2 Although the neural structures responsible for fixation and smooth pursuit are partly different, in our study two kinds of eye movement disturbances were strongly correlated. In schizophrenic patients, a correlation was also observed between eye movement disturbances and impairment on neuropsychological tests, especially those measuring working memory.23,24 Recently, an association was found between the functional polymorphism of catechol-O-methyl-transpherase (COMT) gene and performance on the Wisconsin Card Sorting test in both schizophrenic patients and healthy control subjects.25,26 This was interpreted in terms of an altered activity of the dopaminergic system in prefrontal cortex. Egan et al25 hypothesised that the val allele of val158met polymorphism of COMT gene connected with poorer performance of the working memory test may increase the risk for schizophrenia. According to Holzman16 who has been an unques-

Table 3 Allele frequencies and genotype distributions of DRD3 in schizophrenic patients with none-minimal and moderatesevere eye movement disturbances DRD3 genotype

Fixation 0–1 n = 46 Fixation 2–3 n = 73 Smooth pursuit 0–1 n = 31 Smooth pursuit 2–3 n = 88

Allele frequency n (%)

Genotype distribution

Ser (1)

Gly (1)

Ser-Ser n (%)

Ser-Gly n (%)

Gly-Gly n (%)

50 (54.3%) 113 (77.4%) 33 (41.8%) 130 (73.9%)

42 (45.7%) 33 (22.6%) 46 (58.2%) 46 (26.1%)

11 (23.9%) 43 (58.1%) 8 (25.8%) 46 (52.3%)

28 (60.9%) 27 (37.0%) 17 (54.8%) 38 (43.2%)

7 (15.2%) 3 (4.1%) 6 (19.4%) 4 (4.5%)

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Table 4 The intensity of eye movement disturbances (assessed on a 0–3 scale) in schizophrenic patients with various DRD3 genotypes. Values given as means ± SD DRD3 genotype

n

Fixation

Smooth pursuit

1–1 (Ser-Ser) 1–2 (Ser-Gly) 2–2 (Gly-Gly)

54 55 10

2.1 ± 0.8 1.6 ± 0.9* 1.0 ± 0.8*

2.5 ± 0.8 1.8 ± 0.9** 1.4 ± 0.8*

Difference vs Ser-Ser genotype significant: **P ⬍ 0.001 (Tukey test for unequal numbers).

*P = 0.01;

tionable expert on movement dysfunctions in schizophrenia, these disturbances may touch the essence of this illness. In our study, however, an association of the DRD3 gene polymorphism was observed with this neurophysiological endophenotype related to schizophrenia but not with schizophrenia itself. The one explanation may be that the association between DRD3 and schizophrenia, even that obtained in the metaanalytic studies suggesting the allele 1 (Ser) homozygosity as a possible risk factor for the illness6,7 has been fairly weak. Therefore, the number of subjects studied by us had not enough power to detect such an effect. Furthermore, a higher risk for the occurrence of eye movement disturbances in the general population (5–8%) than that for schizophrenia (1%) may also suggest that schizophrenia as an illness is less penetrant than these disturbances and abnormal eye tracking can be viewed as one pleiotropic manifestation of this illness.16 A similar phenomenon was initially observed with another neurophysiological endophenotype associated with schizophrenia: diminished inhibition of the P50 auditory evoked response to repeated stimuli and its relation with the ␣7 nicotinic acetylcholine receptor subunit gene mapped to chromosome 15q13– 14.27 However, in a recent study, using parent-child triads, a linkage disequilibrium was found supporting this region as a location involved in the genetic transmission of schizophrenia.28 The parent–child triads with schizophrenia are now being studied by us with molecular genetic and neurophysiological methods

and we hope that the results may throw more light on the phenomenon of association between the DRD3 gene polymorphism, the intensity of eye movement disturbances and schizophrenia.

Methods Participants Schizophrenic subjects (74 male, 45 female), aged 18– 63 years (mean 30 years) were inpatients at the Department of Adult Psychiatry, University of Medical Sciences, Poznan in the years 1998–2000. Their diagnoses were established by psychiatrists, using DSM-IV and ICD-10 criteria. Control subjects (40 male, 45 female), aged 22–60 years (mean 27 years) not related to schizophrenic patients were recruited from clinical and laboratory staff, Department of Psychiatry, and from medical students of the University of Medical Sciences in Poznan. All control subjects were somatically healthy without any history of major psychiatric illness. Eye movement measurements Eye trackings were measured by the infrared reflectometry method, using Ober II system and Grenoble 96 computer program. Investigated subjects were seated in a darkened room, in front of the computer screen with goggles (with infrared detectors) on their eyes, the distance between the 17-inch computer screen and subject’s eye being 60 cm. The conditions for measurements were a sampling rate of 400 Hz and a measurement time 20 s, during two tasks: (1) point fixation on a central position on the computer screen; and (2) smooth pursuit at the moving point on Lisajou curves. The reason for choosing the Lisajou curves in the Ober II system for the measurement of smooth pursuit movement was that these curves have constant mathematical characteristics but, unlike regular sinusoidal curves, the subject investigated can not predict the direction of the moving point. The subjects were instructed to keep their eyes fixed on the point during the point fixation task and to observe the moving point during the smooth pursuit task.

Table 5 Allele frequencies and genotype distributions of DRD3 in healthy subjects without (score 0) and with (score 1–3) eye movement disturbances DRD3 genotype

Fixation 0 n = 73 Fixation 1–3 n = 21 Smooth pursuit 0 n = 70 Smooth pursuit 1–3 n = 24

Molecular Psychiatry

Allele frequencies n (%)

Genotype distributions

Ser (1)

Gly (1)

Ser-Ser n (%)

Ser-Gly n (%)

Gly-Gly n (%)

106 (72.6%) 36 (85.7%) 102 (72.9%) 40 (83.3%)

40 (27.4%) 6 (14.3%) 38 (27.1%) 8 (16.7%)

37 (50.7%) 17 (81.0%) 35 (50.0%) 19 (79.2%)

32 (43.8%) 2 (9.5%) 32 (45.7%) 2 (8.3%)

4 (5.5%) 2 (9.5%) 3 (4.3%) 3 (12.5%)

DRD3 gene polymorphism and eye movement disturbances JK Rybakowski et al

Eye movement data were analyzed with the CED Cambridge Spike 2 program, with script for eye movement analysis. The main indexes of eye movement disturbances were the frequency (numbers per second) of saccades (catch-up, back-up and intrusive saccades) occurring during each task and frequency (numbers per second) of square wave jerks (SWJ), regardless of their amplitude. The periods of artifacts or eye blinks were detected and not included in the analysis. The intensity of eye movement disturbances during fixation and during smooth pursuit was assessed on a 0–3 scale. The following assessment criteria were applied: for fixation disturbances: 0 = no disturbances; 1 = minimal disturbances: intrusive saccades frequency: 0.01–0.03; 2 = moderate disturbances: intrusive saccades frequency: 0.04–0.07; and/or any SWJ; 3 = severe disturbances: intrusive saccades frequency ⬎0.07 and/or any SWJ. For smooth pursuit disturbances: 0 = no disturbances; 1 = minimal disturbances: intrusive saccades frequency ⬍0.005; and/or catch-up and back-up saccades frequency ⬍0.005; no SWJ; 2 = moderate disturbances: intrusive saccades, frequency 0.005–0.026; and/or catch-up and back-up saccades frequency 0.003–0.05; and/or SWJ frequency ⬍0.005; 3 = severe disturbances: intrusive saccades frequency 0.026–0.08; and/or catch-up and back-up saccades frequency 0.06– 0.13; and SWJ frequency ⬎0.005. The examples of disturbances of eye tracking recording during fixation and smooth pursuit task, according to the 0–3 scale are shown in Figures 1 and 2.

Genotyping The DNA was extracted from 10 ml of EDTA anticoagulated whole blood using the salting out method.29 A 462-basepair fragment of the first exon of DRD3 gene was amplified by PCR with primer pair described by Lannfelt et al4 in a PTC-100 (MJ Research, Watertown, MA, USA) thermal cycler. A 25-␮l amplification mixture contained 150–300 ng DNA, 0.5 ␮M of each primer, 0.2 of each dNTP, 1.75 mM MgCl2, 100 mM Tris-HCl, 500 mM KCl, 0.8% NP40, 0.5 U of Taq DNA polymerase (MBI Fermentas, Vilnius, Lithuania). Cycling conditions were: denaturation of 95°C for 2 min, 35 cycles with a profile of 94°C for 30 s, 60°C for 30 s, 72°C for 30 s, followed by final elongation at 72°C for 5 min. A volume of 5 ␮l of PCR products was then digested overnight in a total volume of 7.5 ␮l at 37°C with 0.5 U of MlsI restriction endonuclease (MBI Fermentas) – izoschizomer of BalI. Digestion products were electrophoresed on 2.5% agarose gel (Prona, Spain) and visualised by ethidium bromide staining. Band sizes were compared with DNA ladder (MBI Fermentas). For allele 1 (Ser-9), 304-bp fragment and for allele 2 (Gly-9) 206-bp and 98-bp fragments were observed. Additionally two constant bands of 111 bp and 47 bp were detected resulting from two non-polymorphic restriction sites. The genotyping was performed without knowledge of the subject’s clinical status.

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Acknowledgements Supported by the Polish Committee of Scientific Research (KBN), grant No. 4P05B 09316.

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Correspondence: JK Rybakowski, MD, Department of Adult Psychiatry, University of Medical Sciences, ul.Szpitalna 27/33, 60-572 Poznan, Poland. E-mail: rybakows얀wlkp.top.pl Received 5 February 2001; revised and accepted 14 March 2001