J Hum Genet (2001) 46:26–31 © Jpn Soc Hum Genet and Springer-Verlag 2001 ORIGINAL ARTICLE Hiroshi Mitsuyasu · Naotsugu Hirata · Yasuyoshi Sakai Hiroki Shibata · Yasuhiko Takeda · Hideaki Ninomiya Hiroaki Kawasaki · Nobutada Tashiro Yasuyuki Fukumaki Association analysis of polymorphisms in the upstream region of the human dopamine D4 receptor gene (DRD4) with schizophrenia and personality traits Received: August 28, 2000 / Accepted: October 25, 2000 Abstract The human dopamine D4 receptor (DRD4) is of tinct dopamine receptor genes have been identified, and major interest in molecular studies of schizophrenia and have been classified into D1-like receptors (D1 and D5) and personality traits. We examined the association of schizo- D2-like receptors (D2, D3, and D4) according to their struc- phrenia and polymorphisms in the upstream region of the tural and pharmacological characteristics. Of these, the DRD4 gene (2768G.A in the negative modulator region; human dopamine D4 receptor (DRD4) is considered to be 2521C.T, 2376C.T, and 2291C.T in the cell type- a candidate for involvement in schizophrenia and personal- specific promoter region; and 2616C.G between the two ity traits. DRD4 mRNA expression was observed in the regions) in 208 schizophrenic patients and 210 normal con- human frontal cortex, amygdala, thalamus, cerebellum, trols. No significant difference in genotype and allele fre- pituitary, entorhinal cortex, and hippocampus (Hartman quencies was observed between the two groups, indicating and Lanau 1997). An autoradiographic study with a D4- that these polymorphisms do not make a major contribution selective agent, [3H]NGD-94-1, suggested high levels of to the pathogenesis of schizophrenia. We also studied the radioligand binding in the normal post-mortem human hip- association of polymorphisms in the upstream region and a pocampus and entorhinal cortex, and low levels in the cau- 48-bp repeat polymorphism in exon III of the DRD4 gene date, putamen, nucleus accumbens, and substantia nigra with personality traits in 173 Japanese individuals who (Tarazi and Baldessarini 1999). Clozapine, an antipsychotic completed the temperament and character inventory (TCI). for the management of socially withdrawn and treatment- The 2768G.A polymorphism was significantly associated resistant schizophrenics, has a relatively high affinity to with reward dependence (P 5 0.044), while no significant DRD4 (Van Tol et al. 1991). The density of DRD4 was association was observed between novelty seeking and sixfold elevated in the brains of schizophrenic patients polymorphisms in the upstream region or the exon III (Seeman et al. 1993). Elevation of DRD4 mRNA level was repeat polymorphism of the DRD4 gene. found in the frontal cortex of schizophrenic patients post- mortem compared with levels in controls (Stefanis et al. Key words Dopamine D4 receptor · Polymorphism · 1998). These observations suggest that allelic variations Association · Schizophrenia · Personality trait · TCI affecting transcription levels of the DRD4 gene could be involved in the pathogenesis of schizophrenia. Twin studies demonstrate that personality traits, Introduction measured by self-report questionnaires such as the tridi- mensional personality questionnaire (TPQ), are partially inherited, and 30%–60% of observed variance can be ac- The dopaminergic neurotransmission system attracts atten- counted for genetically (Cloninger 1987; Bouchard 1994). tion because of its possible involvement in the etiology of In the temperament and character inventory (TCI) that several psychotic disorders and personality traits. Five dis- was developed from the TPQ, personality traits are divided into four temperaments: novelty seeking; harm avoidance; H. Mitsuyasu · N. Hirata · Y. Sakai · H. Shibata · Y. Takeda · reward dependence; and persistence, based on a biopsy- Y. Fukumaki (*) chosocial view of personality. Association of the exon Division of Disease Genes, Institute of Genetic Information, Kyushu III repeat polymorphism in the DRD4 gene with the University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan personality trait of novelty seeking was reported in 1996 Tel. 181-92-642-6168; Fax 181-92-632-2375 e-mail: [email protected] (Ebstein et al. 1996; Benjamin et al. 1996); however, conflicting results were also reported (Paterson et al. H. Mitsuyasu · Y. Takeda · H. Ninomiya · H. Kawasaki · N. Tashiro Department of Neuropsychiatry, Graduate School of Medical 1999). Association analysis of polymorphisms in the Sciences, Kyushu University, Fukuoka, Japan upstream region of the DRD4 gene with personality B. Jochimsen et al.: Stetteria hydrogenophila 27 traits may be useful to evaluate this interesting with a final extension step of 5min at 74°C, in a GeneAmp observation. 2400 Thermocycler (PE Biosystems, Foster City, CA, Our previous study of DRD4 gene expression, using USA). The PCR products were electrophoresed on a 1.5% cultured cells, revealed that the region between 2591 and agarose gel containing 1 3 Tris-borate/ethylenediamine 2123 relative to the first nucleotide of the initiation codon tetraacetic acid (EDTA) electrophoresis buffer (TBE), and contains the promoter of the DRD4 gene, which is respon- visualized by ethidium bromide staining. sible for the cell type-specific expression of the gene, and We have done genotyping of nine polymorphisms out of the negative modulator region is located between 2770 and ten in the upstream region of the DRD4 gene previously 2679 (Kamakura et al. 1997). We have identified ten reported: they are 2809G.A, 2768G.A, 2616C.G, polymorphisms in the upstream region of the DRD4 gene, 2603T.del, 2602G.del, 2600G.C, 2521C.T, spanning the negative modulator and the cell type-specific 2376C.T, and 2291C.T (Mitsuyasu et al. 1999). The promoter regions (Mitsuyasu et al. 1999). In the present genotypes of the 173 men from the Self-Defense Forces study, we investigated the association between schizophre- personnel were determined by direct DNA sequencing. nia and polymorphisms in the upstream region of the DRD4 Cycle sequencing was done in a volume of 20µl containing gene. We also examined the association between personal- 50–100ng of concentrated PCR product DNA, with a ity traits and polymorphisms in the upstream region and an Microcon YM-100 unit (Millipore, Bedford, MA, USA), exon III repeat polymorphism of the DRD4 gene. with 3.2pmol of either primer D4neg3 (sense) or D4neg4 (antisense) in the thermocycler with a BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Biosystems). Subjects and methods The cycle sequencing protocol was as recommended by the manufacturer, except that 28 cycles were performed. Se- quences were analyzed in an ABI PRISM model 377 DNA Subjects Sequencer (PE Biosystems). The genotypes of the rest of the controls and the schizo- The Ethics Committee of the Faculty of Medicine of phrenic patients were determined by the PCR-restriction Kyushu University approved the study protocol. Two fragment length polymorphism method, as previously hundred and eight schizophrenic patients (116 men and described (Mitsuyasu et al. 1999). The PCR products 92 women) were recruited from seven hospitals in the were digested with NarI, AvaII, BssHII, RsaI, and AvaI to Fukuoka area, one hospital in the Nagasaki area, and one determine the genotypes of the following polymorphisms: hospital in the Oita area, Japan. The diagnosis of schizo- 2768G.A, 2616C.G, 2521C.T, 2376C.T, and phrenia was made according to DSM-IV criteria (American 2291C.T, respectively. Electrophoreses of the digests Psychiatric Association, 1994). The mean age of the schizo- were carried out on a 2% agarose gel and visualization was phrenic patients was 51.5 years (SD, 13.9). There were 210 with ethidium bromide staining, except for AvaI digests, controls (120 men were recruited from the personnel of the which were electrophoresed on a 3% agarose gel. Japanese Self-Defense Forces and 90 women volunteers We examined the exon III repeat polymorphism of the were recruited from the staff of two hospitals). The mean DRD4 gene in the Self-Defense Forces personnel using a age of the controls was 50.5 years (SD, 5.0). In the associa- previously described method (Muramatsu et al. 1996), with tion study for personality traits, 173 subjects (all men; mean slight modification. PCR amplification was performed in a age, 52.2 years; SD, 0.8) were recruited from the Japanese volume of 25µl containing 50mM KCl; 10mM Tris-HCl Self-Defense Forces personnel. All patients and the control (pH 9.0); 100ng genomic DNA; 10pmol of each primer subjects gave their informed consent. All patients and (sense primer, 59-AGGTGGCACGTCGCGCCAAGCT controls were ethnically Japanese. GCA-39; antisense primer, 59-TCTGCGGTGGAGTCTG GGGTGGGAG-39); 100µM each of dATP, dTTP, dCTP, Genotyping and 7-deaza-29-deoxyguanosine 59-triphosphate; 1.5mM MgCl2; 10% DMSO; and 1 unit of Taq DNA polymerase Genomic DNA was extracted from leukocytes by salting (Promega, Madison, WI, USA). The cycle conditions were out with saturated sodium chloride solution, as described 94°C for 1min, then 30 cycles of 94°C for 15s, 65°C for 30s, by Lahiri and Nurnberger (1991). The fragment spanning and 72°C for 15s, with a final extension step of 7min at between 2947bp and 2156bp of the DRD4 gene was 72°C, in a GeneAmp 2400 thermocycler. The PCR products amplified by polymerase chain reaction (PCR), using the were electrophoresed on a 3% agarose gel containing 1 3 sense primer, D4neg3 (59-CAGGTCACAGGTCACCCCT TBE, and visualized by ethidium bromide staining. CTT-39, 2947/2926) and the antisense primer, D4neg4 (59- TTGCTCATCTTGGAATTTTGCG-39, 2156/2177). The Personality questionnaire PCR was performed in a volume of 100µl containing 100ng genomic DNA, 20pmol of each primer, 10µl of KOD Dash We used the Japanese version of the temperament and 10 3 PCR Buffer, 200µM each of dNTPs, 10% dimethylsul- character inventory (TCI) (Cloninger et al. 1993), which is a foxide (DMSO), and 2.5 units of KOD Dash (Toyobo, 240-item questionnaire measuring seven dimensions of per- Osaka, Japan). The cycle conditions were 94°C for 1min, sonality (Kijima et al.