Converging evidence implicates the dopamine D3 receptor gene in vulnerability to schizophrenia.код для вставкиСкачать
RESEARCH ARTICLE Neuropsychiatric Genetics Converging Evidence Implicates the Dopamine D3 Receptor Gene in Vulnerability to Schizophrenia Fuquan Zhang,1,2 Hua Fan,3 Yong Xu,4 Kerang Zhang,4 Xuezhu Huang,1 Yan Zhu,1 Manqiu Sui,1 Gaoxiang Sun,1 Kun Feng,1 Bo Xu,1 Xiaoqian Zhang,1 Zhonghua Su,5 Chunqing Peng,6 and Pozi Liu1* 1 Department of Psychiatry, Tsinghua University Yuquan Hospital, Beijing, China Institute of Mental Health, Peking University, Beijing, China 2 3 Department of Psychiatry, Beijing Anding Hospital, Capital Medical University, Beijing, China 4 Department of Psychiatry, First Hospital of Shanxi Medical University, Taiyuan, China Second Afﬁliated Hospital of Jining Medical College, Jining, China 5 6 Clinical Psychology Section, First Afﬁliated Hospital of Nanhua University, Hengyang, China Received 19 October 2010; Accepted 25 April 2011 The dopamine D3 receptor has been implicated in the pathophysiology of schizophrenia (SZ). A glycine-to-serine polymorphism at codon 9 of the dopamine D3 receptor gene (DRD3), rs6280, has been widely studied for its association with SZ, but with conﬂicting results. Altered levels of DRD3 mRNA have also been reported in SZ compared with normal controls. Moreover, it has been suggested that DRD3 is subject to recent positive selection in European populations. To explore the potential role of DRD3 in SZ from these various aspects, we conducted a threefold study. First, we tested the genetic association of rs6280 with SZ in 685 SZ patients and 768 normal controls. Second, we examined DRD3 mRNA levels in peripheral leukocytes in a subset of 37 patients and 37 controls. Finally, we investigated the possible recent positive selection on DRD3 in an East Asian population. Consequently, we observed that the genotypic distribution of rs6280 was nominally associated with SZ (P ¼ 0.045), with the ancestral CC genotype being signiﬁcantly over-represented in SZ patients. DRD3 mRNA levels were signiﬁcantly lower in patients than in controls (P ¼ 5.91E5). The derived Callele of rs6280 might have been subject to recent positive selection (P < 0.001) in the East Asian population. Taken together, our results suggest that DRD3, a gene possibly under natural selection, might be involved in vulnerability to SZ in the Han Chinese population. These ﬁndings may further add to the body of data implicating DRD3 as a schizophrenia risk gene. How to Cite this Article: Zhang F, Fan H, Xu Y, Zhang K, Huang X, Zhu Y, Sui M, Sun G, Feng K, Xu B, Zhang X, Su Z, Peng C, Liu P. 2011. Converging Evidence Implicates the Dopamine D3 Receptor Gene in Vulnerability to Schizophrenia. Am J Med Genet Part B 156:613–619. initially proposed as a risk gene because the dopamine D3 receptor binds antipsychotic drugs and is abundant in the limbic system of the brain. Furthermore, DRD3 maps to chromosome 3q13.3, which some linkage analyses have suggested as a region of susceptibility to SZ [Kaneko et al., 2007]. Therefore, DRD3 is considered a promising functional and positional candidate gene for SZ. One of the most extensively investigated polymorphisms within this gene is the single nucleotide polymorphism (SNP) rs6280 (Ser9Gly) in exon 2, resulting in a serine (Ser) to glycine (Gly) substitution at codon 9. Some studies suggested an association of the Ser allele [Ishiguro et al., 2000], the Gly allele [Kennedy et al., 1995], or homozygotes of both types [Crocq et al., 1992; Williams et al., 1998] with SZ. However, other studies failed to detect a positive association for this marker, in agreement with two recent 2011 Wiley-Liss, Inc. Key words: schizophrenia; DRD3; rs6280; positive selection INTRODUCTION Schizophrenia (SZ) is a severe mental disorder, with a heritability as high as 80%. Dopamine signaling is strongly implicated in the pathophysiology of SZ, thus dopaminergic genes have received much attention in the search for the genetic basis of the disorder. Among them is the dopamine D3 receptor gene (DRD3), which was 2011 Wiley-Liss, Inc. Grant sponsor: National Natural Science Foundation of China; Grant numbers: 30570430, 81000583, 81000578; Grant sponsor: Tsinghua YuYuan Medical Sciences Fund; Grant number: 20240000518. Fuquan Zhang, Hua Fan, and Yong Xu contributed equally to this work. *Correspondence to: Pozi Liu, Department of Psychiatry, Tsinghua University Yuquan Hospital, 5 Shijingshan Road, Beijing 100049, China. E-mail: firstname.lastname@example.org Published online 18 May 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/ajmg.b.31203 613 614 meta-analyses [Allen et al., 2008; Ma et al., 2008]. Thus, the correlation between DRD3 and SZ remains controversial, and has yet to be further veriﬁed. In line with the concept that abnormal levels of mRNA and proteins in the brain might be involved in the etiology of SZ [Iwamoto and Kato, 2006], postmortem studies have revealed changes in mRNA and protein levels of DRD3 in the brain of SZ patients [Schmauss et al., 1993; Gurevich et al., 1997; MeadorWoodruff et al., 1997]. On the other hand, Ilani et al.  suggested increased levels of DRD3 mRNA in blood lymphocytes of SZ, while Vogel et al.  reported the downregulation of DRD3 mRNA in SZ. Hence, the signiﬁcance of changes of DRD3 mRNA in peripheral leukocytes in SZ is inconclusive because of the limited number of such studies. Thus, further evidence is also needed in this regard. The notion that alleles increasing disease risk are new mutations (i.e., derived alleles) has been broadly accepted for Mendelian diseases and more or less explicitly extended to common diseases. However, some susceptibility polymorphisms for common diseases ostensibly deviate from this paradigm, in that the alleles increasing risk are ancestral ones, while derived alleles are protective. Recently, the ancestral susceptibility model proposed that the ancestral alleles adapted to past environments may be poorly adapted to new environments associated with the shift in lifestyle of human populations, thus giving rise to an increased risk of some common diseases [Di Rienzo and Hudson, 2005]. Therefore, analyses of functional candidate genes undergoing recent natural selection may derive new common susceptibility (or protective) alleles for human phenotypes. It has been suggested that a common haplotype of DRD3 affected by recent positive selection is associated with protection from SZ in a European population, and that the selection acts most probably on rs6280 [Costas et al., 2009]. However, it remains unknown whether such selective pressure acts on Asian populations. In this work, we tested the genetic association of rs6280 with SZ, investigated changes of DRD3 mRNA expression in blood leukocytes, and explored the possible natural selection on rs6280 within an East Asian population. MATERIALS AND METHODS Subjects The patient group comprised 685 patients with SZ, with a mean age of 30.65 10.24 years ranging from 15 to 70 years. The patients were recruited through clinical settings during the period between March 2006 and February 2008. Clinical diagnosis was made by at least two consultant psychiatrists according to the Diagnostic and Statistical Manual of Mental Disorders Fourth Edition (DSM-IV) criteria for SZ. All patients recruited for this study were also assessed with the Chinese Version of the Modiﬁed Structured Clinical Interview for DSM-IV TR Axis I Disorders Patient Edition (SCID-I/P, 11/2002 revision). We excluded potential participants who were pregnant or had signiﬁcant medical conditions, unstable psychiatric features (e.g., suicidal) or a history of substance abuse or drug addiction within the previous 6 months, with the exception of nicotine dependence. AMERICAN JOURNAL OF MEDICAL GENETICS PART B The control group consisted of 768 healthy volunteers aged 28.13 8.01 years ranging from 16 to 60 years. They were recruited from local communities or were people undergoing routine health checkups. All control subjects were assessed using the SCID. Subjects with relevant physical diseases or a history of major psychiatric disorders or suicidal behavior were excluded, and those who had a ﬁrst-degree relative with a history of severe mental disorder or suicidal behavior were also excluded. Of the above subjects, 37 ﬁrst episode medication-free patients and 37 healthy controls were used to measure expression levels of DRD3 mRNA. All the subjects were Chinese of Han origin and came from the same geographical areas in Northern China. They all gave written informed consent for this study as approved by the Ethical Committee for Medicine of the First Hospital of Shanxi Medical University, China. For natural selection analysis, we downloaded the phasing data from the release two of the phase 3 of the International HapMap Map (HapMap3_r2) genotype data [Frazer et al., 2007], comprising 358 individuals from three populations: 170 Asians from Japanese in Tokyo of Japan and Han Chinese in Beijing of China (ASN), 100 Yoruba in Ibadan, Nigeria (YRI), and 88 Utah residents with ancestry from northern and western Europe (CEU). Sequencing and Genotyping PCR was performed to amplify the polymorphisms. The primer pairs for the polymorphism comprised the forward primer 50 ATACGCACAATCTTCACCCG-30 and the reverse primer 50 ATACCACCCAGGGCATCA-30 . The PCR products were sequenced using an ABI 3730 DNA sequencer, as previously described [Fan et al., 2010]. Quantitative Reverse Transcriptase PCR (qRT-PCR) A 3-ml peripheral blood sample was taken from each of a subset of 37 control subjects and 37 patients. The patients were all ﬁrst diagnosed with no prior psycholeptic treatment. The leukocytes were isolated by centrifugation from the fresh blood sample. Total RNA was extracted from leukocytes with Trizol (Invitrogen, Carlsbad, CA) according to the manufacture’s instruction. The purity and integrity of total RNA were evaluated by ultraviolet spectrometric measurements and denaturing agarose gel electrophoresis, respectively. Expression of the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) was used as an internal control. Expression of DRD3 messenger RNA (mRNA) was measured by relative quantitative analysis using the ABI PRISM 7500 real-time PCR (RT-PCR) system (Applied Biosystems, Foster City, CA). A 20 ml reaction volume containing 10 ml of 2 SYBR Green Mastermix (Applied Biosystems), 1 ml of mixed primers (5 mmol/L), and 2 ml of cDNA was used for RT-PCR ampliﬁcation. All RT-PCR reactions were performed in triplicate. The primers used for real-time PCR included 50 -GGCACATCTGGAGCTGAAG-30 (forward) and 50 CCTCTCTTTTCAACTCTCCTCCT-30 (reverse). The detailed RTPCR conditions comprised 95 C for 10 min, followed by 40 cycles of 95 C for 15 sec and 60 C for 1 min. ZHANG ET AL. 615 The comparative Ct (2DDCt ) method was used for quantiﬁcation of transcripts. Efﬁciencies of cDNA ampliﬁcation of DRD3 and GAPDH were approximately equal. Relative expression of DRD3 mRNA was calculated by normalization with GAPDH gene expression. Statistical Analysis Allelic and genotypic associations were analyzed with PLINK v1.07 [Purcell et al., 2007]. The differences of mRNA levels between the patients and the controls were analyzed by the Mann–Whitney U-test (two-tailed) using SPSS for Windows (version 17.0). The P-value was set at 0.05 for signiﬁcance levels. Natural selection was analyzed using the Sweep program [Sabeti et al., 2002]. It uses the long range haplotype (LRH) test to look for alleles of high frequency with long-range linkage disequilibrium (LD), which suggest the haplotype rapidly rose to high frequency before recombination could break down associations with nearby markers [Sabeti et al., 2002]. All the core haplotypes around DRD3 were identiﬁed. For each haplotype at each block (i.e., the core haplotype), we calculated the extended haplotype homozygosity (EHH). EHH is the probability that two randomly chosen chromosomes carrying the core haplotype of interest are identical by descent for the entire interval from the core region to a speciﬁed distance. We also calculated the relative EHH (rEHH), the factor by which EHH decay on the tested core haplotype compared with the decay of EHH on all other core haplotypes combined [Sabeti et al., 2002]. In doing so, the inﬂuence of different recombination rates along the genome in EHH was corrected using the other haplotypes of the same region. Moreover, it employed another algorithm, cross population EHH (XP-EHH), to detect selective sweeps in which the selected allele has approached or achieved ﬁxation in one population but remains polymorphic in the human population as a whole [Sabeti et al., 2007]. RESULTS Genetic Association The genotypic distributions of rs6280 did not deviate from the Hardy–Weinberg equilibrium (P > 0.01) in either the patient or control group. As can be seen in Table I, genotypic distributions of the SNP were signiﬁcantly associated with SZ (P ¼ 0.045). The frequency of the rs6280 CC genotype was signiﬁcantly higher in the patients (CC vs. TT þ TC) than in the controls (P ¼ 0.028). However, no signiﬁcant allelic association was observed. Expression of DRD3 mRNA qRT-PCR analysis showed that the expression of DRD3 in peripheral leukocytes was signiﬁcantly lower in the patients (DCt ¼ 5.00 8.79, n ¼ 37) than in the controls (DCt ¼ 3.77 3.06, n ¼ 37) (P ¼ 5.91E5). There was no correlation between the expression of DRD3 and the genotypic or allelic frequency of rs6280 (data not shown). Natural Selection Analysis We detected a 16-marker haplotype core in the 170 individuals from ASN, encompassing rs226083, rs167770, rs226082, rs324029, rs6810284, rs10934256, rs6806217, rs324024, rs16822416, rs3732783, rs6280, rs324026, rs7638876, rs7638961, rs9825563, and rs6769975. There were three major core haplotypes, CH1 (GATGGCAAGTTTTTAC), CH2 (GGCAGAAAGTCCCCGC), and CH3 (GGCAGAAAGTCCCCAC), with a frequency of 0.65, 0.25, and 0.02, respectively. Thus, CH1 was the proxy of the rs6280 T allele, and CH2 of the rs6280 C allele. As can be seen in Figure 1, our data did not support the existence of natural selection on these core haplotypes within ASN (P > 0.05). However, the allele frequency of rs6280 varies largely across the world (Fig. 2). Therefore, XP-EHH was employed to detect the signal of natural selection among ASN, CEU, and YRI. The results showed the existence of natural selection on rs6280 between ASN and YRI (P < 0.001), and between CEU and YRI (P < 0.001), but not between ASN and CEU (P ¼ 0.188) (Table II). DISCUSSION The dopamine D3 receptor has long been thought to be involved in the pathogenesis of SZ because of its preferential expression in the limbic areas of the brain, which are associated with various aspects of behavior, emotion, and cognition, as well as its strong afﬁnity to antipsychotic drugs. Of particular interest in SZ is the non-synonymous SNP rs6280, which has been related to altered dopamine binding afﬁnity [Lundstrom and Turpin, 1996]. The dopaminemediated cAMP response is increased with the Gly variant, and the mitogen-associated protein kinase signal is prolonged compared with the Ser variant [Jeanneteau et al., 2006]. The present study detected a signiﬁcant over-representation of the rs6280 Gly/Gly (CC at the DNA level) genotype in SZ compared with the controls, consistent with a recent study from Japan in terms of association direction [Utsunomiya et al., 2008]. Intriguingly, our observation that rs6280 Gly/Gly carriers had a higher risk of developing SZ, resonates with a cognitive function study indicating that rs6280 TABLE I. Association Analyses of rs6280 Between SZ Patients and Controls CTL SZ n 768 685 TT (freq.) 377 (0.491) 345 (0.504) CTL, controls; freq., frequency. TC (freq.) 341 (0.444) 274 (0.400) CC (freq.) 50 (0.065) 66 (0.096) P (df ¼ 2) 0.045 T 1095 (0.713) 964 (0.704) C 441 (0.287) 406 (0.296) P (df ¼ 1) 0.584 OR (95%CI) 1.05 (0.89–1.23) AMERICAN JOURNAL OF MEDICAL GENETICS PART B 616 FIG. 1. Haplotype bifurcation diagrams for each haplotype in the ASN population. A 16-marker haplotype core was detected, and these 16 SNPs deﬁned core haplotypes (denoted CH1–3). Panels 1, 2, and 3 denote CH1, CH2, and CH3, respectively. The core haplotype CH1 carried the rs6280 Tallele, where as CH2 and CH3 carried the rs6280 C allele. CH3 was very rare; thus CH2 was chosen as the proxy of the rs6280 C allele. There was no predominance of one thick branch in each of the haplotype bifurcation diagrams, which would indicate a long-range LD, and correspondingly high EHH. could affect executive functions in healthy and ﬁrst-episode psychosis adolescents, with the Gly/Gly subjects showing poorer performance than the Ser/Ser individuals [Bombin et al., 2008]. Therefore, it is tempting to speculate that rs6280 might affect disease susceptibility through its inﬂuence on cognition. DRD3 mRNA levels were signiﬁcantly higher in the controls than in the medication-free patients, suggesting that the alteration of DRD3 mRNA expression was not due to medication, but may be attributable to the disease process itself. This result was in keeping with a previous ﬁnding [Vogel et al., 2004]. Owing to the limited mRNA samples and the rarity of the minor genotype of rs6280 in our sample, our data did not support a correlation between the altered mRNA levels and the rs6280 genotypes. Nevertheless, the result did suggest the involvement of DRD3 in risk of SZ, although the mechanism underlying the down-regulation of DRD3 in SZ requires further exploration. Detecting recent natural selection would have valuable implications for the study of human diseases. Using Sweep, we sought to explore the genetic imprint of recent positive selection by applying the LRH test to the HapMap Asian samples. Core haplotypes that FIG. 2. Global distribution of rs6280. Worldwide allele-frequency distribution for candidate polymorphisms provide evidence for selection. The rs6280 C is common in Africa, but T is common in Africa, but T is common among most areas elsewhere. ZHANG ET AL. 617 TABLE II. XP-EHH Among ASN, CEU, and YRI ASN vs. CEU ASN vs. YRI CEU vs. YRI FST 9.30E04 0.293 0.240 Contingency, x2 1.737 159.004 91.088 P-value 0.188 <0.001 <0.001 with more genotyped markers, are warranted to clarify the role of DRD3 in SZ. In conclusion, the evidence presented in this study suggests that DRD3 might be involved in vulnerability to SZ in the Han Chinese population, and the ancestral susceptibility of this gene may result from the actions of natural selection. ACKNOWLEDGMENTS show unusually high EHH and a high population frequency indicate the presence of a mutation that rose to prominence in the human gene pool faster than expected under neutral evolution. The more powerful algorithm XP-EHH uses cross-population comparison of haplotype lengths with control for local variation in recombination rates. Although we failed to detect natural selection in ASN by LRH, pairwise comparisons among the three populations revealed the presence of a selective sweep on rs6280. The prominent difference detected here was between Africa and elsewhere, rather than between Europe and Asia. Since YRI is more ‘‘ancient’’ than other populations and thus represents the ‘‘ancestral’’ one, the rising of the rs6280 T allele frequency in ASN and CEU supports the positive selection of rs6280 in these two populations. Costas et al.  suggested that a haplotype containing the derived allele Ser (T at the DNA level) at rs6280 has been under recent positive selection in European populations, leading to its fast increase in frequency, and that rs6280 is most probably the actual polymorphism subject to selection at this haplotype. However, unlike Costas et al. , we failed to ﬁnd a signiﬁcant selective sweep on DRD3 within ASN, possibly because of the limited sample size in our study or population stratiﬁcations across studies. This work may just scratch the surface of natural selection on DRD3; such an endeavor should be enhanced by the generation of expanded data sets. It must be borne in mind that the selective pressure on DRD3 detected herein may not necessarily target the disease phenotype per se, but rather phenotypes resulting from normal biological processes, such as cognition [Laszy et al., 2005; Bombin et al., 2008] or sodium homeostasis [Asico et al., 1998]. Notably, some other SZ candidate genes may be undergoing recent adaptive selection, such as MAOB [Carrera et al., 2009] and GARBRB2 [Lo et al., 2007], which also ﬁt the ancestral susceptibility model. Such genes may provoke another interesting area in the genetic study of human phenotypes, by broadening one’s view of diseases from a virtually static point into a dynamic one which will take the long history and thereby the evolutionary force into consideration. Although we present convergent evidence from various facets to support a role of DRD3 in SZ, the results of our work must be evaluated with caution and viewed in the context of several potential limitations. First, although the tested marker is probably one of the most vital functional loci in DRD3, the action of this gene is far from being fully explained by this single marker, and the signiﬁcance level of 0.045 would not survive a multiple testing correction. Second, the effect size of this polymorphism was quite small. 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