A Novel DRD2 Single-Nucleotide Polymorphism Associated with Schizophrenia Predicts Age of Onset: HapMap Tag-Single-Nucleotide Polymorphism Analysis

Abstract

Background: Dopamine D2 receptor (DRD2) is thought to be critical in regulating the dopaminergic pathway in the brain, which is known to be important in the etiology of schizophrenia. It is, therefore, not surprising that most antipsychotic medication acts on DRD2. DRD2 is widely expressed in the brain; levels are reduced in the brains of patients with schizophrenia, and DRD2 polymorphisms have been associated with reduced brain expression. We have previously identified a genetic variant in DRD2, rs6277 to be strongly implicated in schizophrenia susceptibility. Methods: To identity new associations in the DRD2 gene with disease status and clinical severity, we genotyped seven single-nucleotide polymorphisms (SNPs) in DRD2 by using a multiplex mass spectrometry method. SNPs were chosen by using a haplotype block-based gene-tagging approach; so, the entire DRD2 gene was represented. Results: One polymorphism, rs2734839 was found to be significantly associated with schizophrenia as well as late onset age. Individuals carrying the genetic variation were more than twice as likely to have schizophrenia compared with controls. Conclusions: Our results suggest that DRD2 genetic variation is a good indicator for schizophrenia risk and may also be used as a predictor of age of onset.

Introduction

Schizophrenia is a severe psychiatric illness affecting 1% of the population. The genetic risk for schizophrenia is high with some studies predicting up to 80% heritability (McGuffin and Gottesman, 1999; Sullivan et al., 2003). The etiology of schizophrenia has been associated with brain dopaminergic activity. The dopamine D2 receptor (DRD2) gene is located on chromosome 11q22-23 (Grandy et al., 1989) and is thought to play a pivotal role in the dopamine pathway. A genome-wide linkage meta-analysis has also implicated DRD2 in the risk for schizophrenia (Lewis et al., 2003). We have previously reported the 957C-allele of a DRD2 polymorphism (rs6277) to be significantly associated with schizophrenia (Lawford et al., 2005). Several studies have confirmed this association (Hanninen et al., 2006; Hoenicka et al., 2006; Betcheva et al., 2009; Dubertret et al., 2010; Fan et al., 2010) including a meta-analysis (Monakhov et al., 2008). The T-allele of rs6277 polymorphism has been shown to be associated with decreased DRD2 mRNA translation and stability as well as reduced dopamine-induced up-regulation of D2 receptors (Duan et al., 2003). In healthy individuals, the T-allele is also associated with increased striatal D2 receptor binding (Hirvonen et al., 2005).

There have also been other DRD2 polymorphisms found to be associated with schizophrenia including the well-studied deletion polymorphism rs1799732 in the promoter of DRD2. Functional studies have shown that it is associated with lower DRD2 promoter activity (Arinami et al., 1997). A nonsynonymous polymorphism rs1801028 was one of the first polymorphisms showing association with schizophrenia (Arinami et al., 1994), but other studies have failed to replicate this result. Interestingly, a metaanalysis did support the association (Glatt et al., 2003).

In summary, a number of polymorphisms in DRD2 have been identified that are associated with schizophrenia, but their individual effects on risk for schizophrenia are small. To identify novel polymorphisms associated with schizophrenia and replicate previous association studies, seven single-nucleotide polymorphisms (SNPs) in DRD2 were selected that mark haplotype blocks, covering the entire DRD2 gene. After genotyping in an Australian population, these SNPs were analyzed for their association with schizophrenia.

Materials and Methods

Participants

The study included 157 Caucasian patients with schizophrenia and 250 unrelated Caucasian controls. Subjects were aged between 18 and 65 years. Diagnostic and Statistical Manual of Mental Disorders-IV (DSM-IV) (American Psychiatric Association, 1994) diagnosis of schizophrenia was confirmed by at least two independent psychiatrists. These patients had never been diagnosed as having other psychiatric disorders, including schizoaffective disorder, major depressive episode with psychotic features, or bipolar disorder. No patients were treated with antidepressants, anxiolytic agents, or mood-stabilizing psychotropic medications, and all were maintained on a constant dose of antipsychotic medication for a minimum of three weeks. There were 23 women and 134 men in the group in whom schizophrenia was diagnosed with a mean age of 36.2 years (standard deviation [SD]±12.1 years). The mean age of patients at first diagnosis (onset of psychotic symptoms) was 23.2 years (SD±7.3 years). They were being treated at the Royal Brisbane and Women's Hospital, The Park Psychiatric Unit, and the Valley Community Psychiatric Centre. The clinical sample was composed of 65 inpatients and 92 outpatients. All patients were administered the Positive and Negative Symptom Scale (PANSS) (Peralta and Cuesta, 1994) to assess severity of psychotic features. The PANSS total mean score was 45.13 (SD 13.69). This clinical sample was a group who had lived with schizophrenia for an average of 13 years since diagnosis and continued to experience positive and negative symptoms despite treatment with antipsychotic medication. Mean length of illness was 13.39 years (SD 10.91). In addition, the schizophrenia group contained a high proportion of individuals with a relatively severe history and/or a familial risk for psychosis.

The control group consisted of 102 women and 148 men, with a mean age of 36.8 years (SD±12.8 years). The control group was composed of volunteers from the general public, hospital nursing and medical staff, and university staff and students. Formal screening for schizophrenia or other psychological disorders was not undertaken in the control population. As such, the controls represent an unselected control group. To minimize population stratification bias, both control and clinical subjects were recruited in the Brisbane region (a city of approximately 2 million inhabitants on the East Coast of Australia), and all were of British or European descent.

Ethics approval was obtained from all the institutions involved.

Selection of SNPs

Using the International HapMap Project (HapMap) phase II (Frazer et al., 2007), seven tag-SNPs in DRD2 with a minor allele frequency of 0.15 were selected. Tag-SNPs were identified by using the pair-wise option of Tagger with a threshold of r²>0.8.

Genotyping

Oragene kits were used to extract DNA from saliva samples. Samples were genotyped using a homogeneous MassEXTEND (hME) Sequenom assay performed by the Australian Genome Research Facility. The hME assay is based on the annealing of an oligonucleotide primer (hME primer) adjacent to the SNP of interest. The addition of a DNA polymerase along with a mixture of terminator nucleotides allows extension of the hME primer through the polymorphic site and generates allele-specific extension products, each having a unique molecular mass. The resultant masses of the extension products are then analyzed by matrix-assisted lazer desorption/ionization time-of-flight mass spectrometry, and a genotype is assigned in real time. The hME assay was performed in multiplex with up to 36 reactions in a single well.

The genotyping fail rate was 4% for rs1752947; 6% for rs1760161; 4% for rs1800499; 5% for rs2734839; 3% for rs4245147; 3% for rs6275; and 4% for rs7131056. The genotyping of several other SNPs including DRD2 rs6277 was independently verified by other methods such as real-time PCR with a concordance rate of 96.6%. Genotypes were determined by investigators blinded to clinical diagnoses.

Statistical analysis

A Pearson's chi-squared test was performed to identify statistical associations between alleles/genotype and schizophrenia status. Odds ratios (OR) were also calculated. Tests were performed on both genotype and allele data. Statistical tests were performed using the COMPARE2 program from the WinPepi suite of epidemiology programs (Abramson, 2004) and SPSS version 16.0. Hardy-Weinberg equilibrium (HWE) was computed using Utility Programs for Analysis of Genetic Linkage (Ott, 1988). The analysis of genotypes under a recessive model involved pooling the low-risk homozygotes and the heterozygotes and comparing frequencies with the high-risk homozygotes, that is, OR >1. Correction for multiple testing was conducted using the Benjamini-Hochberg method (Benjamini and Hochberg, 1995). Linkage disequilibrium (LD) was calculated using JLIN version 1.6.0 (Carter et al., 2006).

Results

A comprehensive genotype analysis of DRD2 and its association with schizophrenia was undertaken using a haplotype block-based gene-tagging approach. A total of seven tag-SNPs that span from the 3′-end to the 5′-end of the gene were genotyped in a sample of 157 patients with schizophrenia and 250 controls. One SNP was found to be significantly (p=0.0064) associated with schizophrenia at the allele (Table 1). Rs2734839 was also significant at the genotype level (Table 2). Rs2734839 survived correction for multiple testing (corrected α-value was 0.012).

Table 1.

Allele Association of Seven DRD2 Single-Nucleotide Polymorphisms with Schizophrenia

SNP ID	Allele frequency		Position ^a	SNP details	χ²	p-value^b	OR	95% CI
rs17529477	A	G	112822277	Intron 1	2.139	0.144	1.27	0.91-1.76
control	152	318
SZ	85	225
rs17601612	C	G	112822955	Intron 1	2.740	0.098	1.29	0.94-1.76
control	187	269
SZ	108	200
rs4245147	C	T	112823217	Intron 1	1.162	0.281	1.17	0.87-1.58
control	239	245
SZ	140	168
rs7131056	A	C	112834984	Intron 1	0.131	0.718	1.06	0.78-1.43
control	194	276
SZ	132	178
rs1800499	A	G	112792904	Exon 2	0.085	0.771	1.12	0.48-2.56
control	16	448
SZ	12	300
rs2734839	A	G	112791700	Intron 3	7.433	0.0064	1.50	1.11-2.03
control	288	180
SZ	158	148
rs6275	C	T	112788687	Exon 5	1.083	0.298	1.18	0.85-1.62
control	335	143
SZ	205	103

Nucleotide position on the chromosome 11 reference sequence.

p-Value determined by Pearson's chi-squared test.

SNP, single-nucleotide polymorphism; OR, odds ratio; CI, confidence interval.

Table 2.

Genotype Association of rs2734839 with Schizophrenia

Polymorphism	Genotype counts (%)			p-Value^a
rs2734839	AA	GA	GG
Control	84 (35.9)	120 (51.3)	30 (12.8)	0.016
Schizophrenia	40 (26.1)	78 (51.0)	35 (22.9)	0.005^b
OR^c	1	1.37	2.45
(p-value)		(0.388)	(0.008)

p-Value determined by Pearson's χ² test.

p-Value determined using the extended Mantel-Haenszel test for trend.

OR, with respect to the genotype that was not associated with schizophrenia.

In the control and schizophrenia samples, strong LD was observed between rs2734839 and the previously reported rs6277 SNP (D′=1 in the schizophrenia population; D′=0.98 in the control population).

Genotype frequencies indicated that all polymorphisms were in HWE in both cases and control samples (rs2734839 controls χ²=1.63 p=0.20 schizophrenia χ²=0.066 p=0.80).

Examination of the genotype OR of the rs2734839 SNP suggests a recessive pattern of inheritance. The heterozygous OR and the OR for the low-risk homozygote are approximately one (Table 2). Subsequently, rs2734839 was found to be associated with schizophrenia when analyzed under a recessive model by pooling genotypes (p=0.010).

In order to evaluate whether there was any sexual dimorphisms in the population, the male patients with schizophrenia were analyzed separately from the female patients. At the genotype level, rs2734839 showed association with disease with a slightly weaker p-value (Pearson χ²=7.375 p=0.025) for the men. The male schizophrenia samples remained in HWE. The remaining female cases were too small in number to be meaningfully analyzed.

A number of clinical parameters were also examined for association with DRD2 variation. These included onset age, number of hospital admissions, family history, PANSS subscales, PANSS total, Barnes Akathisia Determination Scale (BARN) subscales, BARN total, Abnormal Involuntary Movement Scale (AIMS) subscales, AIMS total, General Health Questionnaire (GHQ) subscales, GHQ total, and Trail Making Test (A and B). A significant association was found between onset age and rs2734839. No other associations were observed. Analysis of variance revealed significant differences in onset age between genotypes (p=0.011, F=4.614). Patients with the GG and GA genotypes had a mean onset age of 24.03 (95% CI=20.87-27.19) and 24.25 (95% confidence interval [CI]=22.68-25.81) years, whereas patients carrying the AA genotype had a mean onset age of 20.11 years (95% CI=18.14-22.07). Post-hoc pairwise comparisons including Tukey and Bonferroni were undertaken to test differences between groups. Patients with schizophrenia carrying the GA genotype had later schizophrenia onset age than those carrying the AA genotype (Tukey P=0.011, Bonferroni P=0.012).

For the six polymorphisms that did not show a significant association, we did not have enough power (<0.80) to detect association with our sample size using retrospective power calculations for a case-control study in syntax SPSS version, 18 because the OR were so low (between 1.06 and 1.29). In fact, a sample size ranging from 1000 to 19,000 would be required to show any significance for these six polymorphisms with schizophrenia. It is most likely that the OR are low, because there is no biological role for these SNPs.

Discussion

Analysis of seven tag-SNPs in DRD2 revealed associations with schizophrenia at the genotype and allele level for rs2734839 in an Australian schizophrenia cohort. Given the number of discordant gene association studies with schizophrenia, it is likely to be a complex disorder with both environment and genetics playing a role. Although the genetic risk is high at 80% (McGuffin and Gottesman, 1999; Sullivan et al., 2003), it is likely that no one gene accounts for this risk and a number of polymorphisms contribute. In a previous study, we found an association between the rs6277 polymorphism and schizophrenia (Lawford et al., 2005), and this association has been replicated by others (Hanninen et al., 2006; Hoenicka et al., 2006; Monakhov et al., 2008; Betcheva et al., 2009; Dubertret et al., 2010; Fan et al., 2010). The positive association with rs2734839 that we observed in this study appears novel. Interestingly, rs2734839 is in nearly complete LD with rs6277. Although rs6277 does not result in a nonsynonymous polymorphism, it does appear to have functional consequences on the protein translated (Duan et al., 2003; Hirvonen et al., 2005). There is still the possibility that rs2734839 is a functional SNP, as other intronic SNPs in DRD2 have been shown to have functional effects. The rs2283265 SNP and rs1076560, which are both intronic, affect the D2 receptor long isoform and D2 receptor short isoform ratio of splice variants (Zhang et al., 2007). The same SNPs were found to modulate working memory and attention (Zhang et al., 2007). Therefore, the association we found between rs2734839 and schizophrenia first needs to be replicated in another sample set and then tested for functional effects.

Our results are similar to the findings of Dubertret et al. (2010), who identified an SNP (rs2242592) in the intergenic region of DRD2, that is in strong LD with rs6277. Since they observed a stronger association with rs2242592 than with rs6277, they concluded that an unknown functional SNP in LD with rs2242592 is likely to be identified in the future. It is possible that a functional SNP exists in noncoding region and has been overlooked by focus on protein coding regions. Evidence now shows that non-coding RNAs are also regulators of human disease (Taft et al., 2010).

Similar to Fan et al. (2010), we did not find an association between rs6275 and schizophrenia. It is likely that there is no biological role for this SNP in schizophrenia, as there was no indication of a bias toward schizophrenia (OR=1.18). Contrary to our observations, this polymorphism was previously shown to be associated with schizophrenia and the same study found that it interacts with rs4680 in catechol-O-methyl transferase (COMT) to increase schizophrenia susceptibility (Gupta et al., 2009). Earlier studies in an Indian and Russian population also found an association with schizophrenia (Kukreti et al., 2006; Monakhov et al., 2008); however, both studies observed departure from HWE in the case population.

The rs2734839 polymorphism was also found to be significantly associated with later onset age. In patients carrying the associated allele (G), onset age was delayed by an average of 4 years. The average onset age for the AA genotype was only 20 years, thus suggesting that this polymorphism is associated with a youth onset subset of schizophrenia. Other studies have also observed polymorphisms to be associated with onset age in schizophrenia. One of these studies suggested that a polymorphism in the CCR5 gene is a susceptibility factor for schizophrenia with late onset or, alternatively, the polymorphism may act as a modifier by delaying the onset of schizophrenia without affecting disease susceptibility (Rasmussen et al., 2006). Our study is different from this one in that they did not find a significant association between the CCR5 polymorphism and schizophrenia, only onset age. Therefore, in our study it is likely that the rs2734839 polymorphism is associated with schizophrenia but only in a subset of patients with delayed onset. For the early onset group (mean age 20), other genes are likely to be responsible.

Our significant association between the rs2734839 polymorphism and schizophrenia needs to be replicated in a larger sample size and other defined ethnic groups. Our study does not rule out population stratification, but the effects are likely to be small as patients and controls were all Caucasians from Australia. The significant association seen with rs2734839 and schizophrenia onset age should be seen as exploratory until it is tested in a larger sample set of equal numbers of cases and controls. Unfortunately, we did not have access to a defined group of late onset patients (onset age over 40 years). Our patient group was mainly early onset with 23 years being the average age of first diagnosis. Ideally, this study would need to be repeated with a defined set of early and late onset patients.

Conclusions

We have found a significant association between schizophrenia and a polymorphism rs2734839 in the DRD2 gene. This same polymorphism was also shown to be associated with schizophrenia onset age. The DRD2 region has shown consistent evidence for association with schizophrenia, and it is likely that many genetic variants interact together, thereby modulating dopamine signaling.

Footnotes

Acknowledgments

This work was financially supported by the Queensland State Government, the Nicol Foundation, and the Institute of Health and Biomedical Innovation, QUT. J.V. is a Queensland Smart State Fellow.

Disclosure Statement

The authors state that no competing financial interests exist.

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