The results of the present study indicate that DRD2 variants are associated with several schizophrenia phenotypes known to be modulated by dopamine, while no association with diagnosis was evident in this relatively small sample. GG subjects for rs1076560 and for rs2283265 have greater relative D2S mRNA in prefrontal cortex than GT subjects, regardless of diagnosis. The fMRI results demonstrate association of rs1076560 (which is strongly linked to rs2283265) with prefrontal and striatal activity during WM, with opposite effects in patients and controls. In controls, subjects heterozygous for rs1076560 have reduced performance and greater brain activity, i.e. they are inefficient. In patients, who perform worse than controls and have reduced brain activity, heterozygote subjects have reduced performance and reduced activity, i.e. they do not fully engage prefrontal–striatal resources. Thus the GG genotype is advantageous in both controls and patients. Similar analysis with rs1236428 revealed that TT patients have greater BOLD activity than TC patients. Consistent with the single SNP results, the TGG haplotype (rs12364283, rs2283265 and rs1076560) had opposite effects on brain activity in patients and controls. The single SNP and haplotype associations with WM behaviour are consistent with the fMRI data. Finally, the promoter SNP weakly predicted negative symptoms by itself and more strongly when part of a haplotype including the two intronic SNPs (again TGG). These results may further suggest that the T allele in the promoter SNP is advantageous in patients especially when in combination with the G allele from both intronic SNPs. All these results together indicate that genetically determined D2 receptor signalling modulates manifestation of several phenotypes in schizophrenia.
Dopamine modulation of WM is very well known. While a role for D1 receptors has long been recognized, more recent studies have demonstrated that D2 receptors are also involved. Behavioural and electrophysiological experiments in non-human primates indicate that D2 agonists and antagonists may, respectively, increase and decrease tuning of prefrontal neurons to the task at hand, especially during the response phase (Arnsten et al.
; Williams and Goldman-Rakic, 1995
; Wang et al.
). Several studies have demonstrated that D2 agonists and antagonists, respectively, improve and deteriorate WM performance also in humans (Mehta and Riedel, 2006
). Consistently, D2 agonists and antagonists, respectively, reduce and increase activity in prefrontal cortex and striatum during WM (Kimberg et al.
; Mehta et al.
), suggesting that D2 receptor modulation modifies tuning of neuronal resources. D2 receptors are found with different density and localization in prefrontal cortex and in striatum. While D2S receptors are found mainly, but not exclusively, in the pre-synaptic environment with relatively greater abundance in prefrontal cortex; D2L receptors are mainly found postsynaptically, being relatively more abundant in striatum. The effects we have measured in vivo
may have resulted from prefrontal and/or striatal modulation of dopamine signalling. Earlier studies have indicated that post-synaptic D1 receptors are negatively modulated by post-synaptic D2S, while they act in synergy with post-synaptic D2L (Usiello et al.
). Thus, we propose that subjects homozygous for the G allele at the two intronic SNPs have a more favourable modulation of prefrontal D1 receptors based on greater D2S/D2L ratios. This interpretation is consistent with our mRNA data in prefrontal cortex suggesting that a post-synaptic mechanism may be relevant. A prefrontal pre-synaptic mechanism may also be involved with the known modulation by pre-synaptic D2 receptors of NMDA and of GABA A receptors of pyramidal neurons and interneurons (Tseng and O'Donnell, 2007; Tseng et al.
). Moreover, our results may also be explained by modulation of dopamine signalling in the striatum within the cortico-striato-thalamo-cortical network. Whereas the overall D2 receptor number modulates GABA-mediated inhibition of striatal neurons, inhibition of glutamate release preferentially involves the D2S variant (Centonze et al.
, 2004). Therefore, reduced D2S expression is expected to increase excitability of striatal medium spiny neurons. Future studies are needed to resolve the molecular/neuronal mechanisms accounting for our effects. On the other hand, the opposite effects manifested by the intronic SNPs in patients and controls, the interaction between the promoter SNP and diagnosis of schizophrenia, the opposite effects of haplotypes with brain activity during WM are consistent with the assumption that dopamine transmission in schizophrenia is disrupted (Carlsson et al.
). Thus, these interactions may arise from different dopamine activity between patients and controls.
The weak association with negative symptoms of the promoter SNP also may suggest that genetically determined total number of D2 receptors may be clinically relevant, especially in the context of homozygosity for the G allele in the two intronic SNPs. Once again, the T allele of the promoter SNP and the G allele of the intronic SNPs seem to be beneficial in that they are associated with lower negative symptoms. These data are consistent with some evidence in earlier cross-sectional and longitudinal studies (Himei et al.
; Lane et al.
) indicating association between DRD2
polymorphisms and negative symptoms. On the other hand, in this case–control study we did not find any evidence for association of the three variants with diagnosis of schizophrenia. A recent meta-analysis of all case–control studies investigating the DRD2
Ser311Cys (rs1801028) variant has indicated association with schizophrenia with an odds ratio of 1.3 for the Cys allele (Glatt et al.
). As rs1801028 is in strong LD with the intron 5/6 SNPs, we propose that the main functional effects may be exerted by their effect on splicing, rather than the imputed amino acid change caused by the non-synonymous SNP. This meta-analysis also suggested that the effect of this DRD2
polymorphism on schizophrenia risk is reliable and uniform across populations, although the magnitude of its effect is small. Similarly, another recent meta-analysis also suggested that rs1801028 may be implicated in risk for schizophrenia (Allen et al.
). Moreover, another recent family-based sample in Han-Chinese has indicated association of DRD2
SNPs and haplotypes with schizophrenia involving the intron 5 SNP rs2283265 by itself and as part of a haplotype block as well as a haplotype block including rs1801028 (Glatt et al.
, 2008). Of note, these two SNPs are in high LD. Finally, DRD2
has also been implicated in linkage areas suggested in the genome-wide linkage meta-analysis (Lewis et al.
). Thus, these previous studies and the LD structure of the gene suggest that DRD2
may be implicated in risk for schizophrenia and that our inability to demonstrate statistically significant association with diagnosis is related to the relatively small number of cases and controls.
A limitation of the present study has to be acknowledged. Since all our patients were treated with anti-psychotic drugs, we cannot definitively exclude an effect of treatment on the measured phenotypes. However, several factors indicate that the effect of treatment is not a confounder of our results. First, genotype groups did not differ in terms of chlorpromazine equivalents (all P
> 0.1). Second, all patients had been on stable treatment for at least 1 month. Third, there was no correlation between WM performance or brain activity with chlorpromazine equivalents or between lifetime exposure to anti-psychotics and %D2S mRNA (data not shown, all P
> 0.3). Fourth, our prefrontal post-mortem data demonstrate unchanged total D2 mRNA. Previous studies have indicated that treatment with anti-psychotics increases total D2 mRNA thus suggesting that pharmacological treatment is unlikely to account for our results (Martres et al.
; D'Souza et al.
, 1997; Lidow et al.
In conclusion, our results indicate associations of three DRD2
variants with prefronto-striatal phenotypes of relevance to schizophrenia, showing that endo-phenotypes may help identify genetic variants affecting disease presentation (Hall et al.
). The potential utility of these genetic variants as biomarkers in schizophrenia therapy requires further study.