21 December 2007. Dopamine, an essential neurotransmitter in the brain, has been linked to the pathology of numerous neurologic disorders. The dopaminergic hypothesis of schizophrenia (see current hypothesis), for example, postulates that too much neurotransmission via the D2 type of dopamine receptor leads to many of the “positive” symptoms of the disease, including hallucinations and delusions (see SRF related news story). This idea is supported by pharmacological evidence: until the recent clinical trial success of a glutamatergic antipsychotic drug (see SRF related news story), all antipsychotic drugs had one thing in common—blocking the D2 receptor.
So what about the D2 receptor itself? Could subtle genetic variations in the D2 receptor gene (DRD2) have functional sequelae? A study in this week’s PNAS online suggests they do. Researchers led by Wolfgang Sadee at Ohio State University, Columbus, report that gene expression, splicing, and neuronal activity during working memory can all be altered by DRD2 polymorphisms. The findings may help resolve why D2 receptor drugs have different effects in different people, suggested Michael Frank, University of Arizona (see comment below), and they may offer a mechanistic explanation for prior studies linking the DRD2 gene with schizophrenia.
Tens of genetic association studies have probed the possible links between schizophrenia and DRD2 polymorphisms, and the gene currently holds the top spot in the SchizophreniaGene "Top Results" list. However, it is not clear if any of these polymorphisms actually alter the activity of the gene. Research shows that one DRD2 single nucleotide polymorphism (SNP), C957T, alters DRD2 mRNA turnover in vitro (see Duan et al., 2003), but none of the SNPs that associate with schizophrenia have been shown to affect production of D2 receptor mRNA in vivo. The difficulty in making that connection may be related, at least partly, to treatment, since antipsychotics drive upregulation of D2 receptors in patients.
Sadee and colleagues adopted an interesting approach to look for functional effects of DRD2 polymorphisms. First author Ying Zhang and colleagues compared expression of different DRD2 alleles in healthy individuals who have two different copies of the receptor gene. In other words, they are using an internal control rather than trying to compare gene expression among individuals. The researchers first identified 68 different brain tissue samples (54 from prefrontal cortex and 14 from the striatum) that are heterozygous for at least one of three marker SNPs. Finding that 15 of these samples exhibited “allelic expression imbalance,” they set out to determine if the expression differences could be attributed to genetic variation. Zhang and colleagues genotyped 23 known SNPs in the DRD2 gene. They found that only one, SNP2, which lies upstream in the promoter region of the gene, had any effect on DRD2 mRNA levels. The minor allele, occurring in about 7 percent of samples, leads to increased expression of the receptor. That SNP is not one of those that have been previously implicated as a risk factor for schizophrenia.
But the researchers didn’t leave it at that. Knowing that the DRD2 gene is alternatively spliced to make short and long isoforms, the researchers probed to see if these specific isoforms exhibit any allelic expression imbalance and if that might be linked to any of the 23 SNPs. In fact, Zhang found that two variants, SNP17 in intron 5 and SNP19 in intron 6, play a role in splicing of exon 6, with the minor allele (a T instead of a G in both cases) favoring inclusion of the exon and an increase in the ratio of long to short D2 receptor isoform. In the prefrontal cortex and the striatum, regions of predicted dysfunction in schizophrenia, they found that the minor alleles increased DRD2 long forms by about twofold.
What is the significance of this finding? For starters, the short and the long isoforms have different functions. “The short form sits presynaptically, so it is an autoinhibitory receptor—the more dopamine there is, the more autoinhibition you get. The long form sits post-synaptically and has very different functions, may even be facilitating or potentiating D1 receptors. We are not quite sure yet,” said Sadee in an interview with SRF. He explained that if you increase the long-to-short ratio, then you would predict greater dopaminergic activation. In fact, that is exactly what the researchers found. In collaboration with Alessandro Bertolino’s group at the University of Bari, they imaged the brain of healthy humans tackling a test that relies heavily on working memory (the N-Back task) and found that there is much greater activation in specific regions of the brain (for example, the caudate, left middle frontal gyrus, left precentral gyrus, left anterior cingulate, left thalamus) in volunteers carrying one of the minor alleles. “These results suggest that intronic SNP17 and -19 robustly modulate activity of the working memory network, especially striatal firing,” write the authors. They also found that heterozygotes have reduced working memory performance when challenged with tasks that require a high level of attention (see comment below from Frank).
The findings may also offer a mechanistic explanation for prior genetic association studies. Sadee and colleagues found that SNP17 and -19 are in linkage disequilibrium, or co-inherited, with the TaqI-A polymorphism that has been associated with schizophrenia in prior studies (see SchizophreniaGene DRD2 entry, and select Taq1-A from the polymorphisms dropdown menu). That polymorphism sits downstream of the gene, and previous evidence suggests that the polymorphism leads to increased D2 receptor density (for discussion, see recent paper by Klein et al., 2007, which finds that Taq1-A polymorphisms affect learning in normal subjects). This new data by Zhang and colleagues suggests that the TaqI-A polymorphism is simply a marker for the DRD2 intronic SNPs. This speaks to the growing trend of using genomewide association (GWA) studies that rely on SNPs identified by the human HapMap project, according to Sadee. “Even full linkage studies with the 500,000 or so SNPs that are being selected for haplotype analysis would systematically miss our three SNPs,” said Sadee, adding that going forward with clinical association studies with SNPs that are only markers is less than ideal. “I think GWA studies are exciting, but we also need to acknowledge that in nearly all cases where one finds a significant association, the OR (odds ratio) is around 1.5 at best. This has led to the conclusion that in most cases for polygenic complex disorders, multiple genes each contribute just a little. That may well be, and the GWA studies are extremely important for revealing novel pathways, but for clinical utility, as biomarkers, for example, one would like to begin with ORs of 3. That's what we are shooting for and beginning to find in some cases,” he said. Sadee plans to continue to look at the most commonly studied genes, applying his technique to look for functional polymorphisms. He said he also plans to test how these three SNPs are related to schizophrenia, addiction, and other brain disorders.—Tom Fagan.
Zhang Y, Bertolino A, Fazio L, Blasi G, Rampino A, Romano R, Lee M-LT, Xiao T, Papp A, Wang D, Sadee W. Polymorphisms in human dopamine D2 receptor gene affect gene expression, splicing, and neuronal activity during working memory. Proc Natl Acad Sci U S A. 2007 Dec 18;104(51):20552-7. Epub 2007 Dec 11.