This study confirms a robust effect of SNP1/SNP2 on D2S/L splicing in human PFC and putamen autopsy tissues in a cohort of cocaine abusers with a documented history of heavy abuse and age-matched controls. Sequencing the region of DRD2 between exon 5 and 7 failed to reveal any additional candidate SNP affecting D2 splicing. A genetic association analysis revealed that SNP1/SNP2 are strongly associated with cocaine abuse in Caucasians but not African Americans.
The association of SNP1/SNP2 with decreased expression of D2S relative to D2L in human PFC and putamen is consistent with our earlier report (Zhang et al, 2007
), replicating the finding in a separate population with a different pathophysiology. The validation of SNP1/SNP2 as DRD2
variants affecting D2S/D2L splicing is important because only a firm understanding of the molecular genetic effect can guide the interpretation of clinical association studies.
As the importance of dopamine in the pathophysiology of addiction is well established, perturbations in dopamine signaling could impact an individual's response to drugs of abuse and/or risk for addiction. There is ample evidence that D2S and D2L are functionally distinct (Smith et al, 2002
; Usiello et al, 2000
; Wang et al, 2000
; Xu et al, 2002
), and alternative splicing of DRD2
to form D2S and D2L represents a regulatory event that could affect dopamine signaling. Consistent with the compelling evidence for the effects of SNP1/SNP2 on D2S/L splicing, SNP1/SNP2 were strongly associated with cocaine abuse in Caucasians. Allele frequencies were substantially higher in the Caucasian cocaine group (SNP1 24% and SNP2 25%), compared with the controls (SNP1 10% and SNP2 9% (p
=0.003 and 0.001, respectively, ). African Americans having considerably lower SNP1/SNP2 allele frequencies (MAF=7%) were insufficiently represented (n
137 Caucasians) for testing associations (). However, the biological effect of SNP1/SNP2 on D2S/L splicing was recapitulated in both groups (Supplementary Figure 3). To determine whether the relative risk of cocaine abuse increase even further for homozygous carriers of SNP1/SNP2, occurring at much lower frequency, a larger study cohort will be needed.
The D2S/L ratio was not measurably correlated with cocaine abuse in our cohort, although SNP1/SNP2 were associated with both reduced relative expression of D2S and with cocaine abuse. The influence of external trans
-acting factors on transcription and alternative splicing can introduce sufficient noise so that any relationship between splicing and cocaine abuse is no longer apparent in a small cohort. Also, from this study alone we cannot exclude the possibility that cocaine exposure affects D2 splicing in specific brain regions or neurons not analyzed or accessible here. Previous evidence supports extensive trans
effects on splicing. Cocaine abuse has been shown to act as a trans
-factor modulating overall gene expression in humans and animals (reviewed in Lull et al, 2008
) and altering differential expression of specific isoforms of brain-derived neurotrophic factor in human brain (Jiang et al, 2009
). Furthermore, D2S and D2L expression in mouse striatum and ventral tegmental area is differentially affected following treatment with amphetamine, another psychostimulant (Giordano et al, 2006
). A host of other trans
-acting factors, including postmortem decay and RNA isolation from postmortem human tissues could have contributed to increased variability in the measurements of D2S/L splicing, confounding the three-way relationship between genotype, splicing, and cocaine abuse. Further studies are needed to characterize the interaction between SNP1/SNP2 genotype, cocaine abuse, and D2S/L splicing.
Numerous previous studies had implicated several DRD2
variants as risk factors in a spectrum of clinical traits (); however, clinical associations per se
are often not replicated leading to low acceptance of such results. We therefore addressed the question whether some of these candidate SNPs are in LD with SNP1/SNP2, and hence could be considered surrogate markers. A number of SNPs in high LD with SNP1/SNP2 form a large haplotype block () (http://www.broadinstitute.org/mpg/snap/ldplot.php
; Johnson et al, 2008
). For example, a SNP tested in this study, rs1125394, in tight LD with SNP1/SNP2 (D
′=1.0, Supplementary Table 3), had been similarly associated with cocaine abuse (). This same SNP had also been shown to predict clozapine response in schizophrenic patients (Hwang et al, 2005
). With R2
values near 1.0 for several SNPs in the SNP1/SNP2 haplotype block, we can consider these findings () to represent independent validation of pervasive clinical relevance. All of these variants can thus be considered markers for a true genetic effect with demonstrable impact on human behavioral phenotypes (). It is further noted that the LD block extends into the adjacent gene ANNK1
(), including the Taq1A variant used as a marker in numerous clinical association studies (). Although ANNK1
could well represent a risk gene on its own, the strong LD to SNP1/SNP2 in the DRD2
locus must be considered. Finally, the haplotype block is significantly shorter in subjects of African descent when measured using the Yoruban HapMap population (not shown), indicating that strong ethnic differences in association studies can be expected.
Clinical Associations of SNPs in LD (R2>0.6) with rs2283265
Additional contributions must also be considered for other proposed functional DRD2
variants not in high LD with SNP1/SNP2. Candidate variants in the DRD2
gene locus with clinical associations and in vitro
evidence for being functional include rs12364283 (Frank and Hutchison, 2009
, Zhang et al, 2007
), rs6277 (C957T; Duan et al, 2003
; Lawford et al, 2005
; Voisey et al, 2009
; Zai et al, 2007
), and rs1799732 (−141C Ins/Del; Arinami et al, 1997
; Ishiguro et al, 1998
; Lerman et al, 2006
). Any possible contributions from these SNPs cannot be ruled out here; however, our previous results on allelic mRNA expression in putamen and PFC had failed to support a detectable functional effect of rs6277 (thought to alter mRNA turnover) or rs1799732 (transcription) in these brain tissues (Zhang et al, 2007
In summary, we have examined the relationship between DRD2
genotype, D2S/L splicing, and cocaine abuse. The results support a robust effect of SNP1/SNP2 decreasing the relative expression of D2S in human brain as reported previously (Zhang et al, 2007
). Moreover, the minor alleles of SNP1/SNP2 represent apparent strong risk factors in cocaine overdose cases. Clinical penetrance is supported by numerous earlier clinical association results in various disorders with these same SNPs, or surrogate SNPs in high LD with them. Taking all previous studies and the present results together, these two splice SNPs modulate dopamine signaling in human brain, affecting cognitive processes and psychiatric disorders including drug addiction, and response to therapies. The large odds ratios observed for SNP1/SNP2 in the present study for risk of cocaine abuse indicates a large effect size in this population of subjects with documented heavy abuse, leading eventually to cocaine overdose and death.