Adenosine A2A receptor gene: Evidence for association of risk variants with panic disorder and anxious personality

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Abstract

Adenosine A2A receptors are suggested to play an important role in different brain circuits and pathways involved in anxiety reactions. A variant within the corresponding ADORA2A gene (rs5751876) increased the risk for panic disorder (PD), for elevated anxiety during challenge tests in healthy probands and for anxiety-related arousal in blood-injury phobia. These multiple effects may mirror a more general effect of the SNP on basic personality traits. In the present study we therefore aimed to replicate the original finding in a large PD sample and extend it by investigating an additional proband sample characterized for different anxiety-related personality scores. In addition, as rs5751876 is assumed not to be the disease variant itself but to be in linkage disequilibrium (LD) with the true functional polymorphism other SNPs of potentially functional relevance were identified by re-sequencing the whole gene including several newly identified regions of putative regulatory potential and analysed for their impact on PD and anxious personality. We were indeed able to replicate rs5751876 as risk factor for PD, particularly PD with agoraphobia. Rs5751876 and several other variants in high LD (rs5751862, rs2298383 and rs3761422) as well as the corresponding haplotypes were also associated with different anxiety-related personality scores (Bonferroni corrected Pall < 0.05). Of these variants, rs2298383 shows functional potential based on in silico analyses and might therefore represent the true underlying causal variant. Our data provide further support for an important role of ADORA2A variants in the pathogenesis of anxiety disorders and anxious personality reflecting their potential as basic susceptibility factors.

Introduction

Adenosine A2A receptors (A2ARs) mediate the effects of extracellular adenosine and are expressed at high levels in the striatum and olfactory bulb and at lower levels in hippocampus, thalamus and neocortex (Ribeiro et al., 2002). They play an important role in the indirect efferent pathway of the basal ganglia system by fine-tuning glutamatergic information flow and by triggering the cAMP-protein kinase A signaling cascade (Schiffmann et al., 2007). A2ARs control glial function and brain metabolic adaptation, and are involved in different psychiatric disorders, particularly mood disorders and anxiety (Cunha et al., 2008, Freitag et al., 2010). A2ARs are blocked by methylxanthines such as caffeine, which at lower doses elicit increases in alertness, stimulation and arousal. Higher doses of caffeine can produce anxiety up to panic attacks, particularly in panic disorder (PD) patients (Charney et al., 1985) (for this reason PD patients often avoid drinking coffee, tea, or coke), which underlines the importance of A2AR function in anxiety. Further evidence that changes in A2AR function modify anxiety reactions comes from mice lacking the A2AR. They are more aggressive and anxious compared to their wildtype counterparts (Ledent et al., 1997). Accordingly, the corresponding gene (ADORA2A, NM_000675) is discussed as a promising candidate for increased anxiety. ADORA2A is located on chromosome 22q11.23 and has two coding exons spanning about 9 kb (MacCollin et al., 1994, Peterfreund et al.,1996, Le et al., 1996). Mutation screening of these exons revealed several variants (Deckert et al., 1998, Deckert et al., 2001) of which SNP rs5751876 (1976C/T) was associated or linked with PD (Deckert et al., 1998, Hamilton et al.,2004). Rs5751876 was also associated with increased anxiety in healthy probands in different challenge tests, i.e. after the consumption of caffeine (Alsene et al., 2003, Childs et al., 2008) or amphetamine (Hohoff et al., 2005). Further, an impact on sympathetic indicators of anxiety-related arousal in blood-injury phobia was suggested (Hohoff et al., 2009). Together, these studies support the assumption of a more general effect of the gene on the development of different forms of anxiety presumably also including basic anxiety-related personality traits. In most of the above mentioned studies the rs5751876 T-allele was identified as risk variant. It was overrepresented in PD patients (Deckert et al., 1998) and T/T homozygous subjects reported higher anxiety or exhibited increased anxiety-related arousal (Alsene et al., 2003, Childs et al., 2008, Hohoff et al., 2005, Hohoff et al., 2009). However, since rs5751876 does not cause an amino acid exchange, other variants in strong linkage disequilibrium (LD) are presumably the underlying functional variants. The potential of variants to affect gene function is highly related to their position relative to the gene, e.g., SNPs located within intron/exon boundaries, exon splicing enhancers, cis elements, conserved regions and particularly within and around the transcription start site (TSS) have a high potential to alter gene function (Conde et al., 2006, Veyrieras et al.,2008). ADORA2A was originally assumed to have only one TSS immediately upstream the first coding exon based on the initial gene characterizations (MacCollin et al., 1994, Peterfreund et al.,1996, Le et al., 1996). This was reflected by common databases such as HapMap (http://www.hapmap.org) and UCSC (http://genome.ucsc.edu; human Nov. 2002 up to May 2004 assemblies) which listed none or only few SNPs of potentially functional relevance within this restricted region. Further characterization of the genomic organization with a focus on a distinctly extended region upstream revealed several variants of a newly identified noncoding “exon 1” in the expanded 5’UTR which lead to different tissue-specific ADORA2A mRNAs (Yu et al., 2004). In the brain variant (X68486), which spans about 25 kb and includes three noncoding exon 1 variants and the two coding exons, Yu et al. (2004) identified additional three new TSS, their corresponding promoters, several important cis elements and several putative SNPs. Further possibly regulatory regions such as transcription factor binding sites, a CpG island, conserved regions and more than 100 predominantly non-validated SNPs are meanwhile listed within this extended 25 kb ADORA2A region by common databases (e.g. UCSC, human Mar. 2006 assembly). In contrast, Hapmap specifies only 15 validated SNPs within this region which did not include the previously identified SNPs such as rs5751876. This discrepancy underlines the need for a systematic screening and validation of SNPs in the extended 25 kb ADORA2A region to identify the potentially underlying causal variants and to elucidate their role in the pathogenesis of PD and anxious personality.

In the present study therefore we first aimed to replicate the original finding of an association of rs5751876 with PD in a large case–control sample. Additionally we investigated the role of rs5751876 in more general trait anxiety, using a sample of healthy individuals characterized for different anxiety-related personality scores. We hypothesized according to the above mentioned studies an overrepresentation of the T-allele in PD patients and subjects scoring high in anxiety traits. ADORA2A was then re-sequenced and analysed in silico to identify further potentially functionally relevant variants. In a second and third step of analysis possibly functional SNPs in LD with rs5751876 and ADORA2A haplotypes were examined under the hypothesis that they were also associated with PD and anxious personality.

Section snippets

Methods

Our initial PD sample of 89 patients (Deckert et al., 1998) was expanded to currently 457 German PD patients, of whom 341 had additional agoraphobia (Table 1). All patients were diagnosed by experienced psychiatrists according to DSM-IIIR or DSM-IV criteria for panic with or without agoraphobia using structured clinical interviews [SADS-LA (Mannuzza et al., 1986); CIDI (Robins et al., 1988); SKID (Wittchen et al., 1997)] and a review of medical records. Patients with mental retardation,

Results

Genotype frequencies of all investigated ADORA2A SNPs conformed to Hardy–Weinberg equilibrium in the PD, PDAgP and the personality sample (Table 2).

Discussion

In the present study we aimed to further assess the role of ADORA2A variants in anxiety. In line with our hypothesis and previously reported data (Deckert et al., 1998, Hamilton et al.,2004), we replicated the original finding of an association of rs5751876 with PD. This effect was stronger in the agoraphobia subgroup PDAgP, though of smaller size and power, indicating that the finding in the whole PD sample probably originates from this subgroup. However, PDAgP is the clinically more severe

Conflicts of interest statement

The authors declare that they have no conflicts of interest.

Contributors

Authors CH, ELM, SVH, JD and PJR designed the study and wrote the protocol. ELM, SVH, KD, PK, MR, MEK, PGU, AE, CJ, JF, BB, WM, FH, PJR and JD managed the participant interviews and analyses. CH and LCN performed the PCR experiments and sequence analyses and CMF (assisted by CH) undertook the statistical analyses. CH, CMF, PJR and JD contributed to the interpretation of the data and preparation of the manuscript. All authors contributed to and have approved the final manuscript.

Role of the funding source

Funding for this study was provided by UK's Biotechnology and Biological Sciences Research Council (BBS/B/10855), by the Bavarian Ministry of Commerce and the Federal Ministry of Education and Research (BMBF) in the framework of the National Genome Research Network (NGFN), by the Deutsche Forschungsgemeinschaft (SFB-TRR 58) and by the University of Muenster faculty of medicine. The UK’ s funding agency, the Bavarian Ministry of Commerce, the Federal Ministry of Education and Research (BMBF),

Acknowledgements

This research was funded partly by UK Biotechnological and Biological Research Council (BBS/B/10855), the Bavarian Ministry of Commerce, the Federal Ministry of Education and Research (BMBF), the Deutsche Forschungsgemeinschaft (SFB-TRR-58) and the University of Münster Faculty of Medicine. We gratefully acknowledge the skilful technical support by Kathrin Schwarte and Christoph Arolt.

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