ADORA2A variation and adenosine A1 receptor availability in the human brain with a focus on anxiety-related brain regions: modulation by ADORA1 variation

Adenosine, its interacting A1 and A2A receptors, and particularly the variant rs5751876 in the A2A gene ADORA2A have been shown to modulate anxiety, arousal, and sleep. In a pilot positron emission tomography (PET) study in healthy male subjects, we suggested an effect of rs5751876 on in vivo brain A1 receptor (A1AR) availability. As female sex and adenosinergic/dopaminergic interaction partners might have an impact on this rs5751876 effect on A1AR availability, we aimed to (1) further investigate the pilot male-based findings in an independent, newly recruited cohort including women and (2) analyze potential modulation of this rs5751876 effect by additional adenosinergic/dopaminergic gene variation. Healthy volunteers (32/11 males/females) underwent phenotypic characterization including self-reported sleep and A1AR-specific quantitative PET. Rs5751876 and 31 gene variants of adenosine A1, A2A, A2B, and A3 receptors, adenosine deaminase, and dopamine D2 receptor were genotyped. Multivariate analysis revealed an rs5751876 effect on A1AR availability (P = 0.047), post hoc confirmed in 30 of 31 brain regions (false discovery rate (FDR) corrected P values < 0.05), but statistically stronger in anxiety-related regions (e.g., amygdala, hippocampus). Additional effects of ADORA1 rs1874142 were identified; under its influence rs5751876 and rs5751876 × sleep had strengthened effects on A1AR availability (Pboth < 0.02; post hoc FDR-corrected Ps < 0.05 for 29/30 regions, respectively). Our results support the relationship between rs5751876 and A1AR availability. Additional impact of rs1874142, together with rs5751876 and sleep, might be involved in regulating arousal and thus the development of mental disorders like anxiety disorders. The interplay of further detected suggestive ADORA2A × DRD2 interaction, however, necessitates larger future samples more comparable to magnetic resonance imaging (MRI)-based samples.


Introduction
Anxiety is a fundamental emotion eliciting considerable reactions like increased attention, heart rate, and energy metabolism 1 . In case of pathological anxiety/anxiety disorders, dysregulation in brain regions of the "fear network" occurs, e.g., hyperactivation of amygdala, hippocampus, thalamus, and cingulum, as well as decreased interconnectivity between these regions 2,3 . Anxiety and arousal are tightly connected, both counteract sleep, and both are strongly linked to the adenosinergic system [4][5][6][7] . The purine nucleoside adenosine is ubiquitous in body and brain and involved in the regulation of e.g., heart rate, energetic homeostasis, sleep, arousal, and anxiety 4,5,8 . It acts through the four Gprotein coupled adenosine receptors (AR) A 1 AR, A 2A AR, A 2B AR, and A 3 AR, of which particularly the first is widely distributed in the brain and has a high affinity to adenosine [4][5][6]9 . An increase in A 1 AR function was reported to elicit anxiolytic effects 10,11 and quantification of A 1 AR availability via positron emission tomography (PET) demonstrated an A 1 AR increase after sleep loss 12,13 . A further PET investigation revealed a modulation of A 1 AR availability by caffeine 14 , which antagonistically binds to both A 1 AR and A 2A AR 15 . Both receptors are coexpressed, form heteromers, and interact to regulate adenosinergic and glutamatergic neurotransmission in the brain, which, apart from various physiological functions, have been implicated in pathological alterations related to psychiatric and anxiety disorders (e.g., [4][5][6][16][17][18]. Genetic loss of A 1 AR or A 2A AR enhanced anxiety-related behavior in knockout mice 19,20 and variants in the corresponding human genes ADORA1 and ADORA2A were reported to be associated with, for example, sympathetic arousalrelated blood-injury phobia 21 , panic disorder 22,23 , and anxiety-related autism spectrum disorder 24 . Further associations were identified in healthy subjects with caffeine-induced anxiety [25][26][27] and caffeine effects on sleep (e.g., 28 ). In all these studies ADORA2A was reported to have the stronger impact compared with ADORA1. Especially the ADORA2A single-nucleotide variant rs5751876 (1976C/T, formerly 1083 C/T) revealed strongest and most consistent associations, and its rare T-allele repeatedly turned out at risk for elevated or pathological anxiety (e.g., [21][22][23][25][26][27]. Further studies support the notion that there is a relation between anxiety and caffeine challenge in female rs5751876 TT homozygotes, indicating also a female sex influence in increased anxiety 29,30 .
Among this direct ADORA2A rs5751876 effect on anxiety, a previous pilot study reported the T-allele to be associated with increased brain A 1 AR availability in healthy participants as measured by PET 31 , suggesting indirect effects on anxiety via A 1 AR modulation. Although this finding highlights the potential putative role of an interaction between both receptors, the pilot study was restricted to a small sample of 28 male individuals only, allowing only exploratory analyses of few ADORA2A and ADORA1 variants. However, there is evidence to suggest that female sex and additional adenosinergic or dopaminergic genetic variants might as well have a modulatory impact on the rs5751876 effect on A 1 AR availability. Female sex is associated with a higher risk of anxiety/anxiety disorders as well as with a higher risk of sleep-(disruption-) related disorders like insomnia or hypertension (e.g., 29,32 ). A 2B AR, A 3 AR, and the adenosine deaminase as members of the adenosinergic system mediate adenosine actions via receptor-receptor interactions 33 and were linked to anxiety-or arousalrelated behavior in mice 9,34,35 . The dopamine D 2 receptor (D 2 R) functions as major interaction partner of A 2A AR as demonstrated by their colocalization, heteromerization, and cross-desensitization 36,37 . Genetic interaction of DRD2-ADORA2A variants has been shown to affect caffeine-induced anxiety 26 , whereas DRD2 variants alone, notably the DRD2/ANKK1 variant rs1800497 (Taq1A polymorphism), were reported to be associated with anxiety symptoms/disorders (e.g., [38][39][40][41].
Taken together, previous studies suggest that female sex and additional genetic variations might further modulate the ADORA2A rs5751876 effect on brain A 1 AR availability. Therefore, the present study aimed to expand on the original pilot study findings by analyzing a larger set of potentially interacting genetic variants in an independent newly recruited male/female mixed cohort. We hypothesized firstly, that the pilot male-based rs5751876 effect on in vivo brain A 1 AR availability 31 can be observed also in an independent male-female based sample of healthy humans. Second, we hypothesized that genetic variants of A 1 AR, A 2B AR, A 3 AR, ADA, and D 2 R contribute to the modulation of the rs5751876 effect on in vivo brain A 1 AR availability.

Subjects
All subjects were newly recruited via advertisement and a subsequent interview by phone. Forty-three healthy volunteers were finally included in the study (mean age 34.2 ± 14.1 y; 32 males, 11 females; for further details see supplemental material) after obtaining written informed consent for imaging procedure, blood sampling, and genetic testing. Exclusion criteria were neurological or psychiatric disorders, head trauma, systemic diseases, or substance/drug abuse interfering with ARs. All these subjects were phenotypically characterized by a further standardized interview (subjects demographics/characteristics in Table 1) and underwent medical examination before PET imaging at the PET laboratory of the Institute of Neuroscience and Medicine (Forschungszentrum Jülich). All subjects had a history free of psychiatric and neurological diseases. Self-reported habitual sleep duration, current medication, presence of allergies, and consumption of cigarettes, alcohol, coffee and caffeine-containing drinks were obtained by selfrating on a standardized questionnaire routinely used in the PET laboratory (for answers/results see Table 1), which all subjects filled-in before PET imaging. Average sleep duration per night was given in hours and coffee consumption in cups/day (0.15 mL). Whole amount of caffeine-containing drinks per day was assessed in liters. Prior to PET scanning participants were asked to avoid caffeine consumption for at least 24 h. All procedures were approved by the Ethics Committee of the Medical Faculty of the University of Duesseldorf and the German Federal Office for Radiation Protection.

Imaging procedures
PET acquisitions and high-resolution three-dimensional T1-weighted magnetic resonance (MR) images were performed as previously reported 31 while postprocessing for calculations of 18 42 ) binding in cerebral tissues differed slightly. In brief, realignment, coregistrations, segmentation, and normalization of three-dimensional PET and corresponding MRI data were done with PMOD software (version 3.408, PMOD Group, Zürich, Switzerland). For definition of regions of interest, the AALtemplate 43 implemented in the PMOD software was used. Regional time-activity curves (TACs) were corrected for decay and the contribution of intracerebral blood volume. Corrected TACs were used to estimate the A 1 AR availability in terms of the [ 18 F]CPFPX binding potential (BP ND 44 ) via the Logan Plot (t* = 20 min, fixed k2' 45 ) with the cerebellar gray matter as a reference region 46 . Imaging procedures are further detailed in Table 1 and in the supplements. Investigated regions are given in Table 2.
Genotyping DNA from blood samples was isolated and used for genotyping of selected variants as described previously 31 and in detail in the supplements (text and Tabs. S1 and S2). In brief, ADORA2A and ADORA1 variants were the same as used for the pilot sample based on the initially described selection process 31 (using tagging information of the international HapMap Project, functional potential information of the UCSC Genome browser and association information of previous studies [21][22][23][24][25][26][27][28][29][30]. Additional pairwise linkage disequilibrium (LD) analyses performed in the pilot study sample provided LD information and redundancy of several variants 31 . These were dropped, leaving five ADORA2A variants (rs5751862, rs5760405, rs2236624, rs5751876, rs4822492) and seven ADORA1 variants (rs1874142, rs10920568, rs12135643, rs3766566, rs17511192, rs6677137, rs3753472) for the present study (for all see Suppl. Tab S1). All other adenosinergic/dopaminergic variants were selected SD standard deviation, y/n yes/no, n.s. not significant (P > 0.05), subj. subjects, inj. injected, act. activity. 1 Including hypertonia, pancreatitis, benign prostatic hyperplasia, sarcoidosis, or psoriasis. 2 Medications were Metoprololsuccinat, Cadiovan/Ibuprofen, Concor 5 Plus, Pangrol, Teracid, or Timo Stulln 0.5%. 3 Data missing for four subjects. 4 "Coffee" stands for coffee drinks alone while "Caffeine" sums drinks of coffee, cola and black tea, data missing for five subjects. 5 Data missing for nine subjects. 6 Check for potential confounding effect on brain region specific A 1 AR availability. 7 Check for potential confounding effects across genotype groups. Bold face indicates highly significant P values (P < 0.01); n.s. not significant. 1 Benjamini-Hochberg FDR correction for testing 31 brain regions.

Statistical analyses
Hardy-Weinberg equilibrium and pairwise LD were assessed using Haploview v.4.1 as reported before 31 . Genotypes were grouped separately for each variant into carriers of both common alleles (common homozygotes) vs. carriers of at least one rare allele (heterozygotes + rare homozygotes); see supplements (Tab. S1). All other analyses were conducted two-sided using SPSS v.25 (IBM, Chicago, IL, USA).
According to our first hypothesis the impact of ADORA2A rs5751876 (CC homozygotes vs. T-allele carrier; as previously reported 31 ) on A 1 AR availability was investigated in our male-female-based sample in a first analysis model. We started with testing the A 1 AR availability data for mutual dependence (intercorrelation) between brain regions and with testing the participants' demographics/characteristics for potential confounding, both as reported before 31 and as detailed in Table 1 as well as in the supplements. Based on the detected intercorrelation and confounders (see results section below) we then utilized general linear model (GLM) multivariate procedure for simultaneous analysis of multiple (intercorrelated) dependent variables 47 (i.e., the brain region specific A 1 AR availabilities) by genotype group effect with additional confounders in a single model. Thereby the multivariate ADORA2A rs5751876 genotype group effect on global A 1 AR brain availability was analyzed and, if significant, followed by post hoc ANCOVAs to identify single brain regions contributing to the global effect as detailed in the supplements. Significance level alpha was set to 0.05 for all pretests and the global multivariate analysis. The ANCOVAs were further corrected for testing 31 brain regions by controlling the false discovery rate (FDR) following the Benjamini-Hochberg procedure 48 .
According to our second hypothesis the ADORA2A rs5751876 effect on A 1 AR brain availability was investigated under the assumption of additional modulatory effects of adenosinergic or dopaminergic gene variation. In analogy to the first model, GLM multivariate procedures were used to analyze the rs5751876 genotype group effect on A 1 AR availability (same confounders and model composition), but now including one by one each genetic variant as main factor and in interaction with rs5751876. Significance level was adjusted to 0.025 because of the high similarity to the first model. In case of a significant global effect of rs5751876 on A 1 AR availability, again FDR-corrected post hoc ANCOVAs followed to identify single brain regions contributing to this effect. Testing two to seven genetic variants per gene (ADORA1: 6, ADORA2B: 2, ADORA3: 6, ADA: 2, DRD2: 7) remained uncorrected in the second model approach.

Results
All genotype distributions were in Hardy-Weinberg equilibrium (P > 0.05). Several variants in close/complete LD (D' = 1.0 and r 2 > 0.8: rs4822492, rs3766566, rs1076560, rs55900980, rs4936272; supplemental Tab. S3) were excluded from further analysis to avoid redundancy. All ADORA2A variants revealed high pairwise LD (D' ≥ 0.78), thus potential confounding across individual genotype groups was considered relevant for the whole ADORA2A gene and included as confounder for our rs5751876-based analyses.
ADORA2A rs5751876 effect on A 1 AR availability in mixed male/female sample High intercorrelation of A 1 AR availability between brain regions was detected (all P < 0.004, pairwise correlation coefficients: mean = 0.832, range = 0.431-0.983). Analysis of participants' demographics/characteristics revealed the following statistically relevant confounders: sex, age, selfreported habitual sleep, self-reported presence of allergies, self-reported caffeine and alcohol consumption, application mode of radiotracer, injected activity of radiotracer, and total amount of injected substance (P all < 0.05; Table 1). Since sex and sleep had effects on both, A 1 AR availability and across ADORA2A genotype groups, additional interaction terms were formed (rs5751876 × sex; rs5751876 × sleep) and further included as confounders.
Our first analysis model comprising these confounders and interaction terms revealed a significant global multivariate effect of rs5751876 on A 1 AR availability (P = 0.047). Post hoc ANCOVAs confirmed this effect for nearly all (30 of 31) investigated brain regions with nominal P values even robust to Benjamini-Hochberg FDR correction for testing multiple regions (corrected P all < 0.05 (except pallidum); Table 2). Moreover, anxiety-related regions such as amygdala, hippocampus-parahippocampus, cingulate cortex, calcarine fissures, temporal lobe, and thalamus were found strongly associated with nominal P values < 0.01 ( Fig. 1; Table 2).

Modulatory influence of ADORA1 rs1874142 on rs5751876 and rs5751876 × sleep effects
Second model analyses revealed ADORA1 rs1874142 to statistically strengthen the model, i.e. under the influence of rs1874142 stronger significances were detected for the effects of rs5751876 (global P = 0.018; Table 2) and the interaction rs5751876 × sleep (global P = 0.020) on A 1 AR availability. Both these rs5751876-related terms revealed P values ranging below the adjusted significance level of 0.025. Further, both findings could be confirmed post hoc for nearly all investigated brain regions ( Table 2; FDRcorrected P values < 0.05 (except pallidum and anterior cingulum for rs5751876; except pallidum for rs5751876 × sleep)). Again, anxiety-related regions such as amygdala, hippocampus/Parahippocampus, calcarine fissures, and temporal lobe were found strongly associated with nominal P values < 0.01 ( Fig. 2; Table 2).
In addition, ADORA2B rs2535609 seemed to statistically strengthen the model. Under its influence, stronger significances were detected for the multivariate effects of rs5751876 (global P = 0.005) and rs5751876-related interactions (rs5751876 × sleep, global P = 0.005; rs5751876 × rs2535609, global P = 0.012) on A 1 AR availability (Table 3). However, none of these multivariate findings could be confirmed post hoc for individual brain regions, as none of them survived correction for multiple brain region testing (all FDRcorrected P values > 0.05).
All other adenosinergic or dopaminergic gene variants seemed to statistically weaken second model analyses (for all: multivariate global P values > 0.05, see Table 3).

Discussion
Our present study revealed two main findings. First, we were able to corroborate the ADORA2A rs5751876 effect on A 1 AR availability, originally detected in our pilot sample of males only 31 , in an independent male/female sample. This allowed a more-generalized interpretation compared with the pilot model and thus further underlined the important role of ADORA2A interacting with A 1 AR as detailed in the introduction (e.g., [4][5][6][16][17][18] ). Second, our findings showed modulation by ADORA1 rs1874142, resulting in statistically strengthened effects of rs5751876 and rs5751876 × sleep on A 1 AR availability. In these findings, anxiety-related brain regions were particularly involved, underscoring the close link between ADORA2A rs5751876, anxiety, and anxiety disorders. This is in line with the variety of anxiety-relevant investigations reported in the introduction (e.g., [21][22][23][24][25][26][27] ). The recruitment of slightly more individuals compared with the pilot study appears to have increased the power of the rs5751876 effect enough (28 subjects in pilot vs. 43 in present sample), to have resulted in now statistically significant multivariate results in contrast to the pilot study 31 . Furthermore, the identified interactive effect of rs5751876 × sleep on A 1 AR availability under the ADORA1 rs1874142 modulation underscores the close interrelationship between the adenosine system, particularly the interplay of A 1 and A 2A receptors, and sleep as mentioned before 5,8,12,13,28 .
However, though the rs5751876 effect on A 1 AR availability was distinctly stronger in the present sample compared with the pilot study, the direction of the rs5751876 effect disclosed discrepancies. In the pilot study the rs5751876 T-allele increased A 1 AR availability 31 , whereas in the present sample the T-allele predominantly decreased A 1 AR availability (in 31 regions in the first model; in 23 regions in the second model under modulation of ADORA1 rs1874142). Analysis of the underlying genotype effects (rs5751876 CC vs. CT vs. TT) uncovered consistent CT to TT (lower to higher A 1 AR availability, respectively) gene-dose effects in both samples. In contrast, the CC carriers scored lowest (with respect to A 1 AR availability) in the pilot, thereby completing the gene-dose distribution, but high in the present sample (in 23 of 31 brain regions even higher than TTs), thereby forming a U-shaped distribution (Suppl. Fig. S1). Though sex differences exist between both samples (males only vs. mixed males/ females), female sex was not related to the detected discrepancy in the direction of the rs5751876 genotype effect between present and pilot sample (present sample males alone: similar rs5751876 T-allele coupled decrease in A 1 AR availability in 30 of 31 brain regions). Besides intended sex differences, both samples were similarly recruited with regard to demographic data like age, body mass index, history of diseases, and habits like sleep, coffee, and nicotine consumption.
To probe alternative explanations for this discrepancy in the direction of the rs5751876 genotype effect and based on the strong interaction of rs5751876 × sleep, we checked if sleep habit might contribute to the rs5751876 genotype effect discrepancies between present and pilot study 31 . As both samples show comparable sleep habits (mean ± SD self-reported habitual sleep duration of present sample 7.35 ± 0.76 vs. pilot sample 7.32 ± 1.01; P = 0.811). Thus, we dichotomized the variant in the subgroup categories "less sleep than mean" (less sleep) and "more sleep than mean" (more sleep) with comparable subgroup sizes in both samples (N (less/more sleep) in present sample: 21/22; in pilot sample: 14/13). In these subgroups, we qualitatively rechecked the respective rs5751876 genotype dependent distribution of A 1 AR availability values with promising findings. In the less sleep group of the pilot sample most regions showed a linear gene-dose relationship, but however 7 of 19 brain regions (hippocampus, thalamus, caudate, putamen, parietal cortex, occipital cortex, sensorimotor cortex; Suppl. Tab. S4) revealed indeed Ushaped distribution, which resembles the U-shaped distribution in 31 brain regions in our present study (Suppl. Tab. S5). In contrast, the more sleep group of the pilot sample revealed the above mentioned gene-dose distribution in almost all (17 of 19) regions (Suppl. Tab. S4; only posterior cingulate gyrus and putamen with inverted U-shaped distributions). This again resembles the more sleep group of our present study revealing as well genedose genotype distribution in 13 of 31 brain regions, including particularly the anxiety-relevant regions amygdala, hippocampus/Parahippocampus, cingulate cortex Table 3 Putative modulatory effect of additional adenosinergic or dopaminergic gene variants on the effects of ADORA2A rs5751876 on human in vivo A 1 AR availability in a mixed male/female sample.  1 Benjamini-Hochberg FDR correction for testing 31 brain regions.
(anterior, middle), thalamus, temporal lobe, calcarine fissures, and occipital lobe; Suppl. Tab. S5). Taken together, though self-reported and not measured by objective methods like actigraphy or EEG, sleep might explain at least several of the observed discrepancies. This is in line with previous findings of a tight connection between ADORA2A and sleep as well as between sleep and A 1 AR availabilities. ADORA2A variants (including rs5751876) modulate psychomotor vigilance in a rested state and after sleep deprivation 28 , as well as A 1 AR availability increases after prolonged wakefulness 12,13 . Slight A 1 AR availability increases might also occur in our less sleep group participants owing to their shorter habitual sleep duration and therefore slightly prolonged wakefulness. Such slightly increased A 1 AR availability might then have obscured or masked the presumably small effect size of the ADORA2A rs5751876 T-allele effect. In contrast, our more sleep group probably would not have slightly prolonged wakefulness and therefore presumably would have overall lower A 1 AR availabilities. In such cases, the small effect of the rs5751876 T-allele and the resulting slightly upregulated A 1 AR availability might be visible. Such slight but possibly permanent upregulation of A 1 ARs by genotype might provide protection against overly increased arousal or anxiety, or even anxiety disorders. Indeed a protective role of upregulated A 1 AR was reported toward hyperarousal associated with restless legs syndrome 49 . In rodents, upregulation or positive allosteric modulation of A 1 AR caused robust anxiolytic effects 10,11 , whereas lack of A 1 AR enhanced anxiety and arousal 20 . Finally, we checked all 31 additional adenosinergic and dopaminergic gene variants for potentially divergent genotype group distributions in pilot vs. present sample by nonparametric statistics (Fisher's exact test; see also Suppl. Tab. S6 with footnotes), which might further explain the rs5751876 genotype effect discrepancies between both samples. Strikingly, DRD2 variants rs4245146 and rs1800497 were distributed differently between both samples (both P < 0.05; Suppl. Tab. S6), which might contribute to the gene-dose vs. U-shaped rs5751876 effects. Although 15 rs4245146 CC homozygotes existed in the present sample, only one was present in the pilot (Suppl. Tab. S6), and this rs4245146 CC-homozygosity seemed to further modulate ADORA2A rs5751876 effects. The 15 rs4245146 CC homozygotes had increased A 1 AR availabilities in all 31 brain regions in rs5751876 CC carriers (11 regions P < 0.05; 10 regions P < 0.1) but had decreased A 1 AR availabilities in all 31 brain regions in rs5751876 CT-/TTcarriers (one region P < 0.05; one region P < 0.1; Suppl. Tab. S7). Thus, rs4245146 CC-dependent modulation might interplay with the rs5751876 effect on A 1 AR availability in the present sample (Suppl. Fig. S2) but not the pilot sample with only one rs4245146 CC-subject. As well, carriers of the rare rs1800497 A-allele are four times more frequent in the present sample (N = 16) than in the pilot (N = 4; Suppl. Tab. S6). This higher number of A-allele carriers might have lowered individual D 2 R levels, in line with a reduced striatal D 2 R binding already shown in rs1800497 A-allele carriers 50 . As A 2A and D 2 receptors have a tight spatial/functional interplay and interact antagonistically 36,37 , lower D 2 R levels could lead to increased A 2A R levels in our 16 rs1800497 A-allele carriers. Via the similarly tight spatial/functional antagonistic interplay of A 2A and A 1 receptors (c.f. introduction), such putatively increased A 2A R levels in the 16 subjects (=37% of present sample) might have decreased A 1 AR levels and thereby covered or obscured the presumably mild ADORA2A rs5751876 effect on A 1 AR availability. In comparison, Eisenstein et al. 50 reported normal striatal D 2 R specific binding in rs1800497 GG-homozygotes, which might be the precondition to clearly identify the small effect size ADORA2A rs5751876 genotype group effects in our present study (63% of subjects vs. 86% of pilot subjects). Interestingly, both DRD2 variants, rs1800497 and rs4245146, were described to play prominent roles in anxiety before (e.g., [39][40][41], further supporting our more distinctive findings in anxiety-related brain regions. Moreover, dopaminergic genes including DRD2 have been shown to be involved in inverted U-shaped relationships between dopamine signaling and prefrontal cortical function (e.g., 51,52 ), supporting our assumption of DRD2 variants presumably contributing to our sample ADORA2A rs5751876 differences. Unfortunately, the size of our present sample did not allow inclusion of additional genetic variants like rs4245146 or rs1800497 together with rs5751876 and rs1874142, as the model is underpowered then, warranting distinctly larger samples in subsequent investigations.
A next step could be to compare individuals with and without anxiety symptoms and heart rate measurements with regard to their objectively measured sleep duration and genotypes. Such multidimensional approach would demand higher statistical power, which underscores the need to extend future sample size. In our present study it was modest, albeit in the range of comparable recent PET-based studies (e.g., 13,53,54 ) and well-powered enough for our ADORA2A rs5751876-focused analyses to detect the hypothesized effects. It did unfortunately not allow for additional detailed investigation of for example additional interacting effects of DRD2 variants together with ADORA2A rs5751876 and ADORA1 rs1874142 in the same multivariate models as mentioned above. Further, our study provided only cross-sectional data, whereas a longitudinal study might allow prospective insights and maybe the potential for interventional approaches.

Conclusion
Our present findings support the role of ADORA2A rs5751876 on in vivo A 1 AR availability in the human brain. Further analyses suggested additional modulatory effects of ADORA1 rs1874142 on the effects of rs5751876 and rs5751876 × sleep on A 1 AR availability. Together, these effects might contribute to the regulation of the sleep-arousal system and thus might be involved in the development of particularly arousal-related mental disorders such as anxiety disorders. However, transferability of our results between present and pilot samples was difficult due to small effect sizes and complex gene × gene (ADORA2A × ADORA1, ADORA2A × DRD2) and gene × environment (ADORA2A × sleep) interactions, which modulated the direction of the biological outcome, i.e., A 1 AR availability in the brain. This might complicate also comparisons with future samples and therefore necessitates increased sample sizes, more comparable to current MRI-based samples, to allow the detection of such complex underlying and modifying relationships. In the next steps, more in-depth analyses of the ADORA2A rs5751876 × sleep relation are required based on objectively measured individual sleep parameters ideally complemented by more-detailed information of individual anxiety sensitivity, life styles, (night shift) workload, and past or present stressful life events. Further, investigation of samples preferably including also individuals with anxiety symptoms and in a longitudinal design are needed.