Impact of Genetic Variability on Physiological Responses to Caffeine in Humans: A Systematic Review

Emerging research has demonstrated that genetic variation may impact physiological responses to caffeine consumption. The purpose of the present review was to systematically recognize how select single nucleotide polymorphisms (SNPs) impact habitual use of caffeine as well as the ergogenic and anxiogenic consequences of caffeine. Two databases (PubMed and EBSCO) were independently searched using the same algorithm. Selected studies involved human participants and met at least one of the following inclusion criteria: (a) genetic analysis of individuals who habitually consume caffeine; (b) genetic analysis of individuals who underwent measurements of physical performance with the consumption of caffeine; (c) genetic analysis of individuals who underwent measurements of mood with the consumption of caffeine. We included 26 studies (10 randomized controlled trials, five controlled trials, seven cross-sectional studies, three single-group interventional studies and one case-control study). Single nucleotide polymorphisms in or near the cytochrome P450 (CYP1A2) and aryl hydrocarbon receptor (AHR) genes were consistently associated with caffeine consumption. Several studies demonstrated that the anxiogenic consequences of caffeine differed across adenosine 2a receptor (ADORA2A) genotypes, and the studies that investigated the effects of genetic variation on the ergogenic benefit of caffeine reported equivocal findings (CYP1A2) or warrant replication (ADORA2A).


Introduction
Caffeine (1,3,7-trimethylxanthine) is one of the most widely used drugs in the world and is available in many mediums for consumption. The pharmacokinetics and pharmacodynamics of caffeine have been well studied [1]. Caffeine metabolism occurs primarily in the liver via the cytochrome P450 system (CYP1A2) [2]. The CYP1A2 proteins are encoded by the CYP1A2 gene, and CYP1A2 activity is induced when aromatic hydrocarbons bind the aryl hydrocarbon receptor [3]. Caffeine acts as an adenosine antagonist via competitive inhibition [4], and research in mice has demonstrated that blockade of adenosine 2a receptors (encoded via ADORA2A gene) may potentiate dopaminergic neurotransmission [5]. It is biologically plausible that variations in the CYP1A2 and aryl hydrocarbon receptor (AHR) genes impact the metabolism of caffeine and thus subsequent physiological concentrations of caffeine achieved. Further, it can be hypothesized that variations in the ADORA2A gene may impact caffeine-adenosine 2a receptor binding characteristics and thus downstream dopaminergic neurotransmission. Recently, the effects of single nucleotide polymorphisms (SNPs) in the aforementioned genes on caffeine use and metabolism have been investigated [3,6,7].
With the widespread consumption of caffeine-containing beverages, the health consequences of these beverages are of particular interest to researchers. For example, the chronic consumption of coffee has been associated with cognitive performance and cardiovascular health [8,9]. The identification of predictors of habitual caffeine consumption may prove useful to epidemiologists and health professionals. To date, several SNPs, such as the CYP1A2 (rs2472297) and AHR (rs4410790, rs6968554), have been implicated in habitual use [10]. Further, while caffeine is generally well tolerated, some individuals report feelings of anxiety following consumption [11]. Recent investigations have explored the effect of variations in the ADORA2A and CYP1A2 genes as a potential explanation for caffeine's anxiogenic impact in some individuals [6,11,12].
Athletes have long utilized caffeine as an ergogenic aid [13]. Research has demonstrated that 3-6 mg kg −1 of body mass mildly improves exercise/physical performance [14][15][16]. Nonetheless, investigators have reported equivocal findings, with some reporting interindividual variation in ergogenic responses to caffeine within their subject pools [17][18][19]. Earlier work has demonstrated that a Single Nucleotide Polymorphism (SNP) in the CYP1A2 gene (rs762551) led to differing rates of caffeine metabolism across genotypes in smokers [3]. Recently, researchers have examined the influence of this specific SNP and select others on the ergogenic benefit of caffeine [20,21].
To our knowledge, investigators have not systematically recognized studies evaluating the effects of indexed and unknown SNPs in biologically plausible genes on physiological responses to caffeine across scholarly disciplines. Such a systematic review may provide a basis for further interdisciplinary approaches and future directions. Therefore, the purpose of the present review was to systematically investigate the impact of select SNPs on the ergogenic and anxiogenic consequences, and habitual use, of caffeine in humans.

Search Strategy
The Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) guidelines [22][23][24] were followed. Two databases [PubMed and Medline (EBSCO)] were independently searched by two investigators (J.L.F and P.C.D) up until 5 July 2018 using an appropriate algorithm ( Figure S1). Any conflicts in the searching procedure were resolved through consensus, while the searching results were reviewed and sorted to identify relevant publications to the topic under review.

Selection Criteria
The studies included in this review involved human participants and met at least one of the following criteria: (a) genetic analysis of individuals who habitually consume caffeine; (b) genetic analysis of individuals who underwent measurements of physical performance with the consumption of caffeine; (c) genetic analysis of individuals who underwent measurements of mood with the consumption of caffeine. Included studies displayed outcomes regarding SNPs associated with habitual caffeine consumption, relationships between certain SNPs, and relationships between caffeine consumption and mood. We excluded animal studies, reviews, conference proceedings, and editorials; however, we screened the reference lists of such publications and of the retrieved articles for relevant papers. The list of the included studies (n = 26) is available in the data extraction table (Table 1), while the list of the excluded studies (n = 3512) is available in Figure S2. ADORA2A TT genotype reported highest anxiety (VAS) (4.6 ± 1.9) and ADORA2A CC (−7.5 ± 3.7) reported the least anxiety after 150 mg of caffeine but was not significant when data for European-American participants were considered (p = 0.1); caffeine-induced anxiety was associated with dopamine receptor 2 gene (DRD2) polymorphism Cornelis [26] Cross-sectional n = 2735

Risk of Bias Assessment
Two reviewers (J.L.F. and P.C.D.) independently evaluated the risk of bias of the non-randomized controlled trials (RCT) via the 13-item tool developed by the Research Triangle Institute (RTI), Evidence-based Practice Center [24]. This tool has previously shown median interrater agreement of 75% [43] and 93.5% [44]. The risk of bias of the RCTs was assessed via the "Cochrane Collaboration's tool for assessing risk of bias" [45]. Conflicts in the risk of bias assessment were resolved by two independent referee investigators (E.J.R. and A.E.C.).

Data Extraction and Analysis
The results of the data extraction procedure are shown in Table 1. Data extraction was performed independently by two investigators (J.L.F. and E.J.R.), and conflicts were resolved by a referee investigator (A.E.C.). For all included studies, we extracted the first authors' name, year of publication, design of the studies, participants' characteristics (i.e., number, sex, age, health status, and intervention) and the main and secondary outcomes, including results from statistical analyses. A qualitative synthesis of the retrieved evidence was completed thereafter.

Results
The reporting of the available information in this systematic review is shown in a PRISMA checklist in Figure S3.

Searching Procedure Results
The entire search yielded 3532 records. Of these, 2115 were duplicates; therefore, 1417 were initially screened to exclude reviews, conferences and editorials (n = 387). Consequently, 1033 records were assessed for eligibility, with 20 studies meeting the inclusion criteria. Finally, an additional six records were added manually. The searching outcome is presented in a PRISMA flow diagram ( Figure S4).

Characteristics of the Included Studies
The characteristics and the results of the included studies are presented in Table 1. Of the included 26 studies, 10 were RCT (38%), five were controlled trials (CT) (19%), seven were cross-sectional studies (CSS) (27%), three were single-group interventional studies (13%), and one was a case-control study (CCS) (4%).

Risk of Bias Assessment
The risk of bias assessment results can be found in Tables 2 and 3, and a summary of the results are displayed in Figures 1 and 2. For the RTCs, eight showed an unclear risk of bias for random sequence generation [11,12,31,36,38,39] and two showed a low risk of bias [21,33]. For allocation concealment, six studies were classified as showing an unclear risk of bias [6,11,12,20,31,36,38,39] and two showed a low risk of bias [21,33]. Seven studies demonstrated an unclear risk of bias for the blinding of participants and researchers [6,11,12,20,31,33,36], although the studies stated that the participants were "blinded", and three displayed a low risk of bias [21,38,39]. Six studies were categorized as having an unclear risk of bias for the blinding of outcome assessment [6,12,21,31,38,39], and four were categorized as being a low risk of bias [11,20,33,36]. One out of the 10 RCTs [21] showed an unclear risk of bias for incomplete data, while the others displayed a low risk of bias. Finally, all 10 of the RCTs were categorized as having a low risk of bias for selective reporting and other bias.  1 Figure 1. Summary of risk of bias assessment for randomized controlled trials (n = 10). Selection bias (random sequence generation, low risk (n = 2), unclear risk (n = 8) + allocation concealment, low risk (n = 4), unclear risk (n = 6)); Performance bias (blinding of participants and researchers, low risk (n = 3), unclear risk (n = 7)); Detection bias (blinding of outcome assessment, low risk (n = 4), unclear risk (n = 6)); Attrition bias (incomplete outcome data, low risk (n = 9), unclear risk (n = 1)); Reporting bias (selective reporting, low risk (n = 10)); Other bias, low risk (n = 10).
Out of the 16 non-RCTs, one showed a high risk of bias [28], two were non-applicable to the category [7,35], two showed an unclear risk of bias [37,42], and the other 11 showed a low risk of bias [3,10,[25][26][27]29,30,32,34,40,41] for selection bias. Of these studies, only one was non-applicable to the category [7], while the other 15 showed a low risk of bias for performance bias. For detection bias, six studies showed a low risk of bias [25,29,30,32,37,41], and the other 10 studies showed an unclear risk of bias. One of the 16 studies showed an unclear risk of bias for attrition bias [7], and the other 15 studies were non-applicable to the category. All 16 of the studies displayed a low risk of bias for selective outcome. For the confounding category, six of the 16 studies displayed an unclear risk of bias [10,27,28,34,35,37], and the remaining 10 displayed a low risk of bias.

Anxiogenic Consequences
Eight of the included studies reported data on genetic variation and anxiety/side effects of caffeine [6,11,12,[29][30][31]38,39]. Two of the included studies investigated the effects of an SNP in the CYP1A2 (rs762551) gene on self-reported side effects of caffeine following consumption in basketball players [38,39]. The results of these studies demonstrated that self-reported feelings of anxiety were not different across genotypes. Alsene et al. [11] investigated the impact of genetic variation in the ADORA2A gene on anxiety following caffeine consumption in caffeine-naive subjects via a double-blind RCT. The data demonstrated that two polymorphisms (rs5751876 and rs35060421) were associated with self-reported anxiety, with the TT and 2592Tins/Tins genotypes reporting higher anxiety, respectively. In an additional double blind RCT, Childs et al. [6] demonstrated that genetic variation in the ADORA2A (rs5751876, rs2298383, rs4822492) and dopamine receptor DRD2 (rs1110976) genes were associated with anxiety in 102 non-to-moderate caffeine users. Further supporting the findings that individuals with the rs5751876 TT genotype may be prone to anxiety with caffeine, Gajewska et al. [31] and Domschke et al. [29] reported that subjects with the rs5751876 TT genotype exhibited impaired prepulse inhibition (female subjects) and an increased startle reflex (particularly female subgroup) with caffeine, respectively. Nonetheless, one study [12] demonstrated that the anxiogenic effect of caffeine was only apparent in subjects with the rs5751876 TT genotype that were caffeine naive. The authors concluded that tolerance to the anxiogenic impact of caffeine is observed when individuals habitually consume moderate to large doses [12]. Additionally, one study reported that variation in the Neuropeptide S receptor gene (rs324981) may (in conjunction with ADORA2A (rs5751876)) further impact the anxiogenic effects of caffeine [30].

Ergogenic Consequences
Eight of the included studies investigated the effects of genetic variability on the ergogenic consequences of caffeine [20,21,25,32,33,36,39,46]. Five of these studies [20,25,32,33,36] assessed the impact of the CYP1A2 (rs762551) SNP on the ergogenic consequences of caffeine using cycling time trials as a performance measure with disparate findings. Womack et al. [20] reported that male cyclists (n = 35) with the CYP1A2 (rs762551) AA genotype demonstrated greater improvements in cycling performance (40 km time trial) versus C-allele carriers following caffeine consumption (6 mg kg −1 anhydrous caffeine). Similarly, Guest et al. [33] reported that male cyclists (n = 101) with the CYP1A2 (rs762551) AA genotype demonstrated greater improvements in cycling performance (10 km time trial) relative to those with the CYP1A2 (rs762551) CC genotype following caffeine treatment (2 and 4 mg kg −1 ). Equivocally, Pataky et al. [36] demonstrated that recreational cyclists (n = 38) with the CYP1A2 (rs762551) AC genotype derived a more robust ergogenic benefit (3 km time trial) following caffeine treatment (6 mg kg −1 and 6 mg kg −1 plus caffeinated mouth rinse) relative to CYP1A2 (rs762551) AA homozygotes. Algrain et al. [25] demonstrated that subject groups (AA vs. C-allele carriers) responded similarly to caffeine treatment (300 mg in gum vs. placebo gum) with performance measured via a 15-min performance ride. Giersch et al. [32] found that the ergogenic consequences (3 km time trial) of caffeine (6 mg kg −1 anhydrous caffeine) did not differ across CYP1A2 (rs762551) genotype groups (AA vs. C-allele carriers). Algrain et al. [25], Pataky et al. [36], and Giersch et al. [32] all cited methodological differences as a potential explanation for the disparate findings in the literature. Studies investigating the impact of the CYP1A2 (rs762551) SNP on the erogenicity of caffeine utilizing sports-related outcome measures (Wingate test, reaction time, basketball-specific skills) have reported that genotype groups responded equally to caffeine [38,39]. One of the included studies [21] examined the impact of the adenosine receptor ADORA2A (rs5751876) SNP on ergogenic responses to caffeine in females (n = 12) with performance assessed via a 10-min cycling time trial. These data demonstrated that subjects with the ADORA2A (rs5751876) TT genotype derived a larger ergogenic benefit from caffeine relative to heterozygotes or CC homozygotes [21].

Other Outcomes
Three of the included studies investigated the effect of the CYP1A2 (rs762551) genotype on caffeine metabolism [3,25,32]. Sachse et al. [3] reported that, among smokers (n = 51), subjects who possessed the CYP1A2 (rs762551) AA genotype metabolized caffeine (100 mg) faster relative to C-allele carriers while utilizing a 5 h paraxanthine/caffeine ratio as the outcome measure. The authors concluded that the CYP1A2 (rs762551) AA genotype may confer high inducibility and CYP1A2 activity. More recently, confirming the findings above in 20 nonsmokers, Giersch et al. [32] reported that, 1 h following the administration of 6 mg kg −1 anhydrous caffeine, circulating caffeine concentrations were lower in subjects possessing the CYP1A2 (rs762551) AA genotype relative to C-allele carriers. Equivocally, Algrain et al. [25] reported comparable circulating caffeine concentrations over time in nonsmokers across CYP1A2 (rs762551) genotypes. Two of the included studies investigated the effects of the CYP1A2 (rs762551) SNP on the cardiovascular consequences of caffeine [40,41]. Thomas et al. [41] reported that changes in postexercise heart rate variability with caffeine treatment (300 mg) were similar between CYP1A2 genotype groups (AA vs. C-allele carriers). Soares et al. [40] reported that CYP1A2 heterozygotes demonstrated increases in systolic blood pressure, while subjects with the CYP1A2 AA genotype did not following acute caffeine ingestion (6 mg kg −1 ).
Three additional studies were included in the present review [28,35,42]. Djordjevic [28] explored the association of multiple CYP1A2 polymorphisms with the induction of CYP1A2 enzyme activity resultant from heavy caffeine consumption. These data demonstrated that high CYP1A2 enzyme activity was associated with heavy coffee consumption only in subjects possessing the CYP1A2 (rs762551) AA genotype. In a classical twin design study, Luciano et al. [35] demonstrated that genes not typically associated with sleep disturbance were implicated in coffee-attributed insomnia. In a CCS, Urry et al. [42] demonstrated that subjects with type 2 diabetes mellitus exhibited higher estimated CYP1A2 enzyme activity relative to control subjects.

Discussion
The purpose of the present review was to systematically recognize how select SNPs impact habitual use of caffeine as well as the ergogenic and anxiogenic consequences of caffeine. The primary findings of our work will be discussed in the subsections below.

Habitual Use
Genome-wide association scans have implicated several indexed SNPs in caffeine consumption. The aforementioned SNPs occur in genes known to be involved in the pharmacokinetics and pharmacodynamics of caffeine. The results of the present review indicate that SNPs in the CYP1A2 gene and near the AHR gene have been consistently associated with caffeine consumption [7,10,27,34]. Further, less conclusive evidence suggests that SNPs in the ADORA2A gene are associated with caffeine consumption [10,26,27]. Recently, novel genes have been implicated in caffeine consumption, with authors calling for replication of the findings and postulating biological plausibility [10,37].

Anxiogenic Consequences
Our search provided strong evidence that SNPs in the ADORA2A gene (primarily rs5751876) are associated with the anxiogenic impact of caffeine [6,11,29,31]. Particularly, caffeine-naïve females possessing the ADORA2A (rs5751876) TT genotype may be especially prone to experiencing anxiety following caffeine consumption [29,31]. Interestingly, one study demonstrated that self-reported anxiety with caffeine was not apparent in subjects possessing ADORA2A (rs5751876) TT who habitually consume large to moderate doses. Thus, the available evidence suggests that habitual use may lead to tolerance to the anxiogenic consequences of caffeine regardless of select genetic variations.

Ergogenic Consequences
The studies included in this section of the review focused primarily on the CYP1A2 (rs762551) SNP and reported equivocal findings. Two studies reported that the CYP1A2 (rs762551) AA genotype resulted in a more robust ergogenic benefit of caffeine [20,33] relative to C-allele carriers, while one study reported opposing findings [36], and two studies reported comparable ergogenic responses across genotype groups [25,32]. Two studies reported that the CYP1A2 (rs762551) SNP did not influence sport-specific outcomes with and without caffeine [38,39]. Our search included one pilot study that investigated the effects of the ADORA2A (rs5751876) SNP on the erogenicity of caffeine; Loy et al. [21] reported that females with the ADORA2A (rs5751876) TT genotype derived a larger ergogenic benefit with caffeine relative to C-allele carriers. In general, the studies included in this section cited some methodological constraints, such as low sample size and capricious outcome measures. We recommend that future studies increase sample size, utilize more standardized outcome measures, and examine a multitude of biologically plausible SNPs to further elucidate the impact genetic variation has on the ergogenic consequences of caffeine.

Quality of Evidence, Limitations, and Potential Biases in the Review Process
Based on the studies selected for the aim of the current systematic review, we may form adequate conclusions regarding the impact of select SNPs on the ergogenic and anxiogenic consequences and habitual use of caffeine in humans. This is because we identified enough available evidence in the area. The included RCTs displayed unclear and low risk of bias in the selection, performance, and detection biases, while they mostly displayed low risk of bias in the attrition, reporting, and other biases. Similarly, the non-RCTs displayed mostly low risk of bias in the selection, performance, reporting, and other biases, while in the attrition bias, most studies were non-applicable for the category. This indicates that both the RCTs and the non-RCTs may provide fairly good quality evidence.
Our systematic review has a number of strengths. We used the PRISMA guidelines [22][23][24] and appropriate databases and algorithms with standardized indexing terms for our searching procedure. We also used well-established tools to evaluate the included [24,[43][44][45] studies. Furthermore, to minimize bias in our systematic review process, two investigators worked independently on the searching and screening procedure, data extraction, and risk of bias assessment. Finally, we have not excluded studies based on language.
A possible limitation of the current systematic review is that we avoided the use of non-peer-review data (grey literature) and conference papers to test our research question. However, the inclusion of non-peer-review data may have itself introduced bias, given that there was available peer-reviewed evidence [47]. Another possible limitation is the small number of the included studies, especially the RCTs, which indicates the need for additional research of this topic in the future.

Conclusions
In conclusion, our search has provided evidence that the CYP1A2, AHR, and ADORA2A genes are associated with habitual consumption, and further exploration is warranted to clarify how these genes directly or indirectly impact physiological and/or psychological mechanisms responsible for the variability in consumption of caffeine across individuals. The literature also demonstrates that gender, habitual caffeine consumption, and variability in the ADORA2A gene collectively influence individual susceptibility to the anxiogenic consequences of caffeine and that variability in the CYP1A2 gene, in conjunction with environmental factors (heavy coffee drinking, smoking), impact the metabolism of caffeine. Future work is warranted to elucidate the effects of variability in the CYP1A2 and ADORA2A genes on the ergogenic impact of caffeine. We recommend all future studies in this area utilize an interdisciplinary approach as the physiological consequences of caffeine in humans are likely dependent on a complex interaction of genetic, physiological, and behavioral factors.