Caffeine consumption and interaction with ADORA2A, CYP1A2 and NOS1 variants do not influence age at onset of Machado-Joseph disease

Caffeine consumption and interaction with ADORA2A, CYP1A2 and NOS1 variants do not influence age at onset of Machado-Joseph disease | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Caffeine consumption and interaction with ADORA2A, CYP1A2 and NOS1 variants do not influence age at onset of Machado-Joseph disease Ana Carolina Martins, Jordânia dos Santos Pinheiro, Luciana Szinwelski, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4325198/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Background The age at onset (AO) of Machado-Joseph disease (SCA3/MJD), a disorder due to an expanded CAG repeat (CAGexp) in ATXN3 , is quite variable and the role of environmental factors is still unknown. Caffeine was associated with protective effects against other neurodegenerative diseases, and in transgenic SCA3/MJD mouse models. We aimed to evaluate whether caffeine consumption and the interaction with variants of caffeine signaling/metabolization genes impact the AO of this disease. Methods a questionnaire on caffeine consumption was applied to adult patients and unrelated controls living in Rio Grande do Sul, Brazil. AO and CAGexp were previously determined. SNPs rs5751876 ( ADORA2A ), rs2298383 ( ADORA2A ), rs762551 ( CYP1A2 ) and rs478597 ( NOS1 ) were genotyped. AO of subgroups were compared, adjusting the CAGexp to 75 repeats (p < 0.05). Results 171/179 cases and 98/100 controls consumed caffeine. Cases with high and low caffeine consumption (more or less than 314.5 mg of caffeine/day) had mean (SD) AO of 35.05 (11.44) and 35.43 (10.08) years (p = 0.40). The mean (SD) AO of the subgroups produced by the presence or absence of caffeine-enhancing alleles in ADORA2A (T allele at rs5751876 and rs2298383), CYP1A2 (C allele) and NOS1 (C allele) were all similar (p between 0.069 and 0.516). Discussion Caffeine consumption was not related to changes in the AO of SCA3/MJD, either alone or in interaction with protective genotypes at ADORA2A , CYP1A2 and NOS1 . Caffeine consumption ADORA2A CYP1A2 NOS1 spinocerebellar ataxia type 3 Machado-Joseph disease 1. Introduction Machado Joseph disease, also known as spinocerebellar ataxia type 3 (SCA3/MJD), is caused by a CAG repeat expansion (CAGexp) at ATXN3 gene [1]. Symptoms begin gradually in adult life, encompassing incoordination of gait, limb movements, speech, eye movements and swallowing, combined with pyramidal, extrapyramidal, lower motor neuron, and sensory findings. The mean (SD; range) age at onset (AO) in our region, of 34.3 (11.2; 5–59) years of age, is comparable to data obtained worldwide [2, 3]. The CAGexp length is the most important determinant of the AO, explaining 55% of its variability [3]. Knowledge about factors that determine the other 45% is quite limited and comprise some modifying genes. ATXN2 [3], CFAP57 , ACTG1, DIDO1 [4, 5], CAST [6], and FAN1 [7] were related to mild effects over AO by association studies not replicated in more than one cohort, with the exception of ATXN2 . No environmental factors have been identified to date as influencing SCA3/MJD phenotype. Caffeine is one of the most popular psychoactive substances in use nowadays, in products such as coffee, tea, chocolate and cola soft drinks [ 8]. Data from the International Coffee Organization indicate that Brazil is the largest grain producer and the second largest consumer of the drink in the world, behind only the USA [ 9]. Caffeine has complex mechanisms of action over the central nervous system, including a non-selective adenosine A2AR receptor - encoded by ADORA2A [10] - antagonism, mobilization of intercellular calcium, and phosphodiesterase inhibition [8, 11]. Enhancing effects may occur by reducing caffeine metabolism, increasing absorption, or direct agonism. Preclinical studies done in SCA3/MJD transgenic mice linked caffeine and/or A2AR blockade to alleviating neurodegeneration [12, 13]. Similar results were obtained in some preclinical studies on Huntington's disease (HD), another CAGexp disease that might share pathogenetic pathways with SCA3/MJD [14]. CYP1A2 codes for cytochrome P450 1A2, the enzyme responsible for 95% of the metabolism of caffeine in the liver [15]. Caffeine metabolism by the CYP1A2 enzyme shows substantial variation between people, due to genetic and environmental factors [16]. Moreover, some CYP1A2 variants were already associated with enhancing the neuroprotective effect of caffeine [17]. Nitric oxide (NO) performs vital physiological functions in the nervous system under normal concentrations [18]. The neuronal isoform (nNOS) signaling has significant involvement in several neurodegenerative diseases, such as Parkinson's disease (PD), Alzheimer’s disease and HD [19]. Caffeine reduces the expression of NOS , including nNOS [20, 21]. A relationship between NOS1 and protection against PD in individuals who consumed high caffeine was proposed [22]. Epidemiological studies on neurodegenerative disorders have shown contradictory results related to caffeine. Previous meta-analyses and a recent case-control study on more than 18,000 subjects have shown neuroprotective results on PD, in line with preclinical studies [23, 24, 25]. In contrast, caffeine consumption was associated with earlier ages of onset in HD [26, 27]. Since epidemiological studies on potential associations between caffeine consumption and changes in the age of onset of SCA3/MJD are lacking, the present observation aimed to detect a relationship between caffeine consumption and changes in the age of onset of SCA3/MJD; and to examine whether the caffeine effect was mediated by variants at ADORA2A, CYP1A2 and NOS1 already proposed to enhance the effect of caffeine on the central nervous system. 2. Methods 2.1 Design A case-control study was done to determine whether the caffeine consumption among SCA3/MJD patients was different from that of the local, control population. Next, a cross-sectional observation was performed in SCA3/MJD carriers to look for associations between the amount of habitual caffeine consumption and their AO. Finally, AO was compared between SCA3/MJD subjects stratified according to their caffeine consumption and to their genotypes in ADORA2A, CYP1A2 and NOS1. 2.2 Population and clinical procedures Symptomatic subjects with molecular diagnosis were recruited from to the Rio Grande do Sul SCA3/MJD cohort [2], if older than 18 years of age, at the outpatient’s clinic of our institution. Unrelated caregivers who were accompanying patients from the same outpatient clinic were invited to participate as controls. A standardized questionnaire ( Supplemental Materials 1 and 2 ) was administered to subjects who agreed to participate, after obtaining their informed consent forms. Questions to estimate caffeine consumption covered the habit of drinking coffee, teas, soft drinks and chimarrão (or yerba mate , in Spanish speaking populations), a traditional hot beverage consumed in southern Brazil and neighboring countries which is prepared from the leaves of Ilex paraguariensis . Information about habits of smoking were also obtained from all participants, in addition to age, residence, education and professional activities. Clinical and molecular data from the SCA3/MJD carriers such as AO and CAGexp were already available from their electronic files; AO of the first symptom (AOfs) was obtained again by the questionnaire. AOfs was defined by the age at which the subject and/or their family members first identified a motor problem, such as difficulty in walking or speaking, or double vision. This study was approved by the Institutional Ethics Committee (Comissão de Ética em Pesquisa do Hospital de Clínicas de Porto Alegre) by the number CAAE 95633218.3.0000.5327. Written informed consent was obtained from all study participants. 2.3 Genotyping DNA samples were retrieved from the biorepositories of our institution. The CAGexp length was already determined and registered in protected files. Genes and their single nucleotide polymorphisms (SNP) chosen to be genotyped were described in Table 1 . rs5751876 ( ADORA2A ), rs2298383 ( ADORA2A ), rs762551 ( CYP1A2 ) and rs478597 ( NOS1 ) were genotyped using TaqMan SNP Genotyping Assay in a final volume of 8 µL containing 2 ng of DNA, according to assay protocol (Applied Biosystems, Foster City, CA, USA). Amplification was performed in the ABI 7500 Fast Real-Time PCR System® equipment (Applied Biosystems, Foster City, CA, USA) as follows: one cycle of 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Table 1. Single nucleotide polymorphisms chosen as candidate markers for caffeine potentialization effects Genes under study Mechanism SNPs Major allele/ Minor allele MAF** Preliminary evidence on neuroprotective effects ADORA2A Encodes adenosine A2A receptors, which are the main target of caffeine action in the nervous system. This receptor is known to participate in neurogenesis, neuroinflammation, and synaptic plasticity within glutamatergic synapse rs5751876 T*/ C 0.44 Facheris et al., 2008; Loy et al 2015; Gggic et al 2020; Rahimi et al 2023; Virgili et al 2023 rs2298383 T/ C 0.48 Childs et al 2008; Rieck et al 2015; Turčin et al., 2016; Santos-Lobato et al 2019; Fan et al 2020; Erblang et al 2021 CYP1A2 Encodes a member of the cytochrome P450, metabolizer of polycyclic aromatic hydrocarbons present in coffee rs762551 C*/A 0.37 Amin et al ., 2012; Tennent et al 2020 NOS1 Encodes a protein that belongs to the family of nitric oxide synthases. Nitric oxide is a reactive free radical, which acts as a biologic mediator in several processes, including neurotransmission. Preliminary evidence suggested a relationship between nNOS and protection against PD in individuals who consumed high caffeine rs478597 C*/T 0.33 Altmann, 2018; Poon et al., 2021 * Allele proposed to be neuroprotective through caffeine potentialization ** From 1000Genome 2.4 Analyses First, a potential relationship between caffeine and SCA3/MJD was studied by comparing the consumptions of carriers with those of controls. After that, only SCA3/MJD carriers were studied, when the main outcome was their AOfs. Subjects were considered caffeine consumers if they drank two or more cups of coffee per week, for at least 3 months in their lifetime [22]. The amount of caffeine ingested per day was calculated by the sum of the daily beverages reported by subjects, as follows: 137mg and 47 mg of caffeine per cup (150ml) of coffee and tea, respectively [28]; and 135 mg of caffeine per 500ml of chimarrão [29]. Most participants were unsure about reporting their soft drink consumption - for example, the quantity and type of soft drinks consumed in recent weeks - as this would be an expensive and erratic habit, limited to special situations such as parties. Because of this, the consumption of soft drinks was not included in the analysis. Since the great majority of participants were caffeine consumers, and comparisons between consumers and non-consumers were not possible, subjects were dichotomized into high vs. low consumers according to the median (SD) daily consumption of mg of caffeine. Age, sex, schooling, and tobacco consumption were controlled in all subjects included in this study. Subjects were considered nicotine users if they have smoked at least 100 cigarettes in their lifetime. Since tobacco was consumed exclusively through industrial cigarettes, in the present cohort, the usual number of cigarettes smoked were taken ( Supplemental Materials 1 and 2 ). Finally, the CAGexp was controlled for SCA3/MJD carriers. Chi-square, t-test and Mann-Whitney test were used to compare case and control groups, according to the variables under analysis and to their distribution. ANOVA was used to compare the AO between subgroups, adjusting the CAGexp to the median value observed in the group of 75 repeats. Tests with p < 0.05 significance level were accepted. All tests were performed with SPSS version 20 software (SPSS Inc., Chicago, IL, USA) [30]. 3. Results One hundred, seventy-nine symptomatic SCA3/MJD subjects and 100 controls were included. More than 95% of cases and controls consumed caffeine beverages. Cases and controls had similar median caffeine consumptions per day ( Table 2 ). Table 2. General characteristics of the subjects under study SCA3/MJD Controls p Women/all subjects (%) 100/179 (55.9) 53/100 (53.0) 0.707* Caffeine consumers (%) 171 (96%) 98 (98%) 0.801* Tobacco consumers 64 (35.8%) 34 (34%) 0.795* Age at onset of symptoms mean (SD) 35.26 (10.62) - CAGexp length median (range) 75 (65-87) - Age at interview mean (SD) 45.65 (11.64) 44.94 (14.14) 0.671** Years of schooling mean (SD) 11.10 (3.80) 11.16 (4.22) 0.896** mg of caffeine consumption/day mean (SD) 331.85 (215.65) 352.12 (246.37) 0.698*** *Chi-square **t-test ***Mann-Whitney test Patients that consumed more or less than the median consumption of 314.5 mg of caffeine/day had a mean (SD) AOfs of 35.05 (11.44) and 35.43 (10.08) years (p=0.40), for a CAGexp of 75 repeats. Covariates such as sex, tobacco consumption habits and schooling did not add any significant effect over their AOfs (data not shown). The distribution of allele frequencies for each SNP studied was displayed in Table S1 , Supplemental Material 3 . The genotypic subgroups of ADORA2A (rs5751876 and rs2298383), CYP1A2 , and NOS1 , were then studied: no association was found between these genotypes and direct differences in AOfs ( Table S2 , Supplemental material 3 ). Then the effect of caffeine consumption was combined to the presence/absence of caffeine-enhancing alleles at ADORA2A (rs5751876, CYP1A2 , and NOS1. Since there was no clear caffeine-enhancing allele at ADORA2A rs2298383, the three possible genotypes, divided by caffeine consumption, were compared. Four to six subgroups were then compared per gene - according to genotypes plus caffeine consumption. No statistical differences were found in the AOfs of each set of subgroups, per gene under study, after controlling for a CAGexp of 75 repeats ( Table 3 ). Table 3. The potential effects of the combination of polymorphisms under study and caffeine habits over the age at onset of SCA3/MJD Caffeine consumption/ day Gene and polymorphism Ages at onset for carriers of 75 CAGexp repeat length mean (SD) p * Genotypes proposed to be protective Alternative genotype CYP1A2, rs762551 CC or AC AA More than 314.5 mg 34.06 (10.76) n = 31 34.73 (11.96) n = 37 0.255 Equal or less than 314.5 35.14 (10.14) n = 35 35.71 (9.82) n = 52 NOS1 , rs478597 CC or TC TT More than 314.5 mg 33.62 (11.07) n=60 43.14 (10.59) n=7 0.069 Equal or less than 314.5 35.81 (10.12) n=77 31.55 (8.96) n=11 ADORA2A, rs5751876 TT or TC CC More than 314.5 mg 35.37 (10.96) n=51 38.23 (12.34) n=17 0.270 Equal or less than 314.5 34.02 (10.56) n=55 38.23 (7.04) n=22 Genotypes ADORA2A, rs2298383 CC CT TT More than 314.5 mg 28.42 (11.80) n = 12 28.42 (11.80) n = 12 37.26 (10.76) n = 27 0.560 Equal or less than 314.5 39.10 (8.26) n = 20 39.10 (8.26) n = 20 33.74 (9.52) n = 23 *ANOVA 4. Discussion Caffeine consumption did not have any modifying effect on the onset of symptoms of patients with SCA3/MJD. There was no direct effect, nor any effect mediated by functional variants of ADORA2A , CYP1A2 and NOS1 , genes that act in caffeine signaling or metabolism. Caffeine has long been related to protective effects against neurodegenerative disease, in special PD [31, 32, 33], a common multifactorial neurodegenerative disease that shares with CAGexp diseases the start in adulthood. There is increasing evidence relating genes, caffeine consumption, and their interaction with the onset and progression of PD [25]. Studies on PD are large, since PD affects 1–2% of individuals with more than 60 years of age [34], and since these studies seek causality. In contrast, causality is well established for CAGexp diseases, whereas studies on interactions between CAGexp, modifier genes and environmental factors, and/or habits are still very preliminary. For instance, lifestyle characteristics of SCA3/MJD European subjects have been the focus of a recent report, but caffeine consumption was not studied there [35]. Caffeinated drinks, such as coffee and tea, contain a mixture of several substances, among which caffeine is assumed to be the protagonist. Chronic caffeine consumption would be neuroprotective through the antagonism of A2ARs [13, 36]. A2ARs have a key role in controlling synaptic viability, apoptotic neuronal death, astrocytic function, and neuroinflammation [37]. In HD models, A2ARs blockade produced a normalization of striatal glutamatergic transmission. In a SCA3/MJD mouse model, not only synaptotoxicity and gliosis in the striatum were found to be early events predating neurodegeneration, but caffeine administration and A2AR blockade delayed striatal pathology, leading the authors to suggest that caffeine consumption was perhaps a "lifestyle prophylactic strategy to delay the onset" of this disease [13]. These experimental studies provided proof of concept for launching an epidemiologic observation like ours. However, our study showed that caffeine consumption did not delay the onset of SCA3/MJD subjects. We tried to check if the caffeine effect would depend on the interaction with other genetic characteristics. As SCA3/MJD is a quite rare disorder and the information about caffeine consumption should only be obtained by a direct survey, sample size was expected to be limited. In this scenario, the best choice to model this investigation was that of a candidate gene approach - and we chose to study variants at ADORA2A, CYP1A2 and NOS1 , and their interaction with caffeine consumption. ADORA2A , located at Chromosome 22 (24,417,879–24,442,357), was a strong candidate as it encodes a G-protein coupled adenosine receptor. This receptor is known to participate in neurogenesis, neuroinflammation, and synaptic plasticity within glutamatergic synapse [38]. According to a systematic review, ADORA2A might indeed modulate the association between caffeine and brain-related outcomes [39]. rs5751876 has a high sensitivity to caffeine and has been the focus of several observations [16, 40, 41]. A study on this SNP and caffeine intake in PD patients gave negative results [42]. rs2298383 has been studied in the context of sleep [43], anxiety [44], epilepsy [45] and levodopa induced dyskinesias [46, 47]. Two other ADORA2A polymorphisms were inversely associated with PD risk, without a clear-cut interaction with coffee consumption [48]. We did not find any interaction between rs5751876 nor rs2298383 variants, caffeine consumption, and AOfs in SCA3/MJD (Table 3 ). CYP1A2 at chromosome 15 (74,748,845–74,756,607) was another strong candidate, as it encodes the cytochrome P450 CYP1A2 enzyme that is responsible for metabolizing over 90% of caffeine into paraxanthine [49]. The SNP rs762551 is in the noncoding region of the CYP1A2 gene and likely impacts on the levels of expression of the CYP1A2 enzyme by affecting the binding of regulatory proteins to the surrounding gene sequences [15]. More than 40 SNPs have been found in CYP1A2 , and one of the most extensively studied is rs762551 (163 C > A) [50]. Individuals with the rs762551 A > C SNP have lower CYP1A2 inducibility, rendering slower caffeine metabolism. The hypothesis to be tested was the effect of caffeine would differ between individuals with the AA genotype (classified as “fast metabolizers'') and those with CC and AC individuals, characterized as “slow metabolizers'' [15, 50]. However, no effect was seen in the present cohort: subjects carrying genotypes CC and AC at rs762551 that were high caffeine consumers had the same AOfs as low consumers (Table 3 ). The absence of interacting effects of this variant with caffeine has been recently reported for the risk of PD, too [51]. Nitric oxide synthase (NOS) genes ( NOS1, NOS2A , and NOS3 ) account for most of the production of nitric oxide (NO) in the nervous system required for synaptic transmission and neuroplasticity. Excess of NO may contribute to neurodegeneration in Parkinson's disease (PD) [18]. A recent review concluded that NOS signaling has significant involvement in several disease pathologies, including protection against PD [19]. NOS effects were related to caffeine consumption in a preclinical study [20], while caffeine suppresses the generation of NO in microglia [21]. rs478597 at NOS1 has been related to Attention-Deficit/Hyperactivity Disorder (ADHD) [52, 53]. In a Brazilian PD cohort study, CC rs478597 carriers who were high consumers of caffeine presented lower PD risk than those who were low consumers [22]. Our results on rs478597 and caffeine consumption discarded any effect over SCA3/MJD AOfs (Table 3 ). If caffeine consumption per se was not related to modification of the onset of symptoms in SCA3/MJD, was it futile to study the effect of candidate genes? Why did we choose to test these associations? First, caffeine exerted neuroprotection in models of SCA3/MJD. Second, SCA3/MJD have some similarities with PD, a disease where caffeine is related to neuroprotection. Third, SCA3/MJD has even more similarities with HD, a CAGexp disease where earlier AOfs were seen among consumers than among non-consumers of caffeine. We did not know the exact direction of the caffeine effect, if any would exist over SCA3/MJD. AOfs in SCA3/MJD carriers living in our region were expected to be lower, according to CAGexp lengths observed in the RS cohort [3]. This intriguing finding points to the presence of some local effect that is neuroprotective to the onset of symptoms. Among the cultural characteristics of our region is the intake of chimarrão , a drink very rich in caffeine. Then we speculated whether the intake of chimarrão could be neuroprotective to SCA3/MJD. Our negative results also propose an additional reflection. SCA3/MJD and other polyQ disorders might have some similarities with PD, as said before, but of course imbalances between neurotransmitters might not be as important in CAGexp diseases as they are in PD. In PD, the neurotransmitter imbalance is a direct consequence of the disease, is paramount for PD signs and symptoms, and is the phenomenon to be corrected for the available treatments. Caffeine can act as a neurotransmitter, and its neuroprotection can reach intracellular processes mediated by neuroreceptors, structures very involved in the chain of events between cause and manifestations in PD. This poses questions about differences in all steps between etiology, cellular biology, neuronal webs, topography and clinical phenomenology of CAGexp disorders and PD. Differences in these steps may explain the lack of association and even divergent effects across these diseases. Finally, we need to stress that our study had limitations - although the sample size was apparently not among them, as the p-values obtained quite convincingly discarded the hypotheses tested. In contrast, the design of studies on consumption habits seems to be the main limitation: it is unsatisfactory, at least in the context of progressive and long-lasting diseases. Habits are defined through questionnaires about consumption in the last few days. This information is then generalized to the person's entire life cycle. There is no way to test cumulative effects of this habit over AO, since we would confuse causes with consequences. After all, those with later AO would have had a greater chance of consuming more coffee before the AO, and we could not conclude anything about this cumulative consumption. Our questionnaire asked about the beginning of the caffeine habit, but not about changes in this habit over the years. Therefore, it was not possible to define whether the habit changed after the onset of symptoms. This information would be interesting as caffeine - a central nervous system (CNS) stimulant - may theoretically alleviate some symptoms. However, SCA3/MJD patients did not consume more caffeine than controls, which might rule out for now the hypothesis that caffeine is consumed by our patients because it has any symptom-mitigating effect. Finally, the choice of molecular variants under study is always a concern. We may not have chosen the best SNPs in our candidate genes. It is always important to emphasize that results on variants should be interpreted with caution, as they might not be generalizable to the genes in question. 5. Conclusion Caffeine consumption and the interaction with variants of genes related to caffeine receptors and caffeine degradation had no effect over the onset of symptoms of SCA3/MJD. The role of other habits and environmental factors should continue to be pursued in this disease. Declarations a. Acknowledgements We are grateful to all study participants. This study was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil, grant number 405963/2021-1; and by Financiamento e Incentivo à Pesquisa, Hospital de Clínicas de Porto Alegre (FIPE HCPA) grants numbers 2018-0660 and 2018-0661. ACM, JSP, LS, ERC, DS, LDP, BAA, MLSP and LBJ were supported by CNPq. b. Conflicts of Interest The authors have no competing interests to disclose. c. Availability of data and material (data transparency) The data that support the findings of this study are available from the corresponding author upon reasonable request. d. 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Ann Neurol 95:677-687 Simonin C, Duru C, Salleron J, Hincker P, Charles P, Delval A et al . (2013) Association between caffeine intake and age at onset in Huntington's disease. Neurobiol Dis. 58:179-82. Wang M, Cornelis MC, Zhang Z, Liu D, Lian X (2021) Mendelian randomization study of coffee consumption and age at onset of Huntington's disease. Clin Nutr. 40:5615-5618. Palacios N, Gao X, McCullough ML, Schwarzschild MA, Shah R, Gapstur S, Ascherio A (2012) Caffeine and risk of Parkinson's disease in a large cohort of men and women. Mov Disord. 27:1276-82. Bastos, Deborah & Fornari, Ana & Queiroz, Yara & Soares-Freitas, Rosana & Torres, Elizabeth. (2005). The Chlorogenic Acid and Caffeine Content of Yerba Maté (Ilex paraguariensis) Beverages. Acta Farmaceutica Bonaerense. 24: 91-95. IBM Corp. Released 2011. IBM SPSS Statistics for Windows, Version 20.0. Armonk, NY: IBM Corp. Saaksjarvi K, Knekt P, Rissanen H, et al. (2008) Prospective study of coffee consumption and risk of Parkinson’s disease. Eur J Clin Nutr 62:908–915. Bakshi R, Macklin EA, Hung AY, et al (2020). Associations of lower caffeine intake and plasma urate levels with idiopathic Parkinson’s disease in the Harvard biomarkers study. J Parkinsons Dis 10:505–510. Gabbert, C., König, I.R., Lüth, T. et al. (2022) Coffee, smoking and aspirin are associated with age at onset in idiopathic Parkinson’s disease. J Neurol 269: 4195–4203 Dorsey ER, Elbaz A, Nichols E, et al. (2018). Global, regional, and national burden of Parkinson’s disease, 1990–2016: a systematic analysis for the global burden of disease study Lancet Neurol 17: 939–953. Hengel H, Martus P, Faber J, Garcia-Moreno H, Solanky N, Giunti P, et al (2022) Characterization of Lifestyle in Spinocerebellar Ataxia Type 3 and Association with Disease Severity. Mov Disord. 37: 405-410. Cunha RA, Agostinho PM. (2010) Chronic caffeine consumption prevents memory disturbance in different animal models of memory decline. J Alzheimers Dis 20(suppl 1): S95–S116. Gomes CV, Kaster MP, Tome AR, et al. (2011) Adenosine receptors and brain diseases: neuroprotection and neurodegeneration. Biochim Biophys Acta 1808:1380–1399. Cunha RA. (2016) How does adenosine control neuronal dysfunction and neurodegeneration? J Neurochem. 139:1019-1055. Kapellou A, King A, Graham CAM, Pilic L, Mavrommatis Y. (2023) Genetics of caffeine and brain-related outcomes - a systematic review of observational studies and randomized trials. Nutr Rev 81:1571-1598. Grgic J, Pickering C, Bishop DJ, Del Coso J, Schoenfeld BJ, Tinsley GM, Pedisic Z (2020). ADORA2A C Allele Carriers Exhibit Ergogenic Responses to Caffeine Supplementation. Nutrients 12:741. Rahimi MR, Semenova EA, Larin AK, Kulemin NA, Generozov EV, Łubkowska B, Ahmetov II, Golpasandi H. (2023) The ADORA2A TT Genotype Is Associated with Anti-Inflammatory Effects of Caffeine in Response to Resistance Exercise and Habitual Coffee Intake. Nutrients. 15:1634. Facheris MF, Schneider NK, Lesnick TG, de Andrade M, Cunningham JM, Rocca WA, Maraganore DM. (2008) Coffee, caffeine-related genes, and Parkinson's disease: a case-control study. Mov Disord. 23:2033-40. Erblang M, Drogou C, Gomez-Merino D, Metlaine A, Boland A, Deleuze JF, Thomas C, Sauvet F, Chennaoui M. (2019) The Impact of Genetic Variations in ADORA2A in the Association between Caffeine Consumption and Sleep. Genes (Basel) 10:1021. Childs E, Hohoff C, Deckert J, Xu K, Badner J, de Wit H. (2008) Association between ADORA2A and DRD2 polymorphisms and caffeine-induced anxiety. Neuropsychopharmacology. 33: 2791-800. Fan X, Chen Y, Li W, Xia H, Liu B, Guo H, Yang Y, Xu C, Xie S, Xu X. (2020) Genetic Polymorphism of ADORA2A Is Associated with the Risk of Epilepsy and Predisposition to Neurologic Comorbidity in Chinese Southern Children. Front Neurosci. 14: 590605. Rieck M, Schumacher-Schuh AF, Callegari-Jacques SM, Altmann V, Schneider Medeiros M, Rieder CR, Hutz MH. (2015) Is there a role for ADORA2A polymorphisms in levodopa-induced dyskinesia in Parkinson's disease patients? Pharmacogenomics. 16:573-82. Santos-Lobato BL, Bortolanza M, Pinheiro LC, Batalhão ME, Pimentel ÂV, Capellari-Carnio E, Del-Bel EA, Tumas V. (2022) Levodopa-induced dyskinesias in Parkinson's disease increase cerebrospinal fluid nitric oxide metabolites' levels. J Neural Transm (Vienna) 129: 55-63. Popat RA, Van Den Eeden SK, Tanner CM, Kamel F, Umbach DM, Marder K, Mayeux R, Ritz B, Ross GW, Petrovitch H, Topol B, McGuire V, Costello S, Manthripragada AD, Southwick A, Myers RM, Nelson LM. (2011) Coffee, ADORA2A, and CYP1A2: the caffeine connection in Parkinson's disease. Eur J Neurol. 18: 756-65. Schwarzschild MA, Xu K, Oztas E, Petzer JP, Castagnoli K, Castagnoli N Jr, Chen JF. (2003) Neuroprotection by caffeine and more specific A2A receptor antagonists in animal models of Parkinson's disease. Neurology. 61(Suppl 6): S55-61. Nikrandt G, Mikolajczyk-Stecyna J, Młodzik-Czyżewska M, Chmurzynska A. (2022) Functional single-nucleotide polymorphism (rs762551) in CYP1A2 gene affects white coffee intake in healthy 20- to 40-year-old adults. Nutr Res. 105:77-81. Kim IY, O'Reilly ÉJ, Hughes KC, Gao X, Schwarzschild MA, McCullough ML, Hannan MT, Betensky RA, Ascherio A. (2018) Interaction between caffeine and polymorphisms of glutamate ionotropic receptor NMDA type subunit 2A (GRIN2A) and cytochrome P450 1A2 (CYP1A2) on Parkinson's disease risk. Mov Disord. 33:414-420 Salatino-Oliveira A, Wagner F, Akutagava-Martins GC, Bruxel EM, Genro JP, Zeni C, Kieling C, Polanczyk GV, Rohde LA, Hutz MH. (2016) MAP1B and NOS1 genes are associated with working memory in youths with attention-deficit/hyperactivity disorder. Eur Arch Psychiatry Clin Neurosci. 266: 359-66 Salatino-Oliveira A, Akutagava-Martins GC, Bruxel EM, Genro JP, Polanczyk GV, Zeni C, Kieling C, Karam RG, Rovaris DL, Contini V, Cupertino RB, Mota NR, Grevet EH, Bau CH, Rohde LA, Hutz MH. (2016) NOS1 and SNAP25 polymorphisms are associated with Attention-Deficit/Hyperactivity Disorder symptoms in adults but not in children. J Psychiatr Res. 75: 75-81. Tables Tables 4 to 6 are not available with this version. Table 4. The ADORA2A polymorphism at rs2298383 and caffeine habits versus age at onset of SCA3/MJD Table 5. The CYP1A2 polymorphism at rs762551 and caffeine habits versus age at onset of SCA3/MJD Table 6. The NOS1 polymorphism at rs478597 and caffeine habits versus age at onset of SCA3/MJD Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4325198","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":296048249,"identity":"3cc3148b-f58e-4f9b-b60d-79c041851184","order_by":0,"name":"Ana Carolina Martins","email":"","orcid":"","institution":"Universidade Federal do Rio Grande do Sul","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"Carolina","lastName":"Martins","suffix":""},{"id":296048252,"identity":"bf53b92f-eb66-4c6f-a74a-080282e3ff87","order_by":1,"name":"Jordânia dos Santos Pinheiro","email":"","orcid":"","institution":"Universidade Federal do Rio Grande do Sul","correspondingAuthor":false,"prefix":"","firstName":"Jordânia","middleName":"dos Santos","lastName":"Pinheiro","suffix":""},{"id":296048254,"identity":"66fbaf17-5992-4156-a922-75c2d7e550d5","order_by":2,"name":"Luciana Szinwelski","email":"","orcid":"","institution":"Universidade Federal do Rio Grande do Sul","correspondingAuthor":false,"prefix":"","firstName":"Luciana","middleName":"","lastName":"Szinwelski","suffix":""},{"id":296048255,"identity":"4221e7fa-d9a5-48ee-ba8f-a24b80a55f04","order_by":3,"name":"Eduardo Rockenbach Cidade","email":"","orcid":"","institution":"Universidade Federal do Rio Grande do Sul","correspondingAuthor":false,"prefix":"","firstName":"Eduardo","middleName":"Rockenbach","lastName":"Cidade","suffix":""},{"id":296048256,"identity":"dbf67524-6655-4551-bdac-4d5987134e41","order_by":4,"name":"Danilo Fernando Santin","email":"","orcid":"","institution":"Universidade Federal do Rio Grande do Sul","correspondingAuthor":false,"prefix":"","firstName":"Danilo","middleName":"Fernando","lastName":"Santin","suffix":""},{"id":296048257,"identity":"43132f4e-68cd-4820-a823-cffd2234e1ec","order_by":5,"name":"Laura Damke Proença","email":"","orcid":"","institution":"Universidade Federal do Rio Grande do Sul","correspondingAuthor":false,"prefix":"","firstName":"Laura","middleName":"Damke","lastName":"Proença","suffix":""},{"id":296048258,"identity":"960e40a6-44fc-4782-bb85-57b968c1f59f","order_by":6,"name":"Bruna Almeida Araújo","email":"","orcid":"","institution":"Universidade Federal do Rio Grande do Sul","correspondingAuthor":false,"prefix":"","firstName":"Bruna","middleName":"Almeida","lastName":"Araújo","suffix":""},{"id":296048259,"identity":"04b2a2c0-c958-4cf6-a053-88d3310517d7","order_by":7,"name":"Maria Luiza Saraiva-Pereira","email":"","orcid":"","institution":"Universidade Federal do Rio Grande do Sul","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"Luiza","lastName":"Saraiva-Pereira","suffix":""},{"id":296048261,"identity":"4a5564e4-8f08-49da-b6f9-ce822cbad8c0","order_by":8,"name":"Laura Bannach Jardim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYBACPoYDDAw8BgxyBhA+MwODBAEtbFAtxqRoAQIeBobEDcRrYTz87MObgm3p29nPHvzAUGGd2CDd+4CALceMZ84xuJ27sycvWYLhTHpig8xxA0J+MWbmAWrZcIPHQIKx7XBig0QaIb8c/wzSkm5wg8f4B+M/orScAduSANRiJsHYQJyWYkagXww3nMlLs0g4lm7cJnMMvxZ+ieObGd78uS1vcPzs4Rsfaqxl+6Xb8GthkDgAYwFjJ4EBGlP4rWlA0jIKRsEoGAWjABsAAAsaRFchJoAgAAAAAElFTkSuQmCC","orcid":"","institution":"Universidade Federal do Rio Grande do Sul","correspondingAuthor":true,"prefix":"","firstName":"Laura","middleName":"Bannach","lastName":"Jardim","suffix":""}],"badges":[],"createdAt":"2024-04-25 15:46:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4325198/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4325198/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55756614,"identity":"1af0d198-d8ae-4e70-82b4-dfc258a6a33a","added_by":"auto","created_at":"2024-05-02 17:18:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":729386,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4325198/v1/282d1486-85a9-4820-8322-9d0bd26debd0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Caffeine consumption and interaction with ADORA2A, CYP1A2 and NOS1 variants do not influence age at onset of Machado-Joseph disease","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMachado Joseph disease, also known as spinocerebellar ataxia type 3 (SCA3/MJD), is caused by a CAG repeat expansion (CAGexp) at \u003cem\u003eATXN3\u003c/em\u003e gene [1]. Symptoms begin gradually in adult life, encompassing incoordination of gait, limb movements, speech, eye movements and swallowing, combined with pyramidal, extrapyramidal, lower motor neuron, and sensory findings. The mean (SD; range) age at onset (AO) in our region, of 34.3 (11.2; 5\u0026ndash;59) years of age, is comparable to data obtained worldwide [2, 3]. The CAGexp length is the most important determinant of the AO, explaining 55% of its variability [3]. Knowledge about factors that determine the other 45% is quite limited and comprise some modifying genes. \u003cem\u003eATXN2\u003c/em\u003e [3], \u003cem\u003eCFAP57\u003c/em\u003e, \u003cem\u003eACTG1, DIDO1\u003c/em\u003e [4, 5], \u003cem\u003eCAST\u003c/em\u003e [6], and \u003cem\u003eFAN1\u003c/em\u003e [7] were related to mild effects over AO by association studies not replicated in more than one cohort, with the exception of \u003cem\u003eATXN2\u003c/em\u003e. No environmental factors have been identified to date as influencing SCA3/MJD phenotype.\u003c/p\u003e \u003cp\u003eCaffeine is one of the most popular psychoactive substances in use nowadays, in products such as coffee, tea, chocolate and cola soft drinks [ 8]. Data from the International Coffee Organization indicate that Brazil is the largest grain producer and the second largest consumer of the drink in the world, behind only the USA [ 9].\u003c/p\u003e \u003cp\u003eCaffeine has complex mechanisms of action over the central nervous system, including a non-selective adenosine A2AR receptor - encoded by \u003cem\u003eADORA2A\u003c/em\u003e [10] - antagonism, mobilization of intercellular calcium, and phosphodiesterase inhibition [8, 11]. Enhancing effects may occur by reducing caffeine metabolism, increasing absorption, or direct agonism.\u003c/p\u003e \u003cp\u003ePreclinical studies done in SCA3/MJD transgenic mice linked caffeine and/or A2AR blockade to alleviating neurodegeneration [12, 13]. Similar results were obtained in some preclinical studies on Huntington's disease (HD), another CAGexp disease that might share pathogenetic pathways with SCA3/MJD [14].\u003c/p\u003e \u003cp\u003e \u003cem\u003eCYP1A2\u003c/em\u003e codes for cytochrome P450 1A2, the enzyme responsible for 95% of the metabolism of caffeine in the liver [15]. Caffeine metabolism by the CYP1A2 enzyme shows substantial variation between people, due to genetic and environmental factors [16]. Moreover, some \u003cem\u003eCYP1A2\u003c/em\u003e variants were already associated with enhancing the neuroprotective effect of caffeine [17].\u003c/p\u003e \u003cp\u003eNitric oxide (NO) performs vital physiological functions in the nervous system under normal concentrations [18]. The neuronal isoform (nNOS) signaling has significant involvement in several neurodegenerative diseases, such as Parkinson's disease (PD), Alzheimer\u0026rsquo;s disease and HD [19]. Caffeine reduces the expression of \u003cem\u003eNOS\u003c/em\u003e, including nNOS [20, 21]. A relationship between \u003cem\u003eNOS1\u003c/em\u003e and protection against PD in individuals who consumed high caffeine was proposed [22].\u003c/p\u003e \u003cp\u003eEpidemiological studies on neurodegenerative disorders have shown contradictory results related to caffeine. Previous meta-analyses and a recent case-control study on more than 18,000 subjects have shown neuroprotective results on PD, in line with preclinical studies [23, 24, 25]. In contrast, caffeine consumption was associated with earlier ages of onset in HD [26, 27].\u003c/p\u003e \u003cp\u003eSince epidemiological studies on potential associations between caffeine consumption and changes in the age of onset of SCA3/MJD are lacking, the present observation aimed to detect a relationship between caffeine consumption and changes in the age of onset of SCA3/MJD; and to examine whether the caffeine effect was mediated by variants at \u003cem\u003eADORA2A, CYP1A2\u003c/em\u003e and \u003cem\u003eNOS1\u003c/em\u003e already proposed to enhance the effect of caffeine on the central nervous system.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1 Design\u003c/h2\u003e\n \u003cp\u003eA case-control study was done to determine whether the caffeine consumption among SCA3/MJD patients was different from that of the local, control population.\u003c/p\u003e\n \u003cp\u003eNext, a cross-sectional observation was performed in SCA3/MJD carriers to look for associations between the amount of habitual caffeine consumption and their AO. Finally, AO was compared between SCA3/MJD subjects stratified according to their caffeine consumption and to their genotypes in \u003cem\u003eADORA2A, CYP1A2\u003c/em\u003e and \u003cem\u003eNOS1.\u003c/em\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2 Population and clinical procedures\u003c/h2\u003e\n \u003cp\u003eSymptomatic subjects with molecular diagnosis were recruited from to the Rio Grande do Sul SCA3/MJD cohort [2], if older than 18 years of age, at the outpatient\u0026rsquo;s clinic of our institution. Unrelated caregivers who were accompanying patients from the same outpatient clinic were invited to participate as controls.\u003c/p\u003e\n \u003cp\u003eA standardized questionnaire (\u003cstrong\u003eSupplemental Materials 1\u003c/strong\u003e and \u003cstrong\u003e2\u003c/strong\u003e) was administered to subjects who agreed to participate, after obtaining their informed consent forms. Questions to estimate caffeine consumption covered the habit of drinking coffee, teas, soft drinks and \u003cem\u003echimarr\u0026atilde;o\u003c/em\u003e (or \u003cem\u003eyerba mate\u003c/em\u003e, in Spanish speaking populations), a traditional hot beverage consumed in southern Brazil and neighboring countries which is prepared from the leaves of \u003cem\u003eIlex paraguariensis\u003c/em\u003e. Information about habits of smoking were also obtained from all participants, in addition to age, residence, education and professional activities. Clinical and molecular data from the SCA3/MJD carriers such as AO and CAGexp were already available from their electronic files; AO of the first symptom (AOfs) was obtained again by the questionnaire. AOfs was defined by the age at which the subject and/or their family members first identified a motor problem, such as difficulty in walking or speaking, or double vision.\u003c/p\u003e\n \u003cp\u003eThis study was approved by the Institutional Ethics Committee (Comiss\u0026atilde;o de \u0026Eacute;tica em Pesquisa do Hospital de Cl\u0026iacute;nicas de Porto Alegre) by the number CAAE 95633218.3.0000.5327. Written informed consent was obtained from all study participants.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3 Genotyping\u003c/h2\u003e\n \u003cp\u003eDNA samples were retrieved from the biorepositories of our institution. The CAGexp length was already determined and registered in protected files. Genes and their single nucleotide polymorphisms (SNP) chosen to be genotyped were described in \u003cstrong\u003eTable\u0026nbsp;1\u003c/strong\u003e. rs5751876 (\u003cem\u003eADORA2A\u003c/em\u003e), rs2298383 (\u003cem\u003eADORA2A\u003c/em\u003e), rs762551 (\u003cem\u003eCYP1A2\u003c/em\u003e) and rs478597 (\u003cem\u003eNOS1\u003c/em\u003e) were genotyped using TaqMan SNP Genotyping Assay in a final volume of 8 \u0026micro;L containing 2 ng of DNA, according to assay protocol (Applied Biosystems, Foster City, CA, USA). Amplification was performed in the ABI 7500 Fast Real-Time PCR System\u0026reg; equipment (Applied Biosystems, Foster City, CA, USA) as follows: one cycle of 50\u0026deg;C for 2 min, 95\u0026deg;C for 10 min, followed by 40 cycles of 95\u0026deg;C for 15 s and 60\u0026deg;C for 1 min.\u003c/p\u003e\n \u003cp\u003eTable 1. Single nucleotide polymorphisms chosen as candidate markers for caffeine potentialization effects\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"1058\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.421550094517958%\"\u003e\n \u003cp\u003e\u003cstrong\u003eGenes under study\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.37429111531191%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMechanism\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.979206049149338%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSNPs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.909262759924385%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMajor allele/\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eMinor allele\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.601134215500945%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eMAF**\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.714555765595463%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePreliminary evidence on neuroprotective effects\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.421550094517958%\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eADORA2A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.37429111531191%\" rowspan=\"2\"\u003e\n \u003cp\u003eEncodes adenosine A2A receptors, which are the main target of caffeine action in the nervous system.\u0026nbsp;This receptor is known to participate in neurogenesis, neuroinflammation, and synaptic plasticity within glutamatergic synapse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.979206049149338%\" valign=\"top\"\u003e\n \u003cp\u003ers5751876\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.909262759924385%\" valign=\"top\"\u003e\n \u003cp\u003eT*/ C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.601134215500945%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.714555765595463%\" valign=\"top\"\u003e\n \u003cp\u003eFacheris \u003cem\u003eet al.,\u0026nbsp;\u003c/em\u003e2008;\u0026nbsp;Loy et al 2015; Gggic et al 2020; Rahimi et al 2023; Virgili et al 2023\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.627450980392158%\" valign=\"top\"\u003e\n \u003cp\u003ers2298383 \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.705882352941178%\" valign=\"top\"\u003e\n \u003cp\u003eT/ C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.84313725490196%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.8235294117647%\" valign=\"top\"\u003e\n \u003cp\u003eChilds et al 2008; Rieck et al 2015; \u0026nbsp;Turčin et al., 2016; Santos-Lobato et al 2019; Fan et al 2020; Erblang et al 2021\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.421550094517958%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eCYP1A2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.37429111531191%\" valign=\"top\"\u003e\n \u003cp\u003eEncodes a member of the cytochrome P450, metabolizer of polycyclic aromatic\u0026nbsp;hydrocarbons present in coffee\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.979206049149338%\" valign=\"top\"\u003e\n \u003cp\u003ers762551\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.909262759924385%\" valign=\"top\"\u003e\n \u003cp\u003eC*/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.601134215500945%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.714555765595463%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;Amin \u003cem\u003eet al\u003c/em\u003e\u003cem\u003e.,\u0026nbsp;\u003c/em\u003e2012; Tennent et al 2020\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.421550094517958%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eNOS1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.37429111531191%\" valign=\"top\"\u003e\n \u003cp\u003eEncodes a protein that belongs to the family of nitric oxide synthases. Nitric oxide is a reactive free radical, which acts as a biologic mediator in several processes, including neurotransmission. Preliminary evidence suggested a relationship between nNOS and protection against PD in individuals who consumed high caffeine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.979206049149338%\" valign=\"top\"\u003e\n \u003cp\u003ers478597\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.909262759924385%\" valign=\"top\"\u003e\n \u003cp\u003eC*/T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.601134215500945%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.714555765595463%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;Altmann, 2018; Poon \u003cem\u003eet al.,\u0026nbsp;\u003c/em\u003e2021\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e* Allele proposed to\u0026nbsp;be\u0026nbsp;neuroprotective through caffeine potentialization\u003c/p\u003e\n \u003cp\u003e** From 1000Genome\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4 Analyses\u003c/h2\u003e\n \u003cp\u003eFirst, a potential relationship between caffeine and SCA3/MJD was studied by comparing the consumptions of carriers with those of controls. After that, only SCA3/MJD carriers were studied, when the main outcome was their AOfs.\u003c/p\u003e\n \u003cp\u003eSubjects were considered caffeine consumers if they drank two or more cups of coffee per week, for at least 3 months in their lifetime [22]. The amount of caffeine ingested per day was calculated by the sum of the daily beverages reported by subjects, as follows: 137mg and 47 mg of caffeine per cup (150ml) of coffee and tea, respectively [28]; and 135 mg of caffeine per 500ml of \u003cem\u003echimarr\u0026atilde;o\u003c/em\u003e [29]. Most participants were unsure about reporting their soft drink consumption - for example, the quantity and type of soft drinks consumed in recent weeks - as this would be an expensive and erratic habit, limited to special situations such as parties. Because of this, the consumption of soft drinks was not included in the analysis.\u003c/p\u003e\n \u003cp\u003eSince the great majority of participants were caffeine consumers, and comparisons between consumers and non-consumers were not possible, subjects were dichotomized into high vs. low consumers according to the median (SD) daily consumption of mg of caffeine.\u003c/p\u003e\n \u003cp\u003eAge, sex, schooling, and tobacco consumption were controlled in all subjects included in this study. Subjects were considered nicotine users if they have smoked at least 100 cigarettes in their lifetime. Since tobacco was consumed exclusively through industrial cigarettes, in the present cohort, the usual number of cigarettes smoked were taken (\u003cstrong\u003eSupplemental Materials 1\u003c/strong\u003e and \u003cstrong\u003e2\u003c/strong\u003e). Finally, the CAGexp was controlled for SCA3/MJD carriers.\u003c/p\u003e\n \u003cp\u003eChi-square, t-test and Mann-Whitney test were used to compare case and control groups, according to the variables under analysis and to their distribution. ANOVA was used to compare the AO between subgroups, adjusting the CAGexp to the median value observed in the group of 75 repeats. Tests with p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 significance level were accepted. All tests were performed with SPSS version 20 software (SPSS Inc., Chicago, IL, USA) [30].\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003eOne hundred, seventy-nine symptomatic SCA3/MJD subjects and 100 controls were included. More than 95% of cases and controls consumed caffeine beverages. Cases and controls had similar median caffeine consumptions per day (\u003cstrong\u003eTable 2\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. General characteristics of the subjects under study\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"870\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"34.367816091954026%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.71264367816092%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSCA3/MJD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.54022988505747%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eControls\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.379310344827585%\" valign=\"top\"\u003e\n \u003cp\u003ep\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"34.367816091954026%\" valign=\"top\"\u003e\n \u003cp\u003eWomen/all subjects (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.71264367816092%\" valign=\"top\"\u003e\n \u003cp\u003e100/179 (55.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.54022988505747%\" valign=\"top\"\u003e\n \u003cp\u003e53/100 (53.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.379310344827585%\" valign=\"top\"\u003e\n \u003cp\u003e0.707*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"34.367816091954026%\" valign=\"top\"\u003e\n \u003cp\u003eCaffeine consumers (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.71264367816092%\" valign=\"top\"\u003e\n \u003cp\u003e171 (96%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.54022988505747%\" valign=\"top\"\u003e\n \u003cp\u003e98 (98%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.379310344827585%\" valign=\"top\"\u003e\n \u003cp\u003e0.801*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"34.367816091954026%\" valign=\"top\"\u003e\n \u003cp\u003eTobacco consumers\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.71264367816092%\" valign=\"top\"\u003e\n \u003cp\u003e64 (35.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.54022988505747%\" valign=\"top\"\u003e\n \u003cp\u003e34 (34%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.379310344827585%\" valign=\"top\"\u003e\n \u003cp\u003e0.795*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"34.367816091954026%\" valign=\"top\"\u003e\n \u003cp\u003eAge at onset of symptoms\u003c/p\u003e\n \u003cp\u003emean (SD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.71264367816092%\" valign=\"top\"\u003e\n \u003cp\u003e35.26 (10.62)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.54022988505747%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.379310344827585%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"34.367816091954026%\" valign=\"top\"\u003e\n \u003cp\u003eCAGexp length\u003c/p\u003e\n \u003cp\u003emedian (range)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.71264367816092%\" valign=\"top\"\u003e\n \u003cp\u003e75 (65-87)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.54022988505747%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.379310344827585%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"34.367816091954026%\" valign=\"top\"\u003e\n \u003cp\u003eAge at interview\u0026nbsp;\u003c/p\u003e\n \u003cp\u003emean (SD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.71264367816092%\" valign=\"top\"\u003e\n \u003cp\u003e45.65 (11.64)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.54022988505747%\" valign=\"top\"\u003e\n \u003cp\u003e44.94 (14.14)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.379310344827585%\" valign=\"top\"\u003e\n \u003cp\u003e0.671**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"34.367816091954026%\" valign=\"top\"\u003e\n \u003cp\u003eYears of schooling\u003c/p\u003e\n \u003cp\u003emean (SD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.71264367816092%\" valign=\"top\"\u003e\n \u003cp\u003e11.10 (3.80)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.54022988505747%\" valign=\"top\"\u003e\n \u003cp\u003e11.16 (4.22)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.379310344827585%\" valign=\"top\"\u003e\n \u003cp\u003e0.896**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"34.367816091954026%\" valign=\"top\"\u003e\n \u003cp\u003emg of caffeine consumption/day\u003c/p\u003e\n \u003cp\u003emean (SD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.71264367816092%\" valign=\"top\"\u003e\n \u003cp\u003e331.85 (215.65)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.54022988505747%\" valign=\"top\"\u003e\n \u003cp\u003e352.12 (246.37)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.379310344827585%\" valign=\"top\"\u003e\n \u003cp\u003e0.698***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*Chi-square **t-test ***Mann-Whitney test\u003c/p\u003e\n\u003cp\u003ePatients that consumed more or less than the median consumption of 314.5 mg of caffeine/day had a mean (SD) AOfs of 35.05 (11.44) and 35.43 (10.08) years (p=0.40), for a CAGexp of 75 repeats. Covariates such as sex, tobacco consumption habits and schooling did not add any significant effect over their AOfs (data not shown).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe distribution of allele frequencies for each SNP studied was displayed in \u003cstrong\u003eTable S1\u003c/strong\u003e, \u003cstrong\u003eSupplemental Material 3\u003c/strong\u003e. The genotypic subgroups of \u003cem\u003eADORA2A\u003c/em\u003e (rs5751876 and rs2298383), \u003cem\u003eCYP1A2\u003c/em\u003e, and \u003cem\u003eNOS1\u003c/em\u003e, were then studied: no association was found between these genotypes and direct differences in AOfs (\u003cstrong\u003eTable S2\u003c/strong\u003e, \u003cstrong\u003eSupplemental material 3\u003c/strong\u003e). Then the effect of caffeine consumption was combined to the presence/absence of caffeine-enhancing alleles at \u003cem\u003eADORA2A\u003c/em\u003e (rs5751876, \u003cem\u003eCYP1A2\u003c/em\u003e, and \u003cem\u003eNOS1.\u0026nbsp;\u003c/em\u003eSince there was no clear caffeine-enhancing allele at \u003cem\u003eADORA2A\u003c/em\u003e rs2298383, the three possible genotypes, divided by caffeine consumption, were compared. Four to six subgroups were then compared per gene - according to genotypes plus caffeine consumption. No statistical differences were found in the AOfs of each set of subgroups, per gene under study, after controlling for a CAGexp of 75 repeats (\u003cstrong\u003eTable 3\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eThe potential effects of the combination of\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003epolymorphisms under study and caffeine habits over the age at onset of SCA3/MJD\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"680\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.5%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCaffeine consumption/\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eday\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.91176470588235%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGene and polymorphism\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"49.11764705882353%\" colspan=\"4\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAges at onset for carriers of 75 CAGexp repeat length\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003emean (SD)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.470588235294118%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ep *\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"53.89221556886228%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;Genotypes proposed to be protective\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"46.10778443113772%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAlternative genotype\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.5%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.91176470588235%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eCYP1A2,\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;rs762551\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.470588235294116%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eCC or AC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.647058823529413%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eAA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.470588235294118%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.5%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMore than 314.5 mg\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.91176470588235%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.470588235294116%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e34.06 (10.76)\u003c/p\u003e\n \u003cp\u003en = 31\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.647058823529413%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e34.73 (11.96)\u003c/p\u003e\n \u003cp\u003en = 37\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.470588235294118%\" valign=\"top\"\u003e\n \u003cp\u003e0.255\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.5%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eEqual or less than 314.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.91176470588235%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.470588235294116%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e35.14 (10.14)\u003c/p\u003e\n \u003cp\u003en = 35\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.647058823529413%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e35.71 (9.82)\u003c/p\u003e\n \u003cp\u003en = 52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.470588235294118%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.5%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.91176470588235%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eNOS1\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e, rs478597\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.470588235294116%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCC or TC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.647058823529413%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTT\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.470588235294118%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.5%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMore than 314.5 mg\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.91176470588235%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.470588235294116%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e33.62 (11.07)\u003c/p\u003e\n \u003cp\u003en=60\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.647058823529413%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e43.14 (10.59)\u003c/p\u003e\n \u003cp\u003en=7\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.470588235294118%\" valign=\"top\"\u003e\n \u003cp\u003e0.069\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.5%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eEqual or less than 314.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.91176470588235%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.470588235294116%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e35.81 (10.12)\u003c/p\u003e\n \u003cp\u003en=77\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.647058823529413%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e31.55 (8.96)\u003c/p\u003e\n \u003cp\u003en=11\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.470588235294118%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.5%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.91176470588235%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eADORA2A,\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;rs5751876\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.470588235294116%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTT or TC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.647058823529413%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.470588235294118%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.5%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMore than 314.5 mg\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.91176470588235%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.470588235294116%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e35.37 (10.96)\u003c/p\u003e\n \u003cp\u003en=51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.647058823529413%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e38.23 (12.34)\u003c/p\u003e\n \u003cp\u003en=17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.470588235294118%\" valign=\"top\"\u003e\n \u003cp\u003e0.270\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.5%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eEqual or less than 314.5\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.91176470588235%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.470588235294116%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e34.02 (10.56)\u003c/p\u003e\n \u003cp\u003en=55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.647058823529413%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e38.23 (7.04)\u003c/p\u003e\n \u003cp\u003en=22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.470588235294118%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.5%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.91176470588235%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"49.11764705882353%\" colspan=\"4\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGenotypes\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.470588235294118%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.52577319587629%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.944035346097202%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eADORA2A,\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;rs2298383\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.347569955817377%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.347569955817377%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCT\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.347569955817377%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTT\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.487481590574374%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.52577319587629%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMore than 314.5 mg\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.944035346097202%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.347569955817377%\" valign=\"top\"\u003e\n \u003cp\u003e28.42 (11.80)\u003c/p\u003e\n \u003cp\u003en = 12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.347569955817377%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e28.42 (11.80)\u003c/p\u003e\n \u003cp\u003en = 12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.347569955817377%\" valign=\"top\"\u003e\n \u003cp\u003e37.26 (10.76)\u003c/p\u003e\n \u003cp\u003en = 27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.487481590574374%\" valign=\"top\"\u003e\n \u003cp\u003e0.560\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.52577319587629%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eEqual or less than 314.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.944035346097202%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.347569955817377%\" valign=\"top\"\u003e\n \u003cp\u003e39.10 (8.26)\u003c/p\u003e\n \u003cp\u003en = 20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.347569955817377%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e39.10 (8.26)\u003c/p\u003e\n \u003cp\u003en = 20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.347569955817377%\" valign=\"top\"\u003e\n \u003cp\u003e33.74 (9.52)\u003c/p\u003e\n \u003cp\u003en = 23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.487481590574374%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*ANOVA\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eCaffeine consumption did not have any modifying effect on the onset of symptoms of patients with SCA3/MJD. There was no direct effect, nor any effect mediated by functional variants of \u003cem\u003eADORA2A\u003c/em\u003e, \u003cem\u003eCYP1A2\u003c/em\u003e and \u003cem\u003eNOS1\u003c/em\u003e, genes that act in caffeine signaling or metabolism.\u003c/p\u003e \u003cp\u003eCaffeine has long been related to protective effects against neurodegenerative disease, in special PD [31, 32, 33], a common multifactorial neurodegenerative disease that shares with CAGexp diseases the start in adulthood. There is increasing evidence relating genes, caffeine consumption, and their interaction with the onset and progression of PD [25]. Studies on PD are large, since PD affects 1\u0026ndash;2% of individuals with more than 60 years of age [34], and since these studies seek causality. In contrast, causality is well established for CAGexp diseases, whereas studies on interactions between CAGexp, modifier genes and environmental factors, and/or habits are still very preliminary. For instance, lifestyle characteristics of SCA3/MJD European subjects have been the focus of a recent report, but caffeine consumption was not studied there [35].\u003c/p\u003e \u003cp\u003eCaffeinated drinks, such as coffee and tea, contain a mixture of several substances, among which caffeine is assumed to be the protagonist. Chronic caffeine consumption would be neuroprotective through the antagonism of A2ARs [13, 36]. A2ARs have a key role in controlling synaptic viability, apoptotic neuronal death, astrocytic function, and neuroinflammation [37]. In HD models, A2ARs blockade produced a normalization of striatal glutamatergic transmission. In a SCA3/MJD mouse model, not only synaptotoxicity and gliosis in the striatum were found to be early events predating neurodegeneration, but caffeine administration and A2AR blockade delayed striatal pathology, leading the authors to suggest that caffeine consumption was perhaps a \"lifestyle prophylactic strategy to delay the onset\" of this disease [13]. These experimental studies provided proof of concept for launching an epidemiologic observation like ours. However, our study showed that caffeine consumption did not delay the onset of SCA3/MJD subjects.\u003c/p\u003e \u003cp\u003eWe tried to check if the caffeine effect would depend on the interaction with other genetic characteristics. As SCA3/MJD is a quite rare disorder and the information about caffeine consumption should only be obtained by a direct survey, sample size was expected to be limited. In this scenario, the best choice to model this investigation was that of a candidate gene approach - and we chose to study variants at \u003cem\u003eADORA2A, CYP1A2\u003c/em\u003e and \u003cem\u003eNOS1\u003c/em\u003e, and their interaction with caffeine consumption.\u003c/p\u003e \u003cp\u003e \u003cem\u003eADORA2A\u003c/em\u003e, located at Chromosome 22 (24,417,879\u0026ndash;24,442,357), was a strong candidate as it encodes a G-protein coupled adenosine receptor. This receptor is known to participate in neurogenesis, neuroinflammation, and synaptic plasticity within glutamatergic synapse [38]. According to a systematic review, \u003cem\u003eADORA2A\u003c/em\u003e might indeed modulate the association between caffeine and brain-related outcomes [39]. rs5751876 has a high sensitivity to caffeine and has been the focus of several observations [16, 40, 41]. A study on this SNP and caffeine intake in PD patients gave negative results [42]. rs2298383 has been studied in the context of sleep [43], anxiety [44], epilepsy [45] and levodopa induced dyskinesias [46, 47]. Two other \u003cem\u003eADORA2A\u003c/em\u003e polymorphisms were inversely associated with PD risk, without a clear-cut interaction with coffee consumption [48]. We did not find any interaction between rs5751876 nor rs2298383 variants, caffeine consumption, and AOfs in SCA3/MJD (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eCYP1A2\u003c/em\u003e at chromosome 15 (74,748,845\u0026ndash;74,756,607) was another strong candidate, as it encodes the cytochrome P450 CYP1A2 enzyme that is responsible for metabolizing over 90% of caffeine into paraxanthine [49]. The SNP rs762551 is in the noncoding region of the \u003cem\u003eCYP1A2\u003c/em\u003e gene and likely impacts on the levels of expression of the CYP1A2 enzyme by affecting the binding of regulatory proteins to the surrounding gene sequences [15]. More than 40 SNPs have been found in \u003cem\u003eCYP1A2\u003c/em\u003e, and one of the most extensively studied is rs762551 (163 C\u0026thinsp;\u0026gt;\u0026thinsp;A) [50]. Individuals with the rs762551 A\u0026thinsp;\u0026gt;\u0026thinsp;C SNP have lower CYP1A2 inducibility, rendering slower caffeine metabolism. The hypothesis to be tested was the effect of caffeine would differ between individuals with the AA genotype (classified as \u0026ldquo;fast metabolizers'') and those with CC and AC individuals, characterized as \u0026ldquo;slow metabolizers'' [15, 50]. However, no effect was seen in the present cohort: subjects carrying genotypes CC and AC at rs762551 that were high caffeine consumers had the same AOfs as low consumers (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The absence of interacting effects of this variant with caffeine has been recently reported for the risk of PD, too [51].\u003c/p\u003e \u003cp\u003eNitric oxide synthase (NOS) genes (\u003cem\u003eNOS1, NOS2A\u003c/em\u003e, and \u003cem\u003eNOS3\u003c/em\u003e) account for most of the production of nitric oxide (NO) in the nervous system required for synaptic transmission and neuroplasticity. Excess of NO may contribute to neurodegeneration in Parkinson's disease (PD) [18]. A recent review concluded that NOS signaling has significant involvement in several disease pathologies, including protection against PD [19]. NOS effects were related to caffeine consumption in a preclinical study [20], while caffeine suppresses the generation of NO in microglia [21]. rs478597 at \u003cem\u003eNOS1\u003c/em\u003e has been related to Attention-Deficit/Hyperactivity Disorder (ADHD) [52, 53]. In a Brazilian PD cohort study, CC rs478597 carriers who were high consumers of caffeine presented lower PD risk than those who were low consumers [22]. Our results on rs478597 and caffeine consumption discarded any effect over SCA3/MJD AOfs (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIf caffeine consumption per se was not related to modification of the onset of symptoms in SCA3/MJD, was it futile to study the effect of candidate genes? Why did we choose to test these associations? First, caffeine exerted neuroprotection in models of SCA3/MJD. Second, SCA3/MJD have some similarities with PD, a disease where caffeine is related to neuroprotection. Third, SCA3/MJD has even more similarities with HD, a CAGexp disease where earlier AOfs were seen among consumers than among non-consumers of caffeine. We did not know the exact direction of the caffeine effect, if any would exist over SCA3/MJD. AOfs in SCA3/MJD carriers living in our region were expected to be lower, according to CAGexp lengths observed in the RS cohort [3]. This intriguing finding points to the presence of some local effect that is neuroprotective to the onset of symptoms. Among the cultural characteristics of our region is the intake of \u003cem\u003echimarr\u0026atilde;o\u003c/em\u003e, a drink very rich in caffeine. Then we speculated whether the intake of \u003cem\u003echimarr\u0026atilde;o\u003c/em\u003e could be neuroprotective to SCA3/MJD.\u003c/p\u003e \u003cp\u003eOur negative results also propose an additional reflection. SCA3/MJD and other polyQ disorders might have some similarities with PD, as said before, but of course imbalances between neurotransmitters might not be as important in CAGexp diseases as they are in PD. In PD, the neurotransmitter imbalance is a direct consequence of the disease, is paramount for PD signs and symptoms, and is the phenomenon to be corrected for the available treatments. Caffeine can act as a neurotransmitter, and its neuroprotection can reach intracellular processes mediated by neuroreceptors, structures very involved in the chain of events between cause and manifestations in PD. This poses questions about differences in all steps between etiology, cellular biology, neuronal webs, topography and clinical phenomenology of CAGexp disorders and PD. Differences in these steps may explain the lack of association and even divergent effects across these diseases.\u003c/p\u003e \u003cp\u003eFinally, we need to stress that our study had limitations - although the sample size was apparently not among them, as the p-values obtained quite convincingly discarded the hypotheses tested. In contrast, the design of studies on consumption habits seems to be the main limitation: it is unsatisfactory, at least in the context of progressive and long-lasting diseases. Habits are defined through questionnaires about consumption in the last few days. This information is then generalized to the person's entire life cycle. There is no way to test cumulative effects of this habit over AO, since we would confuse causes with consequences. After all, those with later AO would have had a greater chance of consuming more coffee before the AO, and we could not conclude anything about this cumulative consumption. Our questionnaire asked about the beginning of the caffeine habit, but not about changes in this habit over the years. Therefore, it was not possible to define whether the habit changed after the onset of symptoms. This information would be interesting as caffeine - a central nervous system (CNS) stimulant - may theoretically alleviate some symptoms. However, SCA3/MJD patients did not consume more caffeine than controls, which might rule out for now the hypothesis that caffeine is consumed by our patients because it has any symptom-mitigating effect. Finally, the choice of molecular variants under study is always a concern. We may not have chosen the best SNPs in our candidate genes. It is always important to emphasize that results on variants should be interpreted with caution, as they might not be generalizable to the genes in question.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eCaffeine consumption and the interaction with variants of genes related to caffeine receptors and caffeine degradation had no effect over the onset of symptoms of SCA3/MJD. The role of other habits and environmental factors should continue to be pursued in this disease.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003ea. Acknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to all study participants. This study was supported by Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq), Brazil, grant number 405963/2021-1; and by Financiamento e Incentivo \u0026agrave; Pesquisa, Hospital de Cl\u0026iacute;nicas de Porto Alegre (FIPE HCPA) grants numbers 2018-0660 and 2018-0661. ACM, JSP, LS, ERC, DS, LDP, BAA, MLSP and LBJ were supported by CNPq.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb. Conflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec. Availability of data and material (data transparency)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed. Authors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.C.M and L.B.J. contributed to the conception and design of the study; A.C.M, J.S.P., L.S., E.R.C., D.F.S., L.D.P., B.A.A., M.L.S.P., and L.B.J. contributed to the acquisition and analysis of data; A.C.M. and L.B.J. contributed to drafting the text and preparing the tables. All authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eKawaguchi Y, Okamoto T, Taniwaki M, Aizawa M, Inoue M, Katayama S, Kawakami H, Nakamura S, Nishimura M, Akiguchi I, et al. (1994) CAG expansions in a novel gene for Machado-Joseph disease at chromosome 14q32.1. Nat Genet 8:221-8.\u003c/li\u003e\n \u003cli\u003eSouza GN, Kersting N, Krum-Santos AC, Santos ASP, Furtado GV, Pacheco D, Gon\u0026ccedil;alves TA, Saute JA, Schuler-Faccini L, Mattos EP et al (2016) Spinocerebellar ataxia type 3/Machado-Joseph disease: segregation patterns and factors influencing instability of expanded CAG transmissions. Clin Genet 90:134\u0026ndash;140.\u003c/li\u003e\n \u003cli\u003ede Mattos E, Musskopf M, Leotti V, Saraiva-Pereira M, Jardim LB. (2019). Genetic risk factors for modulation of age at onset in Machado-Joseph disease/spinocerebellar ataxia type 3: A systematic review and meta-analysis. 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Eur J Neurol. 18: 756-65.\u003c/li\u003e\n \u003cli\u003eSchwarzschild MA, Xu K, Oztas E, Petzer JP, Castagnoli K, Castagnoli N Jr, Chen JF. (2003) Neuroprotection by caffeine and more specific A2A receptor antagonists in animal models of Parkinson\u0026apos;s disease. Neurology. 61(Suppl 6): S55-61.\u003c/li\u003e\n \u003cli\u003eNikrandt G, Mikolajczyk-Stecyna J, Młodzik-Czyżewska M, Chmurzynska A. (2022) Functional single-nucleotide polymorphism (rs762551) in CYP1A2 gene affects white coffee intake in healthy 20- to 40-year-old adults. Nutr Res. 105:77-81.\u003c/li\u003e\n \u003cli\u003eKim IY, O\u0026apos;Reilly \u0026Eacute;J, Hughes KC, Gao X, Schwarzschild MA, McCullough ML, Hannan MT, Betensky RA, Ascherio A. (2018) Interaction between caffeine and polymorphisms of glutamate ionotropic receptor NMDA type subunit 2A (GRIN2A) and cytochrome P450 1A2 (CYP1A2) on Parkinson\u0026apos;s disease risk. Mov Disord. 33:414-420\u003c/li\u003e\n \u003cli\u003eSalatino-Oliveira A, Wagner F, Akutagava-Martins GC, Bruxel EM, Genro JP, Zeni C, Kieling C, Polanczyk GV, Rohde LA, Hutz MH. (2016) MAP1B and NOS1 genes are associated with working memory in youths with attention-deficit/hyperactivity disorder. Eur Arch Psychiatry Clin Neurosci. 266: 359-66\u003c/li\u003e\n \u003cli\u003eSalatino-Oliveira A, Akutagava-Martins GC, Bruxel EM, Genro JP, Polanczyk GV, Zeni C, Kieling C, Karam RG, Rovaris DL, Contini V, Cupertino RB, Mota NR, Grevet EH, Bau CH, Rohde LA, Hutz MH. (2016) NOS1 and SNAP25 polymorphisms are associated with Attention-Deficit/Hyperactivity Disorder symptoms in adults but not in children. J Psychiatr Res. 75: 75-81.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 4 to 6 are not available with this version. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4.\u0026nbsp;\u003c/strong\u003eThe \u003cem\u003eADORA2A\u0026nbsp;\u003c/em\u003epolymorphism at rs2298383 and caffeine habits versus age at onset of SCA3/MJD\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5.\u0026nbsp;\u003c/strong\u003eThe \u003cem\u003eCYP1A2\u0026nbsp;\u003c/em\u003epolymorphism at rs762551 and caffeine habits versus age at onset of SCA3/MJD\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 6.\u0026nbsp;\u003c/strong\u003eThe \u003cem\u003eNOS1\u003c/em\u003e polymorphism at rs478597 and caffeine habits versus age at onset of SCA3/MJD\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"the-cerebellum","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cere","sideBox":"Learn more about [The Cerebellum](http://link.springer.com/journal/12311)","snPcode":"12311","submissionUrl":"https://submission.nature.com/new-submission/12311/3","title":"The Cerebellum","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Caffeine consumption, ADORA2A, CYP1A2, NOS1, spinocerebellar ataxia type 3, Machado-Joseph disease","lastPublishedDoi":"10.21203/rs.3.rs-4325198/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4325198/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe age at onset (AO) of Machado-Joseph disease (SCA3/MJD), a disorder due to an expanded CAG repeat (CAGexp) in \u003cem\u003eATXN3\u003c/em\u003e, is quite variable and the role of environmental factors is still unknown. Caffeine was associated with protective effects against other neurodegenerative diseases, and in transgenic SCA3/MJD mouse models. We aimed to evaluate whether caffeine consumption and the interaction with variants of caffeine signaling/metabolization genes impact the AO of this disease.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003ea questionnaire on caffeine consumption was applied to adult patients and unrelated controls living in Rio Grande do Sul, Brazil. AO and CAGexp were previously determined. SNPs rs5751876 (\u003cem\u003eADORA2A\u003c/em\u003e), rs2298383 (\u003cem\u003eADORA2A\u003c/em\u003e), rs762551 (\u003cem\u003eCYP1A2\u003c/em\u003e) and rs478597 (\u003cem\u003eNOS1\u003c/em\u003e) were genotyped. AO of subgroups were compared, adjusting the CAGexp to 75 repeats (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003e171/179 cases and 98/100 controls consumed caffeine. Cases with high and low caffeine consumption (more or less than 314.5 mg of caffeine/day) had mean (SD) AO of 35.05 (11.44) and 35.43 (10.08) years (p\u0026thinsp;=\u0026thinsp;0.40). The mean (SD) AO of the subgroups produced by the presence or absence of caffeine-enhancing alleles in \u003cem\u003eADORA2A\u003c/em\u003e (T allele at rs5751876 and rs2298383), \u003cem\u003eCYP1A2\u003c/em\u003e (C allele) and \u003cem\u003eNOS1\u003c/em\u003e (C allele) were all similar (p between 0.069 and 0.516).\u003c/p\u003e\u003ch2\u003eDiscussion\u003c/h2\u003e \u003cp\u003eCaffeine consumption was not related to changes in the AO of SCA3/MJD, either alone or in interaction with protective genotypes at \u003cem\u003eADORA2A\u003c/em\u003e, \u003cem\u003eCYP1A2\u003c/em\u003e and \u003cem\u003eNOS1\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Caffeine consumption and interaction with ADORA2A, CYP1A2 and NOS1 variants do not influence age at onset of Machado-Joseph disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-02 17:18:32","doi":"10.21203/rs.3.rs-4325198/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-04T08:38:53+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-29T08:59:08+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-21T12:31:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"33889645253891636139520523530820340189","date":"2024-05-08T20:13:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"293866578571395759394039243517918497444","date":"2024-05-07T08:34:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-07T08:27:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-26T01:35:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-26T01:35:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"The Cerebellum","date":"2024-04-25T15:42:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"the-cerebellum","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cere","sideBox":"Learn more about [The Cerebellum](http://link.springer.com/journal/12311)","snPcode":"12311","submissionUrl":"https://submission.nature.com/new-submission/12311/3","title":"The Cerebellum","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9d7989df-af28-4067-a4ee-5db190716c1b","owner":[],"postedDate":"May 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-06-27T10:14:07+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-02 17:18:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4325198","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4325198","identity":"rs-4325198","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}