ORIGINAL RESEARCH published: 09 June 2020 doi: 10.3389/fphar.2020.00860 Polymorphisms Involved in Platelet Activation and Inflammatory Response on Aspirin-Related Upper Gastrointestinal Bleeding: A Case- Control Study Edited by: Narmeen Mallah 1,2, Maruxa Zapata-Cachafeiro 1,2*, Carmelo Aguirre 3,4,5, José A. G. Agúndez, Eguzkiñe Ibarra-Garcı́a 3,6, Itziar Palacios–Zabalza 3,4, Fernando Macı́as-Garcı́a 7, Institute of Molecular Pathology J. Enrique Domı́nguez-Muñoz 7, Marı́a Piñeiro-Lamas 2,8, Luisa Ibáñez 9, Xavier Vidal 9, Biomarkers, University of Lourdes Vendrell 9, Luis Martin-Arias 10, Marı́a Sáinz-Gil 10, Verónica Velasco-González 10 Extremadura, Spain and Adolfo Figueiras 1,2,8 on behalf of the EMPHOGEN group Reviewed by: 1 Department of Preventive Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain, Francesco Sessa, University of Florence, Italy 2 Consortium for Biomedical Research in Epidemiology and Public Health (CIBER en Epidemiologı´a y Salud Pública - CIBERESP), Shilong Zhong, Carlos III Health Institute, Madrid, Spain, 3 Biocruces Bizkaia Health Research Institute, Pharmacotherapy Group, Bizkaia, Spain, 4 University Hospital of Galdakao-Usansolo. Basque Country Pharmacovigilance Unit, Osakidetza, Spain, 5 Pharmacology Guangdong Provincial People’s Hospital, China Department, Medicine and Nursing Faculty, University of the Basque Country, Barakaldo, Bizkaia, Spain, 6 Osakidetza Basque Health Service, Urduliz Hospital, Pharmacy Department, Urduliz, Spain, 7 Department of Gastroenterology and Hepatology, University *Correspondence: Hospital of Santiago de Compostela, Santiago de Compostela, Spain, 8 Health Research Institute of Santiago de Compostela (IDIS), Maruxa Zapata-Cachafeiro Santiago de Compostela, Spain, 9 Department of Pharmacology, Therapeutics and Toxicology, Catalonian Institute of Pharmacology, maruxa.zapata@usc.es Clinical Pharmacology Service, Vall d’Hebron University Teaching Hospital, Autonomous University, Barcelona, Spain, 10 Centre for Research on Drug Safety (CESME), Valladolid University, Valladolid, Spain Specialty section: This article was submitted to Pharmacogenetics and Background: Despite the wide benefits of aspirin and its cost-effectiveness, aspirin Pharmacogenomics, prescriptions have been reduced due to idiosyncratic responses in susceptible a section of the journal Frontiers in Pharmacology individuals. Low-dose aspirin and single-nucleotide polymorphisms (SNPs) are Received: 06 April 2020 independently associated with increased risk of gastrointestinal hemorrhage; however, Accepted: 26 May 2020 to-date, no studies investigated the SNP-aspirin interaction effect on upper Published: 09 June 2020 gastrointestinal hemorrhage (UGIH). Therefore, we aimed to evaluate the role of 25 Citation: Mallah N, Zapata-Cachafeiro M, SNPs in multiple genes involved in platelet activation, angiogenesis and inflammatory Aguirre C, Ibarra-Garcı´a E, response in aspirin-related UGIH. Palacios–Zabalza I, Macı´as-Garcı´a F, Domı´nguez-Muñoz JE, Methods: A multicenter, full case–control study was conducted in patients exposed and Piñeiro-Lamas M, Ibáñez L, unexposed to aspirin. Three hundred twenty-six cases diagnosed with UGIH were Vidal X, Vendrell L, Martin-Arias L, Sáinz-Gil M, matched with 748 controls (1:3) by age, gender, health center, and recruitment date. Velasco-González V and Figueiras A Only adults of European origin were included. Participants were stratified by aspirin (2020) Polymorphisms Involved in exposure and genotype [(Aspirin(−), wild-type), (Aspirin(+), wild-type), (Aspirin(+), genetic Platelet Activation and Inflammatory Response on Aspirin-Related Upper variation), (Aspirin(−), genetic variation)]. For each SNP, the Odds Ratio of UGIH and their Gastrointestinal Bleeding: 95% confidence intervals were estimated in each subgroup by using the generalized linear A Case-Control Study. Front. Pharmacol. 11:860. mixed models for dependent binomial variables. SNP-aspirin interaction effect was doi: 10.3389/fphar.2020.00860 estimated through Relative Excess Risk due to Interaction (RERI) measures. Frontiers in Pharmacology | www.frontiersin.org 1 June 2020 | Volume 11 | Article 860 Mallah et al. Polymorphism, Aspirin, and Gastrointestinal Bleeding Results: We observed two categories of SNPs that might modify the risk magnitude of UGIH in aspirin consumers. Seven SNPs (rs1387180 A > G, rs2238631 T > C, rs1799964 T > C, rs5050 T > C/T > G, rs689466 T > C, rs1799983 T > A/T > G, and rs7756935 C > A) were “positive modifiers” associated with an excess of risk from aspirin exposure and carrying that genetic variation (1.75 ≤ RERI ≤ 4.95). On the contrary, the following nine SNPs (rs2243086 G > T, rs1131882 G > A, rs4311994 C > T, rs10120688 G > A, rs4251961 T > C, rs3778355 G > C, rs1330344 C > T, rs5275 A > G/A > T, and rs3779647 C > T) were “negative modifiers” and associated with a reduced risk in aspirin users (−2.74 ≤ RERI ≤ −0.95). Conclusion: This preliminary study suggests that polymorphisms in genes involved in platelets activity, angiogenesis and inflammatory response might modify the risk of aspirin- related UGIH. Further studies with larger sample size and in different populations are needed to confirm our findings. If confirmed, this might have great impact on public health, thanks to aspirin’s prophylactic properties in diseases of high incidence and severity. Keywords: aspirin, upper gastrointestinal haemorrhage, genetic polymorphism, pharmacogenomics, platelet, interaction INTRODUCTION (UGIH) in aspirin users (Zhang et al., 2011; Andersen et al., 2011; Wu et al., 2016). Aspirin is one of the most commonly used medicines worldwide In specific, it was suggested that single-nucleotide due to its broad spectrum of health benefits such as analgesic, polymorphisms (SNPs) mainly those present in genes involved anti-inflammatory and antiplatelet properties (Throat and in drug metabolism, platelet activation and inflammatory Cuzick, 2015). Some recent studies also suggested that aspirin response, might increase the risk of gastrointestinal bleeding in may have protective effects against cancer; a disease that affects users of low-dose aspirin (Shiotani et al., 2010). However, the all communities and contributes substantially to the global studies were limited by their small sample size and by the disease burden by impinging on the lives of tens of millions of assessment of exposed cases, exclusively (Shiotani et al., 2013; individuals each year (Global Burden of Disease Cancer Shiotani et al., 2014; Wu et al., 2016; Cho et al., 2016; Milanowski Collaboration, 2019). Moreover, there is extensive evidence et al., 2017). The non-inclusion of individuals unexposed to about the benefits of aspirin use for the secondary prevention aspirin in the design of these studies made it impossible to assess against cardiovascular diseases (Godley and Hernandez- the direct effect of genetic polymorphisms on gastrointestinal Vila, 2016). bleeding. Therefore, it could not be ascertained whether the On the other hand, patients on aspirin treatment may previously reported high risk of bleeding in low-dose aspirin manifest idiosyncratic reactions to this drug and may be at risk users was a consequence of aspirin consumption, the presence of of bleeding, which consequently limit the widespread use of a genetic variation, or a combination of both factors. aspirin as prophylactic of many diseases, despite its effectiveness Accordingly, in the current “full” case-control study, we and low cost (Throat and Cuzick, 2015). Gastrointestinal aimed at exploring the association between 25 SNPs in genes bleeding is a frequent clinically relevant adverse effect in involved in platelet activation, angiogenesis and inflammatory patients on low-dose aspirin treatment. Several cohort studies response and UGIH in a population of exposed and unexposed and meta-analyses have demonstrated that low-dose aspirin cases and controls. In addition, we intended to determine any increases the risk of gastrointestinal bleeding between 37% and possible interaction or modification of effect between these SNPs 85% (Sutcliffe et al., 2013; Whitlock et al., 2015; Whitlock et al., and aspirin intake on the risk of upper gastrointestinal bleeding. 2016; Raju et al., 2016; Luo et al., 2019; Haykal et al., 2019). The idiosyncratic response to aspirin could be related to the genetic susceptibility of the individuals. In fact, genetic variations influence patients’ reactions to drugs (Madian et al., 2012). METHODS Likewise, several lines of evidence indicated a possible association between predisposing genetic factors and Study Population and Study Design gastrointestinal disorders (Shiotani et al., 2013; Shiotani et al., A full case-control study that included 326 cases and 748 2014; Wu et al., 2016; Cho et al., 2016; Milanowski et al., 2017). controls was conducted. The subjects were adults of at least 18 For instance, previous studies associated the genetic variant years of age. They were recruited in two time periods (2004–2007 rs689466 with an increased risk of ulcerative colitis and gastric and 2013–2015) from four health centers located in four different cancer, and rs2238631 with upper gastrointestinal hemorrhage Spanish cities: Barcelona, Galdakao, Santiago de Compostela, Frontiers in Pharmacology | www.frontiersin.org 2 June 2020 | Volume 11 | Article 860 Mallah et al. Polymorphism, Aspirin, and Gastrointestinal Bleeding and Valladolid. The ethics committee of each involved hospital counter drugs. Second, information was obtained about the approved the study protocol and each subject signed a written frequent symptoms for which aspirin was prescribed, and the informed consent before participating in the study. treatments used to reduce these symptoms. Third, prompt cards Cases consisted of hospitalized patients with surgical or of the most common commercial aspirin boxes were shown to endoscopic diagnosis of UGIH. Cases were considered eligible the participants in order to facilitate the recall process. Fourth, if the diagnosis revealed any of the following ulcers (pyloric, subjects who could not remember a specific information were re- cardia, duodenal, or gastric), erosions (duodenitis pyloric, or interviewed later during their hospitalization, or were telephoned cardia), erosive duodenitis, and/or acute gastric mucosal lesions. in case they were discharged before repeating the interview. Hospitalized patients who presented signs of a recent stomach When a patient was in a poor health condition and could not bleeding were also considered as eligible cases, even if the main answer the whole interview, the accompanying persons who were motive of hospitalization was not UGIH. The signs of recent in charge of the patient’s medication (healthcare assistants or bleeding were determined according to Forrest classification direct relatives) were allowed to help him/her complete the (Forrest et al., 1974). Cirrhotic patients who did not have interview, however only the information confirmed by the variceal esophageal bleeding were included if the endoscopic patient was considered. In case the patient had doubts or was diagnosis showed any sign of recent bleeding. Patients with not certain about the answers, the given information was endoscopic diagnoses different from those mentioned above confirmed by reviewing the medical records of the patient. were excluded. Moreover, patients who doubted about the name or the dose Three controls were matched to each case according to their of the consumed medicine were telephoned once they discharged age (+/− 5 years), gender and clinical center. To prevent selection from the hospital and requested to check this information in the bias due to high aspirin consumption, controls were selected corner drugstore. from outpatients or were recruited from the preoperative units At the end of the interview, the interviewer rated the among patients who had scheduled non-painful mild surgeries perceived reliability of each interview by using 0–10 Likert that were not related to aspirin intake. The included surgeries scale. Interviews with a zero score were considered completely are: prostatic adenoma, prostatic hyperplasia, inguinal or unreliable and excluded from the analysis. The scores of the umbilical hernia (strangulated or programmed), eye cataract, included interviews were then inspected for their influence on phimosis, ear pinning, tubal ligation, plastic surgery, lipoma, the estimated measure of effect as explained later in the statistical vocal cord cyst, septoplasty, varicotomy, thyroid nodules, and analysis section. thyroglossal cyst (euthyroid). All participants were not An index date of aspirin intake was established in order to biologically related. They were recruited from patients and ensure that exposure happened prior to any symptom of UGIH. outpatients of the same health centers in order to ensure that For the cases, the index date was set as the day of occurrence of they originate from the same population. Controls that had a the first signs or symptoms of UGIH. For the controls, the index history of UGIH or that experienced intrahospital UGIH were date was defined as the interview date. In line with earlier studies, excluded. Cases and controls with non-white race were excluded we considered an etiological window of 7 days from the index from the analysis in order to control for bias due to population day. Aspirin intake that occurred after the index date was not stratification. Participants living outside the study area; having a considered as exposure in the statistical analysis. history of liver cirrhosis or coagulopathy and/or neoplasia; and/ or unreliably answered the interview questions were also Determination of Helicobacter pylori (H. excluded. A detailed list of exclusion and inclusion criteria is available in supplementary data online (see Table S1 of the pylori) Infection The presence of anti–H. pylori immunoglobulin G was tested in supplementary materials attached to this article). human serum using the Enzyme Linked Immunosorbent Assay (ELISA) commercial kits [Human Anti-Helicobacter pylori IgG Data Collection ELISA Kit (ab108736, Abcam, Cambridge, England), and A comprehensive questionnaire was designed in a previous study Captia™ H. pylori IgG EIA (ref: 2346400, Trinity Biotech of ours that shares the same protocol (Figueiras et al., 2016). The Captia, Co. Wicklaw, Ireland)] according to the manufacturer’s questionnaire was administered by experienced health personnel protocol. To avoid obtaining any false positive result due to an who interviewed the subjects. The collected data included: old infection, patients were asked whether they had been sociodemographic characteristics; smoking, alcohol and previously treated against H. pylori infection. caffeine consumption; clinical history; reason for current hospitalization; medicines’ intake; and past episodes of gastric disorders. In addition, cases were asked about the underlying Selection of SNPs and Genotyping symptomatology, and controls were inquired about the motive of An extensive literature review was carried out until April 2017 in the planned surgery. order to identify SNPs associated with gastrointestinal disorders, Complete pharmacological anamnesis was generated using the function of the corresponding genes and the clinical the following four complementary strategies. First, direct significance of the genetic variation. Subsequently, related questions were raised to the patients about the name, the daily SNPs were selected, and samples were genotyped using the dose, the indication of use and the source of prescription of the iPlex® Gold chemistry and MassARRAY platform, according medicines consumed in the past two months, including over-the- to manufacturer’s instructions (Agena Bioscience, San Diego, Frontiers in Pharmacology | www.frontiersin.org 3 June 2020 | Volume 11 | Article 860 Mallah et al. Polymorphism, Aspirin, and Gastrointestinal Bleeding EEUU). Genotyping assays were designed using the Agena each SNP (Knol and VanderWeele, 2012). S was obtained from Bioscience MassARRAY Assay Designer 4.1 software and were the ratio of the combined effects to the sum of each of the performed in 384-well plates. The quality of the genotyping was individual effects of aspirin and polymorphisms. RERI was checked by including negative controls and a trio of Coriell estimated by contrasting the effects of aspirin exposure and samples. In addition, the reproducibility of 7% of the samples polymorphism together to the sum of each factor considered was tested between and/or within plates. Finally, all clusters plots separately: RERI = OR[(genetic variation; aspirin(+)] – OR were checked manually by trained personnel using MassArray [(wild-type; aspirin(+)] – OR[(genetic variation; aspirin(−)] +1 Typer software. (Hosmer and Lemeshow, 1992; Andersson et al., 2005). The Possible bias in the selected controls was assessed by checking confidence intervals of the interaction terms were calculated by the compliance of the genotyped SNPs with Hardy-Weinberg applying the method developed by Figueiras et al. (Figueiras equilibrium (HWE). HWE test was performed using the et al., 1998). SNPassoc Library of the R software package (Version 1.9-2). Statistical Analysis The participants were stratified according to aspirin exposure RESULTS [aspirin(+); aspirin(−)] and to genetic profile of each of the 25 SNPs (wild-type; genetic-variation). Accordingly, for each SNP, Study Population the patients were grouped into four categories [aspirin(−); wild- Of 5,896 interviewed subjects, 326 cases and 748 controls were type], [aspirin(+); wild-type], [aspirin(−); genetic variation] and eligible to be included in the final analysis. The most common [aspirin(+); genetic variation]. The adjusted Odds Ratios (ORs) exclusion criteria were ineligible endoscopic diagnosis (1,590 of UGIH were calculated in each group in comparison with the cases) and unavailability of biological material (328 cases and 748 category [aspirin(−); wild-type]. Subsequently, any potential controls). The flow diagram of cases and controls enrollment in interaction effect upon the co-presence of genetic the study and the reasons of exclusion are presented in polymorphisms and aspirin exposure was explored by supplementary data online (see Figure S1 and Table S1 of the estimating the Synergism index (S) (also called effect supplementary materials attached to this article). Table 1 modification) and the Relative Excess Risk due to Interaction summarizes the demographic and clinical characteristics of the (RERI) along with their 95% CI. study population. The ORs and their 95% confidence intervals (CI) were estimated using the generalized linear mixed models for Single-Nucleotide Polymorphisms dependent binomial variables (Gonzá lez et al., 2014). To build Genotyping up the statistical models, the following four levels were All the 1,074 biologically unrelated patients were genotyped. considered consecutively: study subjects, strata (each case and High quality of genotyping testing was obtained as revealed by its three matched controls), recruitment center, and period of the reproducibility analysis (100%) and genotype recall rate patients’ enrolment. (≥98%). In addition, the controls were in equilibrium with A bivariate analysis was carried out to test the effect of the respect to the corresponding SNPs as confirmed by the manual potentially confounding variables. Covariables with a P-value < inspection of the clusters’ plots and the HWE analysis (p < 0.001) 0.2 was selected for multivariate logistic regression analysis, (see Table S2 of the supplementary materials attached to while those with higher levels of statistical significance were this article). eliminated successively from the original model, provided that the coefficients of the main exposure variables did not change by Risk Estimation and Interaction Analysis more than 10%, and that Schwartz’s Bayesian Information Aspirin consumption substantially increases the risk of Criterion was improved (Schwarz, 1978; Mickey and occurrence of UGIH by around six folds [adjusted OR: 5.82 Greenland, 1989; Bates et al., 2015). The covariables that were (95% CI: 2.2–10.08)]. retained in the final model are: age, Body Mass Index (BMI), Stratifying the analysis by genotype (wild-type vs. genetic gender, history of arthrosis, Helicobacter Pylori infection, variation) and aspirin exposure yielded the following two distinct gastrointestinal disorders (ulcer or bleeding), source of groups of SNPs. The first group comprises SNPs that increased information (patients or health assistants/direct relatives), the risk magnitude of aspirin-related UGIH and was called number of conducted interviews, answers’ reliability score, and “positive modifiers”. The second group consists of SNPs that exposure to each of the following medicines: Nonsteroidal Anti- showed protective effect by decreasing the risk value and was Inflammatory Drugs (NSAIDs) except aspirin, inhibitors of the named “negative modifiers”. proton pump, antiaggregant and anticoagulants. The models were estimated using the lmer function of the lme4 R package SNPs as Positive Modifiers of the Effect of (version 1.1-21) (Brown and Prescott, 2006). The analysis was Aspirin-Related UGIH restricted to individuals with complete data on the variables In the absence of aspirin exposure, no association was observed included in the model. The recommendations of Knol M.J, 2012 between carrying the following genetic variations and the were followed to represent the interaction between aspirin and occurrence of UGIH (rs1387180 A > G, rs2238631 T > C, Frontiers in Pharmacology | www.frontiersin.org 4 June 2020 | Volume 11 | Article 860 Mallah et al. Polymorphism, Aspirin, and Gastrointestinal Bleeding TABLE 1 | Description of cases and controls. RERI estimations that indicates the presence of positive effect Characteristic Cases Controls modification of aspirin use across the strata of genotypes (1.75 ≤ (N = 326) (N = 748) RERI ≤ 4.95), though these estimations were not statistically № (%) № (%) significant (Table 2). Age <45 41 (12.6%) 95 (12.7%) SNPs as Negative Modifiers of the Effect 45–65 117 (35.9%) 271 (36.2%) of Aspirin-Related UGIH >65 161 (49.4%) 370 (49.5%) Furthermore, carrying any of the following nine SNPs was not missing 7 (2.1%) 12 (1.6%) associated with the risk of UGIH when the patients were not BMI exposed to aspirin [rs2243086 G > T, rs1131882 G > A, Underweight 10 (3.1%) 24 (3.2%) Normal weight 114 (35.0%) 204 (37.3%) rs4311994 C > T, rs10120688 G > A, rs4251961 T > C, Overweight 128 (39.3%) 374 (50.0%) rs3778355 G > C, rs1330344 C > T, rs5275 A > G/A > T, and Obese 68 (20.9%) 144 (19.3%) rs3779647(C > T)] (Table 3). However, on the contrary to the Missing 6 (1.8%) 2 (0.3%) pattern of association observed earlier, when exposed to aspirin, Gender Male 236 (72.4%) 559 (74.7%) carriers of the wild-type genotypes of these SNPs are at higher Female 87 (26.7%) 189 (25.3%) risk of occurrence of UGIH in comparison with carriers of the Missing 3 (0.9%) 0 corresponding genetic variations. Arthrosis These findings would indicate that the estimated effect on the No 219 (67.2%) 469 (62.7%) additive scale of aspirin use by carriers of these genetic variations is Yes 86 (26.4%) 216 (28.9%) Missing 21 (6.4%) smaller than the effect of aspirin use by carriers of the corresponding Helicobacter pylori wild-type genotypes. The interaction analysis revealed that there is No or inconclusive 27 (8.3%) 138 (18.4%) negative effect modification of aspirin use across the strata of Yes 276 (84.7%) 574 (76.7%) genotypes on an additive scale (−2.74 ≤ RERI ≤ −0.95); however, Missing 23 (7.1%) 36 (4.8%) this modification was not statistically significant. Source of information Patients 259 (79.4%) 672 (89.8%) Finally, no modification of effect was observed for the SNP Healthcare assistants/direct relatives 67 (20.6%) 76 (10.2%) rs2990510 T > G [OR wild-type: 3.98 (95% CI: 1.24–12.76) vs. Interview Variables OR genetic-variation: 4.26 (95% CI: 1.58–11.47); S: 1.19 (95% CI: Number of interviews conducted 0.15–9.42); RERI: 0.51 (95% CI: −5.58, 6.61)]. In addition, no 1 274 (84.0%) 644 (86.1%) conclusive interpretation could be done about the following 8 ≥2 52 (16.0%) 104 (13.9%) Reliability of the interview SNPs due to the limited number of cases and controls who were <5 13 (4.0%) 20 (2.7%) aspirin users and carriers of these genetic variations (< 5 subjects 5–7 36 (11%) 78 (10.4%) in one of the groups): rs2502488, rs1800629, rs361525, 7–9 134 (41.1%) 310 (41.4%) rs1143627, rs16944, rs3842787, rs3842788 and rs5788 (see ≥9 143 (43.9%) 340 (45.5%) Personal history of gastrointestinal disorders Table S3 of the supplementary materials attached to this article). None or dyspepsia 208 (63.8%) 647 (86.5%) Ulcer 48 (14.7%) 56 (7.5%) Bleeding 70 (21.5%) 45 (6.0%) Exposure to other medications DISCUSSION All Nonsteroidal anti-inflammatory drugs 131 (40.2%) 112 (15.0%) (NSAIDs) To our knowledge, this is the first and largest case-control study All NSAIDs except aspirin 111 (34.0%) 96 (12.8%) NSAIDs metabolized by CYP2C9 58 (17.8%) 37 (4.9%) that assesses the effect of a large number of SNPs in multiple NSAIDs (M01A group) 74 (22.7%) 48 (6.4%) genes involved in platelet aggregation angiogenesis and Inhibitors of COX2 3 (0.9%) 6 (0.8%) inflammatory response on the risk of aspirin-related UGIH, Nonsteroidal anti-inflammatory agents 1 (0.3%) 1 (0.1%) and that explores the excess of risk from SNPs-aspirin Aspirin and its derivatives 32 (9.8%) 19 (2.5%) interaction. We tested the presence of genetic variations at the Analgesics not narcotics 54 (16.6%) 56 (7.5%) Inhibitors of the proton pump 36 (11.0%) 66 (8.8%) DNA sequence level, and our preliminary data suggested that Antiaggregant 65 (19.9%) 86 (11.5%) variations in these genes might alter the risk magnitude of UGIH Anticoagulants 35 (10.7%) 32 (4.3%) in aspirin users. These results are of a high clinical interest as they indicate that the likelihood of occurrence of the rs1799964 T > C, rs5050 T > C/T > G, rs689466 T > C, rs1799983 idiosyncratic bleeding response to aspirin depends on the T > A/T > G, and rs7756935 C > A) (Table 2). However, when patient’s genotype. exposed to aspirin, carriers of the wild-type genotypes of these We identified a group of seven SNPs that act as “positive SNPs are at lower risk of occurrence of UGIH in comparison modifiers” by increasing the risk magnitude of UGIH in aspirin with carriers of the corresponding genetic variations. The users up to more than three folds. Among these SNPs, rs1387180 estimated effect on the additive scale of aspirin use by carriers A > G, rs2238631 T > C, and rs689466 T > C yielded the greatest of these genetic variations is larger than the effect of aspirin use excess of risk. A hypothesis that could explain these effects is that by carriers of the wild-type genotypes. This was also revealed by SNPs belonging to this group “positive modifiers” might cause a Frontiers in Pharmacology | www.frontiersin.org 5 June 2020 | Volume 11 | Article 860 Mallah et al. Polymorphism, Aspirin, and Gastrointestinal Bleeding TABLE 2 | Odds ratios (OR) for upper gastrointestinal bleeding associated with aspirin intake and SNPs of the positive modifiers category, and their interaction assessment represented by Synergism Index (S) and Relative Excess Risk due to Interaction (RERI). Single-Nucleotide Polymorphism Wild-type genotype Genetic Variation OR* (95% CI) for RERI S (Reference number) any genetic (95% CI) (95% CI) N cases/ OR* (95% CI); N cases/ OR* (95% CI); variation controls p-value controls p-value within strata of aspirin intake; p-value rs1387180 A > G 143/358 1.00 119/278 0.84 (0.60–1.18) 0.84 (0.60–1.18) 4.28 3.33 Aspirin intake (No) P = 0.3233 p = 0.3278 (−4.41–12.97) (0.4–27.59) Aspirin intake (Yes) 10/12 2.99 (1.07–8.37) 14/5 7.12 (2.22–22.83) 3.26 (0.44–29.51) P = 0.0369 P = 0.2298 OR (95% CI) for Aspirin intake within strata 3.19 (1.12–9.08) 8.74 (2.61–29.24) of genotype; p-value P = 0.0293 P = 0.0001 rs2238631 T > C 74/168 1.00 188/468 0.97 (0.67–1.41) 0.97 (0.67–1.42) 2.22 2.08 Aspirin intake (No) P = 0.8765 P = 0.8932 (−4.17–8.61) (0.18–24.13) Aspirin intake (Yes) 6/5 3.08 (0.69–13.81) 18/12 5.28 (2.12–13.14) 1.57 (0.17–14.97) P = 0.1414 P = 0.0004 P = 0.6935 OR (95% CI) for Aspirin intake within strata 3.78 (0.82–17.43) 5.44 (2.25–13.17) of genotype; p-value P = 0.0881 P = 0.0002 rs1799964 T > C 150/372 1.00 112/264 1.19 (0.84–1.66) 1.19 (0.84–1.66) 3.29 2.13 Aspirin intake (No) P = 0.3251 P = 0.3265 (−5.33–11.91) (0.36–12.73) Aspirin intake (Yes) 12/10 3.73 (1.34–10.40) 12/7 7.20 (2.38–21.79) 2.17 (0.35–13.38) P = 0.0120 P = 0.0005 P = 0.4027 OR (95% CI) for Aspirin intake within strata 3.86 (1.36–10.92) 5.73 (1.84–17.81) of genotype; p-value P = 0.011 P = 0.0026 rs5050 T > C/T > G 85/188 1.00 177/448 0.82 (0.57–1.18) 0.82 (0.57–1.18) 1.75 1.84 Aspirin intake (No) P = 0.2765 P = 0.2746 (−4.19–7.71) (0.22–15.6) Aspirin intake (Yes) 9/8 3.26 (1.03–10.34) 15/9 4.83 (1.72–13.60) 1.22 (0.22–6.90) P = 0.0444 P = 0.0028 P = 0.819 OR (95% CI) for Aspirin intake within strata 2.75 (0.78–9.73) 6.05 (2.22–16.53) of genotype; p-value P = 0.1174 P = 0.0004 rs689466 T > C 170/393 1.00 92/243 0.85 (0.60–1.20) 0.84 (0.59–1.20) 4.95 3.18 Aspirin intake (No) P = 0.3583 P = 0.3431 (−6.39–16.26) (0.43–23.29) Aspirin intake (Yes) 16/12 3.42 (1.40–8.36) 8/5 8.22 (2.14–31.59) 2.98 (0.37–23.96) P = 0.0069 P = 0.0022 P = 0.3036 OR (95% CI) for Aspirin intake within strata 3.72 (1.49–9.26) 7.68 (2.01–29.36) of genotype; p-value P = 0.0048 P = 0.0029 rs1799983 T > A/T > G 126/302 1.00 136/336 1.28 (0.92–1.80) 1.29 (0.92–1.81) 3.40 2.03 Aspirin intake (No) P = 0.1462 P = 0.1361 (−5.91–12.72) (0.36–11.6) Aspirin intake (Yes) 14/10 4.01 (1.43–11.20) 10/7 7.69 (2.49–23.74) 4.42 (0.54–36.33) P = 0.0081 P = 0.0004 P = 0.1665 OR (95% CI) for Aspirin intake within strata 4.13 (1.35–12.57) 4.89 (1.62–14.77) of genotype; p-value P = 0.0126 P = 0.0048 rs7756935 C > A 150/347 1.00 112/289 1.02 (0.72–1.44) 1.02 (0.73–1.44) 1.84 1.61 Aspirin intake (No) P = 0.9239 P = 0.9012 (−5.70–9.38) (0.25 – 10.17) Aspirin intake (Yes) 14/9 4.02 (1.48–10.93) 10/8 5.88 (1.90–18.21) 1.82 (0.31–10.85) P = 0.0064 P = 0.0022 P = 0.5091 OR (95% CI) for Aspirin intake within strata 3.86 (1.42–10.50) 6.67 (2.11–21.62) of genotype; p-value P = 0.0081 P = 0.0013 (*): Odds Ratio adjusted for: period of patients´ recruitment, previous history of arthrosis, infection with Helicobacter pylori, gastrointestinal disorders (ulcer and bleeding), exposure to NSAIDs except ASA, exposure to inhibitors of the proton pump, exposure to antiaggregant, exposure to anticoagulants, and the interview variables (the number and the reliability of the interview). reduction in platelets’ activity, and therefore contribute to with previous studies which associated rs689466 with an increasing the risk of bleeding. Platelets play a fundamental increased risk of gastrointestinal disorders such as ulcerative role in homeostasis and platelet rich plasma are suggested to colitis and gastric cancer (Andersen et al., 2011; Zhang et al., treat specific hemorrhages such as hemorrhagic cystitis (Masieri 2011). We also coincide with Yun Wu who reported that et al., 2019). In fact, the genetic variant rs1387180 was associated rs2238631 is a risk factor of upper gastrointestinal bleeding in earlier with decreased platelets activity in diabetic patients on aspirin users (Wu et al., 2016). Nevertheless, our results go aspirin treatment (Postula et al., 2013). rs689466 belongs to beyond that of Wu by showing that aspirin users who are COX-2, a gene involved in the production of prostaglandins carriers of the wild-type genotype of rs2238631 are not at which plays a role in platelets aggregation and protects against significant risk of UGIH, and that this genetic variation gastric damage (Caughey et al., 2001). Our findings are in line presents an additional risk of bleeding. Frontiers in Pharmacology | www.frontiersin.org 6 June 2020 | Volume 11 | Article 860 Mallah et al. Polymorphism, Aspirin, and Gastrointestinal Bleeding TABLE 3 | Odds ratios (OR) for upper gastrointestinal bleeding associated with aspirin intake and SNPs of the negative modifiers category, and their interaction assessment represented by Synergism Index (S) and Relative Excess Risk due to Interaction (RERI). Single-Nucleotide Wild-type genotype Genetic Variation OR* (95% CI) RERI S Polymorphism for any genetic (95% CI) (95% CI) (Reference number) N cases/ OR* (95% CI); N cases/ OR* (95% CI); variation within controls p-value controls p-value strata of aspirin intake; p-value rs2243086 G > T 172/403 1.00 89/232 0.96 (0.67–1.36) 0.95 (0.67–1.35) −0.95 0.76 Aspirin intake (No) P = 0.7987 P = 0.7811 (−7.84–5.94) (0.1–5.99) Aspirin intake (Yes) 18/12 5.02 (2.01–12.54) 6/5 4.03 (1.07–15.23) 0.24 (0.03–2.18) P = 0.0005 P = 0.0398 P = 0.2029 OR (95% CI) for Aspirin intake within strata 5.41 (2.12–13.83) 3.71 (0.94–14.65) of genotype; p-value P = 0.0001 P = 0.0615 rs1131882 G > A 183/431 1.00 79/205 0.97 (0.68–1.40) 0.97 (0.67–1.40) −2.74 0.43 Aspirin intake (No) P = 0.8778 P = 0.8705 (−9.42–3.94) (0.05–3.69) Aspirin intake (Yes) 18/10 5.86 (2.26–15.17) 6/7 3.09 (0.87–10.93) 0.01 (0.00–0.54) P = 0.0003 P = 0.0798 P = 0.0264 OR (95% CI) for Aspirin intake within strata 5.68 (2.18–14.81) 4.69 (1.19–18.55) of genotype; p-value P = 0.0001 P = 0.0276 rs4311994 C > T 190/454 1.00 72/182 0.97 (0.67–1.40) 0.96 (0.66–1.40) −1.78 0.58 Aspirin intake (No) P = 0.8688 P = 0.8472 (−8.42–4.87) (0.06–5.46) Aspirin intake (Yes) 18/12 5.25 (2.15–12.82) 6/5 3.44 (0.84–14.07) 0.24 (0.01–4.57) P = 0.0003 P = 0.0855 P = 0.3399 OR (95% CI) for Aspirin intake within strata 5.40 (2.21–13.22) 3.56 (0.80–15.92) of genotype; p-value P = 0.0002 P = 0.096 rs10120688 G > A 135/305 1.00 127/331 0.94 (0.68–1.32) 0.95 (0.68–1.32) −2.76 0.49 Aspirin intake (No) P = 0.7369 P = 0.7454 (−11.41–5.90) (0.07–3.35) Aspirin intake (Yes) 10/5 6.52 (1.88–22.54) 14/12 3.70 (1.41–9.71) 0.44 (0.07–2.95) P = 0.0031 P = 0.0078 P = 0.4018 OR (95% CI) for Aspirin intake within strata 5.98 (1.73–20.63) 4.94 (1.77–13.73) of genotype; p-value P = 0.0047 P = 0.0022 rs4251961 T > C 115/305 1.00 147/331 1.20 (0.86–1.69) 1.21 (0.86–1.69) −2.59 0.54 Aspirin intake (No) P = 0.2780 P = 0.2751 (−10.37–5.18) (0.09–3.35) Aspirin intake (Yes) 13/8 6.43 (2.30–18.03) 11/9 4.05 (1.32–12.39) 0.52 (0.09–2.90) P = 0.0004 P = 0.0144 P = 0.4543 OR (95% CI) for Aspirin intake within strata 6.22 (2.19–17.68) 3.25 (1.04–10.08) of genotype; p-value P = 0.0006 P = 0.0418 rs3778355 G > C 133/326 1.00 129/3100 1.0 (0.72–1.40) 1.01 (0.72–1.40) -1.26 0.71 Aspirin intake (No) P = 0.9998 P = 0.9752 (-8.28–5.75) (0.11–4.56) Aspirin intake (Yes) 13/8 5.38 (1.85–15.65) 11/9 4.12 (1.41–12.00) 0.67 (0.11–4.17) P = 0.0020 P = 0.0095 P = 0.6697 OR (95% CI) for Aspirin intake within strata 5.80 (1.95–17.26) 3.93 (1.35–11.44) of genotype; p-value P = 0.0016 P = 0.0121 rs1330344 C > T 160/389 1.00 102/247 1.09 (0.78–1.54) 1.10 (0.78–1.55) -1.44 0.68 Aspirin intake (No) P = 0.6050 P = 0.5802 (−8.41–5.54) (0.09–4.98) Aspirin intake (Yes) 15/12 5.36 (2.13–13.49) 9/5 4.02 (1.11–14.54) 0.66 (0.11–3.83) P = 0.0004 P = 0.0341 P = 0.6408 OR (95% CI) for Aspirin intake within strata 5.38 (2.11–13.75) 3.42 (0.90–12.96) of genotype; p-value P = 0.0004 P = 0.0706 rs5275 A > G/A > T 119/292 1.00 129/338 1.0 (0.71–1.40) 1.0 (0.71–1.41) −1.66 0.65 Aspirin intake (No) P = 0.9964 P = 0.9966 (−9.93–6.62) (0.09–4.71) Aspirin intake (Yes) 7/6 5.72 (1.53–21.33) 15/10 4.06 (1.54–10.73) 0.24 (0.02–2.49) P = 0.0094 P = 0.0047 P = 0.2307 OR (95% CI) for Aspirin intake within strata 6.31 (1.71–23.24) 4.33 (1.62–11.59) of genotype; p-value P = 0.0056 P = 0.0036 rs3779647 (C > T) 129/323 1.0 133/313 1.07 (0.77–1.49); 1.07 (0.77–1.5); -1.57 0.66 Aspirin intake (No) p = 0.6930 p = 0.6733 (-8.56, 5.43) (0.1–4.41) Aspirin intake (Yes) 13/11 5.53 (2.08–14.69); 11/6 4.03 (1.24–13.12); 0.67 (0.11–4.24); p = 0.0006 p = 0.0205 p = 0.6722 OR (95% CI) for Aspirin intake within strata 6.10 (2.27–16.43); 3.61 (1.10–11.87); of genotype; p-value p = 0.0003 p = 0.0345 (*): Odds Ratio adjusted for: period of patients´ recruitment, previous history of arthrosis, infection with Helicobacter pylori, gastrointestinal disorders (ulcer and bleeding), exposure to NSAIDs except ASA, exposure to inhibitors of the proton pump, exposure to antiaggregant, exposure to anticoagulants, and the interview variables (the number and the reliability of the interview). Frontiers in Pharmacology | www.frontiersin.org 7 June 2020 | Volume 11 | Article 860 Mallah et al. Polymorphism, Aspirin, and Gastrointestinal Bleeding We also detected another group of nine SNPs “negative be further confirmed by (1) other studies with larger sample size modifiers” that were found to decrease the risk magnitude of before their application in clinical settings; and (2) meta-analysis aspirin-related UGIH. As opposed to the “positive modifiers” of other studies about modification effect from aspirin-SNPs SNPs, the genetic variants in this category might have a role in interaction on UGIH. Due to the limited sample size, we were enhancing platelets activity which would therefore aid in not able to determine the dose-response relationship as well as lowering the odds of occurrence of bleeding. In this regard, an the effect of some SNPs. In addition, we could not analyze the association between rs1131882, rs4311994 and rs3779647 and effect of carrying more than one SNP on aspirin-related UGIH increased platelet activity in diabetic patients on aspirin therapy due to the limited number of cases and/or controls in some has already been reported (Postula et al., 2011; Postula et al., combinations of aspirin exposure and genetic profile (data not 2013). Studies also indicated that the presence of genetic shown). In spite of these limitations this study can help designing variations in the COX-2 gene such as rs5275 boosts COX-2 future pharmacogenomic ones in order to better understand the expression and therefore aid in reducing the risk of myocardial interaction effect between genetics and aspirin use on UGIH. infarction and ischemia; cardiac conditions in which platelet In conclusion, our preliminary findings suggest that certain activation makes an important contribution (Haybar et al., genetic variations might modify the risk magnitude of aspirin- 2018). rs4251961 was associated with higher expression of related UGIH. Future studies with larger sample size and fibrinogen that plays a key role in hemostasis, and thus additional gene expression analyses are needed to confirm our participates in the prevention of bleeding (Wassel et al., 2011). results and explain the biological effect of these SNPs on aspirin- In addition, Postula and colleagues, reported that rs10120688 is related UGIH. If confirmed, these findings would have high associated with decreased platelets activity in aspirin users impact at the clinical and public health levels, because they will (Postula et al., 2011). In this study, we also found that aspirin permit personalized aspirin prescriptions to prevent diseases users who are carriers of rs10120688 are at an increased risk of with high incidence and mortality rates. UGIH. However, additionally, we showed that aspirin consumers who are carriers of the wild-type of rs10120688 are at higher risk of developing UGIH than the consumers who are carriers of the genetic variation. DATA AVAILABILITY STATEMENT In a similar fashion, previous reports also suggested that The datasets presented in this study can be found in online rs2243086, rs3778355 and rs1330344 are possible risk factors repositories. The names of the repository/repositories and of gastric mucosal injury induced by aspirin (Wu et al., 2016). In accession number(s) can be found below: https://doi.org/10. the present study, we also detected that carriers of these genetic 6084/m9.figshare.12030606.v1. variations are at increased odds of developing UGIH, and found that the risk magnitude is even higher in carriers of the wild- type genotypes. Our study has its strengths and limitations. A main strength ETHICS STATEMENT of this study is adjusting the measure of effect to factors that were The studies involving human participants were reviewed and known to increase the risk of gastrointestinal bleeding such as ́ rea De approved by Comité É tico de Investigació n Clı́ nica, A comedication with non-steroidal anti-inflammatory drugs, Salud Valladolid - ESTE (CEIC-VA-ESTE-HCUV; protocol proton-pump inhibitors, anticoagulants, and previous history number: PI-14-142) Comité É tico de Investigació n Clı́ nica De of bleeding or peptic ulcer. Memory bias was reduced by Galicia (CEIC-G; protocol number: 2013/263), Comité É tico de demonstrating prompt cards of the most frequent aspirin Investigació n Clı́ nica De Euskadi (CEIC-E; protocol number: commercial boxes to the participants. The exclusion of non- PI2013101), and Comité É tico de Investigació n Clı́ nica De white patients also allowed to prevent bias due to racial Barcelona (CEIC; protocol number: Es38121226Z). The differences between populations. Moreover, conducting the patients/participants provided their written informed consent study in biologically unrelated patients, exclusively, avoided to participate in this study. ascertainment bias due to over-representation of SNPs within families (Malomane et al., 2018). Nonetheless, due to the genetic differences between races, our results cannot be generalized to populations that are not Caucasian. Another major limitation of AUTHOR CONTRIBUTIONS our study is the insufficient sample size. This caused the 95% confidence intervals of RERI and S to be very wide, and CA, LI, XV, and AF conceived the research idea, designed the consequently the null hypothesis could not be rejected. This study and supervised and administered the project. NM did the occurred despite the fact that we present the largest case-control literature review and conceptualized and wrote the manuscript. on this topic. Indeed, low statistical power is a common NM and MP-L analyzed the data. NM interpreted the data and limitation across candidate gene studies (Salanti et al., 2005; participated in the genetic laboratory testing. AF supervised the Dumas-Mallet et al., 2017). We therefore consider that our data analysis and interpretation. MZ-C, EI-G, IP–Z, FM-G, findings should be treated as preliminary ones and they should JD-M, LV, LM-A, MS-G, and VV-G were involved in patients’ Frontiers in Pharmacology | www.frontiersin.org 8 June 2020 | Volume 11 | Article 860 Mallah et al. Polymorphism, Aspirin, and Gastrointestinal Bleeding recruitment and data registration. All authors contributed to the service was carried out at CEGEN-PRB3-ISCIII; Instituto de article and approved the submitted version. Salud Carlos III and ERDF (PT17/0019, of the PE I+D+i 2013-2016). FUNDING This work was supported by a grant from Instituto de Salud ACKNOWLEDGMENTS Carlos III (PI12/02414)/Plan Estatal de I+D+I 2012-2016; The authors would like to thank the health personnel who helped Fondo Europeo de Desarrollo Regional (FEDER); the in recruiting and interviewing the patients. Novartis, Pfizer and Dr Esteve pharmaceutical companies; the Health Research Fund/Fondo de Investigació n Sanitaria (PI021512, PI021364, PI020661, and PI021572); Ministry of Health & Consumer Affairs, Spain (SAF2002-04057); Galician SUPPLEMENTARY MATERIAL Regional Authority, Spain (PGIDIT03PXIC20806PN); Department of Health of the Basque Country (03/11092 and The Supplementary Material for this article can be found online 11/111103); Fundacion Vasca de innovacion e investigacion at: https://www.frontiersin.org/articles/10.3389/fphar.2020. sanitarias (OSIBG19/002 and OSIBG18/105). The genotyping 00860/full#supplementary-material REFERENCES Haybar, H., Khodadi, E., Zibara, K., and Najmaldin, S. (2018). Platelet Activation Polymorphisms in Ischemia. Cardiovasc. Hematol. Disord. Drug Targets 18, Andersen, V., Nimmo, E., Krarup, H. B., Drummond, H., Christensen, J., Ho, G. T., 153–161. doi: 10.2174/1871529X18666180326121239 et al. (2011). 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Suppl 4, Conflict of Interest: The authors declare that the research was conducted in the 47–52. doi: 10.1111/jgh.12770 absence of any commercial or financial relationships that could be construed as a Sutcliffe, P., Connock, M., Gurung, T., Freeman, K., Johnson, S., Kandala, N. B., potential conflict of interest. et al. (2013). Aspirin for prophylactic use in the primary prevention of cardiovascular disease and cancer: a systematic review and overview of Copyright © 2020 Mallah, Zapata-Cachafeiro, Aguirre, Ibarra-Garcıa,́ Palacios–Zabalza, reviews. Health Technol. Assess. 17, 1–253. doi: 10.3310/hta17430 ́ Macıas-Garcı a,́ Domınguez-Muñoz, ́ Piñeiro-Lamas, Ibáñez, Vidal, Vendrell, Martin-Arias, Thorat, M. A., and Cuzick, J. (2015). Prophylactic Use of Aspirin: Systematic Sáinz-Gil, Velasco-González and Figueiras. This is an open-access article distributed under Review of Harms and Approaches to Mitigation in the General Population. the terms of the Creative Commons Attribution License (CC BY). The use, distribution or Eur. J. Epidemiol. 30, 5–18. doi: 10.1007/s10654-014-9971-7 reproduction in other forums is permitted, provided the original author(s) and the copyright Wassel, C. L., Lange, L. A., Keating, B. J., Taylor, K. C., Johnson, A. D., Palmer, C., owner(s) are credited and that the original publication in this journal is cited, in accordance et al. (2011). Association of genomic loci from a cardiovascular gene SNP array with accepted academic practice. No use, distribution or reproduction is permitted which with fibrinogen levels in European Americans and African-Americans from six does not comply with these terms. Frontiers in Pharmacology | www.frontiersin.org 10 June 2020 | Volume 11 | Article 860