ANNALS OF MEDICINE 2022, VOL. 54, NO. 1, 379–392 https://doi.org/10.1080/07853890.2021.2016940 ORIGINAL ARTICLE Synergism interaction between genetic polymorphisms in drug metabolizing enzymes and NSAIDs on upper gastrointestinal haemorrhage: a multicenter case-control study Narmeen Mallaha,b,c,d, Maruxa Zapata-Cachafeiroa,e, Carmelo Aguirref,g,h, Eguzkin ~e Ibarra-Garcıaf,i, f,g j ~eiro-Lamas , Luisa Iban Itziar Palacios-Zabalza , Fernando Macıas-Garcıa , Marıa Pin e,k ~ezl, Xavier Vidall, Lourdes Vendrell , Luis Martin-Arias , Marıa Sainz-Gil , Vero l m m nica Velasco-Gonzalez , m Manuel Bacariza-Cortin ~asn, Angel Salgadoa, Ana Estany-Gestalk and Adolfo Figueirasa,e,k; on behalf of the EMPHOGEN Group a Department of Preventive Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain; bWHO Collaborating Centre for Vaccine Safety, Santiago de Compostela, Spain; cGenetics, Vaccines and Pediatric Infectious Diseases Research Group (GENVIP), Instituto de Investigacion Sanitaria de Santiago de Compostela, Santiago de Compostela, Spain; dCentro de Investigacion Biomedica en Red de Enfermedades Respiratorias (CIBER-ES), Carlos III Health Institute, Madrid, Spain; eConsortium for Biomedical Research in Epidemiology and Public Health (CIBER en Epidemiologıa y Salud P ublica-CIBERESP), Carlos III Health Institute, Madrid, Spain; fPharmacotherapy Group, Biocruces Bizkaia Health Research Institute, Barakaldo, Spain; gBasque Country Pharmacovigilance Unit, University Hospital of Galdakao-Usansolo, Osakidetza, Spain; hPharmacology Department, Medicine and Nursing Faculty, University of the Basque Country, Barakaldo, Spain; iOsakidetza Basque Health Service, Pharmacy Department, Urduliz Hospital, Urduliz, Spain; jDepartment of Gastroenterology and Hepatology, University Hospital of Santiago de Compostela, Santiago de Compostela, Spain; kHealth Research Institute of Santiago de Compostela (IDIS), Santiago de Compostela, Spain; lDepartment of Pharmacology, Therapeutics and Toxicology, Catalonian Institute of Pharmacology, Clinical Pharmacology Service, Vall d’Hebron University Teaching Hospital, Autonomous University, Barcelona, Spain; mCentre for Research on Drug Safety (CESME), Valladolid University, Valladolid, Spain; nCentro de Saude de Vite, Santiago de Compostela, Spain ABSTRACT ARTICLE HISTORY Background: Interindividual genetic variations contribute to differences in patients’ response to Received 26 August 2021 drugs as well as to the development of certain disorders. Patients who use non-steroidal anti- Revised 24 November 2021 inflammatory drugs (NSAIDs) may develop serious gastrointestinal disorders, mainly upper gastro- Accepted 5 December 2021 intestinal haemorrhage (UGIH). Studies about the interaction between NSAIDs and genetic varia- KEYWORDS tions on the risk of UGIH are scarce. Therefore, we investigated the effect of 16 single nucleotide Aspirin; genetic variation; polymorphisms (SNPs) involved in drug metabolism on the risk of NSAIDs-induced UGIH. interaction; non-steroidal Materials and methods: We conducted a multicenter case-control study of 326 cases and 748 anti-inflammatory drugs; controls. Participants were sub-grouped into four categories according to NSAID exposure and upper gastrointestinal genetic profile. We estimated odds ratios (ORs) and their 95% confidence intervals (CI) using haemorrhage generalized linear mixed models for dependent binomial variables and then calculated the measures of interaction, synergism index (S), and relative excess risk due to interaction (RERI). We undertook stratified analyses by the type of NSAID (aspirin, non-aspirin). Results: We observed an excess risk of UGIH due to an interaction between any NSAID, non- aspirin NSAIDs or aspirin and carrying certain SNPs. The greatest excess risk was observed for car- riers of: rs2180314:C>G [any NSAID: S ¼ 3.30 (95%CI: 1.24–8.80), RERI ¼ 4.39 (95%CI: 0.70–8.07); non-aspirin NSAIDs: S ¼ 3.42 (95%CI: 1.12–10.47), RERI ¼ 3.97 (95%CI: 0.44–7.50)], and rs4809957:A>G [any NSAID: S ¼ 2.11 (95%CI: 0.90–4.97), RERI ¼ 3.46 (95%CI: 0.40–7.31)]. Aspirin use by carriers of rs6664:C>T is also associated with increased risk of UGIH [ORaspirin(þ),wild-type: 2.22 (95%CI: 0.69–7.17) vs. ORaspirin(þ),genetic-variation: 7.72 (95%CI: 2.75–21.68)], yet larger sample size is needed to confirm this observation. Conclusions: The joint effect of the SNPs s2180314:C>G and rs4809957:A>G and NSAIDs are more than three times higher than the sum of their individual effects. Personalized prescriptions based on genotyping would permit a better weighing of risks and benefits from NSAID consumption. KEY MESSAGES  Multicenter case-control study of the effect of genetic variations involved in drug metabolism on upper gastrointestinal haemorrhage (UGIH) induced by NSAIDs (aspirin and non-aspirin).  There is a statistically significant additive synergism interaction between certain genetic poly- morphisms and NSAIDs on UGIH: rs2180314:C>G and rs4809957:A>G. The joint effect of CONTACT Narmeen Mallah narmeen.mallah@usc.es Department of Preventive Medicine, Faculty of Medicine, University of Santiago de Compostela, c/San Francisco s/n., 15 786 Santiago de Compostela, A Coru~ na, Spain ß 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 380 N. MALLAH ET AL. each of these single nucleotide polymorphisms and NSAIDs on UGIH is more than three times higher than the sum of their individual effects.  Genetic profiling and personalized prescriptions would be useful in managing the risks and benefits associated with NSAIDs. 1. Introduction polymorphisms involved in drug metabolism on the risk of NSAIDs-related UGIH. As a secondary objective Adverse effects of non-steroidal anti-inflammatory of this study, we investigated the modification effect drugs (NSAIDs) were associated with heavy health and of those 16 genetic polymorphisms on the risk of non- economic burdens [1,2]. Upper gastrointestinal haem- aspirin NSAIDs-related UGIH as well as on aspirin- orrhage (UGIH) is a frequent adverse effect of NSAID related UGIH. treatment that can be life-threatening [3]. Nonetheless, NSAIDs continue to be the most pre- scribed drugs worldwide [4]. 2. Materials and methods Furthermore, it is well-established that aspirin plays 2.1. Study settings and design an important prophylactic role against highly incident diseases that are associated with elevated mortality This study represents a continuation of a previous full rates, such as several types of cancer and cardiovascu- case-control study (i.e. case-control encompassing lar events [5–12]. Nevertheless, the association of exposed and non-exposed patients to NSAIDs, on the aspirin with gastrointestinal bleeding has discouraged contrary to other partial case-control studies that the adoption of this drug as a general prophylactic include only exposed patients) [30], published else- measure against disorders with great public health where, and shares the same protocol [31,32]. Patients impact [13]. In addition, gastrointestinal symptoms in were recruited from four hospitals in Spain (Barcelona, patients who used aspirin to protect against cardiovas- Galdakao, Santiago de Compostela, and Valladolid), cular events had led to treatment interruption [14], between January 2004 and November 2007 and and consequently to an increase in cardiovascular between January 2013 and October 2015. The study risk [15,16]. protocol was approved by the ethics committee of Marked interindividual differences with respect to each participating centre (Barcelona: CEIC protocol their response to NSAIDs have long been recognized number: Es38121226Z; Euskadi: CEIC-E protocol num- and attributed to many factors including genetic varia- ber: PI2013101; Galicia: CEIC-G protocol number: 2013/ tions in metabolizing enzymes [17–20]. Several studies 263 and Valladolid: CEIC-VA-ESTE-HCUV protocol num- also reported a possible relationship between genetic ber: PI-14-142). The participants provided written variations in users of NSAIDs and gastrointestinal dis- informed consent before enrolment in the study. orders [21–27]. In this context, genetic pharmacoki- netic factors are of special importance since variations 2.2. Definition of cases and controls in genes involved in drug metabolism might alter their expression and thus increase the risk of undesirable Cases were patients admitted to the hospital with effects like bleeding and cardiovascular events. symptoms of UGIH that were diagnosed surgically or Therefore, identifying patients at risk of UGIH based endoscopically. Eligible cases were included irrespect- on their genetic background and personalized NSAID ive of the grade of UGIH severity. prescriptions might help weigh the risks and benefits For each case, controls matched by the hospital, associated with each type of NSAIDs and thus avoid gender, and age (±5 years) were selected. To avoid adverse effects in susceptible individuals. selection bias due to excessive intake of NSAIDs, con- Currently, there is a lack of knowledge about the trols were either outpatients or patients enrolled from effect of variations in genes involved in drug metabol- the preoperative unit among subjects who were about ism on the risk of UGIH in general, and in NSAID users to undergo any of the following non-painful mild sur- in specific. Taking into account the considerable mor- geries which were unrelated to the use of NSAIDs: bidity and mortality rates of UGIH [28,29], and the plastic surgery, inguinal or umbilical hernia (strangu- wide spectrum of NSAID benefits, we carried out a lated or programmed), lipoma, varicotomy, prostatic multicenter case-control study that primarily aimed at adenoma, prostatic hyperplasia, thyroid nodules and testing the modification effect of 16 genetic thyroglossal cyst (euthyroid), eye cataract, phimosis, ANNALS OF MEDICINE 381 Table 1. Motives of the exclusion of cases and controls from the study. Reasons of exclusion† EMPHOGEN I (2004–2007) EMPHOGEN II (2013–2015) CASES (N ¼ 3731) 3120 611 Primary exclusions (N ¼ 2655) 2147 508 Age < 18 31 2 Excludable endoscopic diagnosis‡ 1213 377 History of UGIH 121 18 Intrahospital UGIH 89 5 UGIH without endoscopic or surgical diagnosis from admission to discharge 121 3 Nasogastric or percutaneous tube carrier 75 2 <3 months’ residence in study area 42 7 Admission time <24 h 208 8 Admission not due to UGIH 154 80 Death 0 2 Other 93 4 Secondary exclusions (N ¼ 744) 646 98 Refusal to sign informed consent form 21 0 Occurred at weekend or vacations period 57 21 Death 11 2 Endoscopy performed more than 48 h after admission 83 39 Discharge from hospital or visit to healthcare facility in the 15 days prior to admission 54 20 Severe condition 7 1 Psychological disorders 12 4 Illiterate 2 0 Deaf or blind 1 0 Lives in a residence or closed institution and does not know the drugs taken 7 1 Refusal to answer or failure to complete the interview 12 5 Impossible to conduct interview within the 15-day period preceding admission 6 4 Admission time <24 h 0 1 Other 373 0 Excluded from analysis (N ¼ 332) 327 5 Non-white patients 4 0 Unavailable biological material 323 5 CONTROLS (N ¼ 1073) 1071 2 Refused to sign informed consent form 45 0 Age < 18 1 0 History of disease 11 1 Intrahospital UGIH 89 0 Nasogastric or percutaneous tube carrier 2 0 <3 months’ residence in study area 1 0 Severe condition 1 0 Psychological disorders 1 0 Deaf or blind 3 0 Refusal to answer or failure to complete the interview 80 0 Impossible to conduct interview within the 15-day period preceding admission 60 0 Date of last admission 0 1 Other 13 0 Non-white patients 15 0 Unavailable biological material 749 0 † Cases and controls were excluded upon presenting one or more exclusion criteria. ‡ Excludable endoscopic diagnosis included gastritis, esophagitis, oesophageal varices, gastric or duodenal neoplasia, Mallory-Weiss syndrome, angiodys- plasia, anastomotic ulcers, diverticulitis, acute alcohol intoxication, hiatal hernia, and papule. Bold values represent the total per reason of exclusion group and study period. ear pinning, vocal cord cyst, tubal ligation, and The inclusion and exclusion criteria of the cases and septoplasty. controls are specified in more detail in Table 1. To ensure that all subjects belong to the same source of population, they were recruited from 2.3. Data collection patients and outpatients attended by the same hospi- tals. All patients were biologically unrelated. The ana- Both cases and controls were thoroughly interviewed lysis was restricted to European participants to control by trained health personnel, using a questionnaire for the risk of stratification bias [33]. We used the specifically designed for this study. The collected data native language of the participants and their parents include participants’ sociodemographic characteristics, as a proxy of ethnicity [34–37]. Patients with a history clinical antecedents, smoking habits, alcohol and caf- of neoplasia, liver cirrhosis, or coagulopathy were feine consumption, the motive for hospital admission, excluded to control for the risk of Berkson’s bias [38]. underlying symptomatology (for cases), the motive for 382 N. MALLAH ET AL. the scheduled surgery (for controls), previous episodes H. pylori IgG EIA (ref: 2346400, Trinity Biotech Captia, of gastric diseases, and exposure to pharmaceutical Co. Wicklaw, Ireland), and following the manufacturer’s drugs (including the medicine’s daily dose and indica- protocol. The participants were inquired if they had tion). Direct relatives or healthcare assistants, who previously been treated against H. pylori infection to took care of the patient’s medication, could attend avoid any false-positive results caused by and participate in the interview, but only data con- old infections. firmed by the patient were considered. When the par- ticipant was not able to remember any of the 2.6. Single nucleotide polymorphisms (SNPs) requested information, the interview was repeated on selection and genotyping a posterior date, or the patient was contacted by tele- phone if s/he had been discharged from the hospital. A comprehensive list of SNPs involved in gastrointes- In case the patient doubted or was uncertain about tinal disorders (bleeding or ulcer) was retrieved by specific information, that information was confirmed reviewing research reports published in MEDLINE until later by consulting the medical records of the patient. April 2017. The reference numbers (rs number) of the Index dates were established to ascertain any selected SNPs were confirmed using PubMed [42]. exposure to NSAIDs. Information on NSAID exposure Subsequently, the function of the corresponding was extracted from patients’ medical records, but the genes and the clinical significance of the genetic varia- researchers were blind to patients’ use of NSAIDs. For tions were identified through a literature review. the cases, the index date was the day of onset of the Finally, SNPs in genes that may influence drug metab- first signs or symptoms of UGIH, while for the controls olism were selected for genotyping [25,26]. it was the day of the interview. NSAIDs exposure was DNA was extracted from blood stored in EDTA considered if the consumption took place in the week tubes using chemagicTM DNA Buffy Coat 200 Kit H96 preceding the index date [39–41]. For ease of recall, a (PerkinElmer, reference number CMG-713) and from catalog of prompt cards of the most consumed NSAID blood spots using chemagicTM DNA Blood 200 Kit H96 boxes was shown to the participants during (PerkinElmer, reference number CMG-717). Extracted the interview. DNA was then quantified using Quant-iTTM The reliability of the interview was rated on a scale PicoGreenTM dsDNA Assay Kits (ThermoFisher of 0–10 as perceived by the interviewer, where zero Scientific, reference number P7589). DNA concentra- means that the answers provided by the patient were tion was normalized at 10–20 ng/ml in a minimum completely unreliable. Patients whose interview was total volume of 40 ml. Samples were genotyped in a phonotype-blind process. iPlexV Gold chemistry and R rated by zero were excluded from the study. A 5 ml blood sample was withdrawn from each par- MassARRAY platform were used according to the man- ticipant and stored in EDTA tubes or as spots on ufacturer’s instructions (Agena Bioscience, San Diego, IsoCode papers at 80  C until genotyping. USA). Genotyping assays were designed using the Agena Bioscience MassARRAY Assay Designer 4.1 soft- ware. All assays were performed in 384-well plates, 2.4. Risk factors associated with UGIH including negative controls and a trio of Coriell sam- The following co-variables which were known to affect ples for quality control. The reproducibility of 7% of the risk of UGIH were considered: (1) previous infec- the samples was also checked between and/or tion with Helicobacter pylori; (2) therapeutic groups, within plates. such as proton pump inhibitors or oral anticoagulants; The compliance of the SNPs with Hardy–Weinberg (3) digestive system disorders classified according to equilibrium was checked using the SNPassoc Library the patient’s history of ulcer and bleeding (none or of the R package (Version 1.9-2) [43–45]. In addition, dyspepsia; ulcer; or bleeding); and (4) the reliability of all cluster plots were manually inspected by trained the interview. personnel using MassArray Typer software. 2.5. Helicobacter pylori determination 2.7. Statistical analysis The presence of anti-H. pylori IgG antibodies in human To determine any interaction between each of the 16 serum were determined using the commercial ELISA SNPs and NSAID exposure on the risk of UGIH, partici- kits: Human Anti-Helicobacter pylori IgG ELISA Kit pants were grouped according to their genotype and (ab108736, Abcam, Cambridge, England), and CaptiaTM NSAID exposure. Stratified analysis by the type of ANNALS OF MEDICINE 383 Figure 1. Flow of the cases and the controls throughout the two stages of the project. NSAID (any NSAID, non-aspirin NSAIDs, and aspirin) 3. Results was carried out. In each analysis, the following four 3.1. Clinical data collection groups of participants were obtained: [group 1: drug(þ), wild-type; group 2: drug(þ), genetic-variation; One thousand and seventy-four patients (326 cases group 3: drug(), genetic-variation; and group 4: and 748 controls) fulfilled the inclusion criteria and drug(), wild-type]. Adjusted odds ratios (ORs) of were included in the final analysis. The flow of UGIH were calculated in each group and then checked subjects and the motives of exclusion are pre- for any potential interaction between the presence of sented in Figure 1 and Table 1. The patients’ a genetic variation and drug exposure. The group of demographic and clinical characteristics are pre- subjects who were not exposed to the studied drug sented in Table 2. category (any NSAID, non-aspirin NSAIDs, or aspirin) and who were carriers of the wild-type genotype of 3.2. Genotyping the analyzed SNP (group 4) was used as the reference category for the estimations of the interactions. All genotyped samples were included in the analysis. ORs and their 95% confidence intervals (CI) were The reproducibility of the 7% replicated random sam- estimated by generalized linear mixed models for ples was 100%. All SNPs showed an acceptable geno- dependent binomial variables [46]. In the construction type call rate: 98%. Both the calculations of the of the models, patients were placed at level 1; the Hardy–Weinberg equilibrium (p < .001) and the man- strata (each case and its matched controls) at level 2; ual inspection of the cluster plots confirmed that the the hospital at level 3; and the period of patients’ controls were in equilibrium in terms of the corre- recruitment at level 4. A random-effects model was sponding polymorphisms (Table 3). used to examine the effect of the patients’ recruitment period, and a nested random-effects model was 3.3. Risk estimation and modification of effect applied for the strata of cases and controls and health centre. The lmer function of the lme4 R package (ver- The odds of UGIH varied according to the genotype sion 1.1-21) was applied in the estimation of the mod- and NASID (aspirin or non-apsirin) exposure. els [47]. Potential confounding variables were introduced in the model if they modified the OR of 3.3.1. Genotypes associated with high excess of risk the main variable by at least 10% and provided that of UGIH the Schwartz’s Bayesian Information Criterion The presence of certain genetic variations increases improved [48]. the odds of UGIH in users of any NSAID, non-aspirin The recommendations given by Knol and col- NSAIDs, or aspirin as compared to users with wild- leagues were followed to explore any potential inter- type genotypes (Table 4). action between NSAIDs and genetic polymorphisms, rs2180314:C>G: Any NSAID use by carriers of whereby we estimated the relative excess risk due to rs2180314:C>G is associated with substantially higher interaction (RERI) and the synergism index (S) along odds of UGIH in comparison with NSAID users carrying with their 95% CI [49–52]. the wild-type genotype [ORdrug(þ),wild-type: 3.17 (95%CI: 384 N. MALLAH ET AL. Table 2. Description of the cases and controls included in the study. Cases (N ¼ 326) Controls (N ¼ 748) Characteristic @ (%) @ (%) OR (95% CI) p-Value Age <45 41 (12.6%) 95 (12.7%) 1 45–65 117 (35.9%) 271 (36.2%) 1.05 (0.68–1.63) .8327 >65 161 (49.4%) 370 (49.5%) 1.01 (0.66–1.54) .9757 missing 7 (2.1%) 12 (1.6%) BMI Underweight 10 (3.1%) 24 (3.2%) 0.74 (0.33–1.62) .4588 Normal weight 114 (35.0%) 204 (37.3%) 1 Overweight 128 (39.3%) 374 (50.0%) 0.61 (0.45–0.83) .0201 Obese 68 (20.9%) 144 (19.3%) 0.85 (0.58–1.24) .8676 Missing 6 (1.8%) 2 (0.3%) Gender Male 236 (72.4%) 559 (74.7%) 1 Female 87 (26.7%) 189 (25.3%) 1.18 (0.87–1.6) .2852 Missing 3 (0.9%) 0 Arthrosis No 219 (67.2%) 469 (62.7%) 1 Yes 86 (26.4%) 216 (28.9%) 0.86 (0.63–1.17) .3303 Missing 21 (6.4%) 63 (8.4%) Helicobacter pylori No or uncertain 27 (8.3%) 138 (18.4%) 1 Yes 276 (84.7%) 574 (76.7%) 2.54 (1.62–3.99) <.0001 Missing 23 (7.1%) 36 (4.8%) Source of information Patients 259 (79.4%) 672 (89.8%) 1 Healthcare assistant/direct relative 67 (20.6%) 76 (10.2%) 2.35 (1.62–3.42) <.0001 Interview variables Number of interviews conducted 1 274 (84.0%) 644 (86.1%) 1 2 52 (16.0%) 104 (13.9%) 1.27 (0.81–1.99) .2927 Reliability of the interview <5 13 (4.0%) 20 (2.7%) 1 5–7 36 (11%) 78 (10.4%) 0.67 (0.29–1.54) .3466 7–9 134 (41.1%) 310 (41.4%) 0.70 (0.33–1.48) .3540 9 143 (43.9%) 340 (45.5%) 0.67 (0.31–1.41) .2855 Personal history of gastrointestinal disorders None or dyspepsia 208 (63.8%) 647 (86.5%) 1 Ulcer 48 (14.7%) 56 (7.5%) 2.74 (1.79–4.21) <.0001 Bleeding 70 (21.5%) 45 (6.0%) 4.79 (3.15–7.27) <.0001 Co-medications with drugs that are not NSAIDs Analgesics not narcotics No 272 (83.4%) 692 (92.5%) 1 Yes 54 (16.6%) 56 (7.5%) 2.74 (1.81–4.15) <.0001 Inhibitors of the proton pump No 290 (89.0%) 682 (91.2%) 1 Yes 36 (11.0%) 66 (8.8%) 1.2 (0.77–1.88) .4202 Antiaggregant No 261 (80.1%) 662 (88.5%) 1 Yes 65 (19.9%) 86 (11.5%) 1.94 (1.34–2.8) .0005 Anticoagulants No 291 (89.3%) 716 (95.7%) 1 Yes 35 (10.7%) 32 (4.3%) 3.09 (1.85–5.15) <.0001 Inhibitors of COX2 No 323 (99.1%) 742 (99.2%) 1 Yes 3 (0.9%) 6 (0.8%) 0.88 (0.21–3.77) .8659 1.79–5.63) vs. ORdrug(þ),genetic variation: 7.30 (95%CI: statistical significance in aspirin category probably due 4.27–12.48)]. The measures of interaction showed a to the limited number of aspirin users. statistically significant high excess risk of UGIH from rs4809957:A>G: Substantially higher ORs of the interaction between NSAID and rs2180314:C>G UGIH were observed for patients carrying [S ¼ 3.30 (95%CI: 1.24–8.80), RERI ¼ 4.39 (95%CI: rs4809957:A>G who are on treatment involving any 0.70–8.07)]. Similar findings were observed when the NSAID [ORwild-type: 4.12 (95%CI: 2.18–7.79) vs. analysis was stratified by the type of NSAID: non- ORgenetic-variation: 7.57 (95%CI: 4.43–12.93)], or non- aspirin NSAIDs [S ¼ 3.42 (95%CI: 1.12–10.47), RERI ¼ aspirin NSAID [ORwild-type: 3.99 (95%CI: 2.06–7.75) vs. 3.97 (95%CI: 0.44, 7.50)] and aspirin [S ¼ 7.65 (95%CI: ORgenetic-variation: 7.15 (95%CI: 4.10–12.46] in com- 0.81, 72.33), RERI ¼ 8.39 (95%CI: 4.20, 20.99)], parison with drug users carriers of the wild type though the interaction estimates did not reach genotype (Table 4). This excess in risk is suggested ANNALS OF MEDICINE 385 Table 3. Prevalence of the studied genotypes and Hardy–Weinberg equilibrium test. Single nucleotide polymorphism Cases Controls Hardy–Weinberg Gene reference number Genotypes N (%) N (%) equilibrium p-value CYP4F11, cytochrome P450 family 4 subfamily F member 11 rs1060463 CC 64 (19.6) 127 (17.0) 0.03 CT 165 (50.6) 398 (53.2) TT 97 (29.8) 223 (29.8) CYP2A6, cytochrome P450 family 2 subfamily A member 6 rs28399433 AA 288 (88.3) 662 (88.5) Not applicable AC 36 (11.0) 80 (10.7) CYP2B6, cytochrome P450 family 2 subfamily B member 6 rs36079186 TT 326 (100.0) 748 (100.0) Not applicable CYP4F11, cytochrome P450 family 4 subfamily F member 11 rs3765070 AA 65 (19.9) 128 (17.1) 0.03 AG 165 (50.6) 398 (53.2) GG 96 (29.4) 222 (29.7) CYP2A7, cytochrome P450 family 2 subfamily A member 7 rs3869579 AA 94 (28.8) 214 (28.6) 0.05 AG 147 (45.1) 346 (46.3) GG 85 (26.1) 188 (25.1) CYP11B2, cytochrome P450 family 11 subfamily B member 2 rs4536 CT 7 (2.1) 22 (2.9) Not applicable TT 318 (97.5) 725 (96.9) CYP24A1, cytochrome P450 family 24 subfamily A member 1 rs4809957 AA 200 (61.3) 450 (60.2) 0.09 AG 108 (33.1) 271 (36.2) GG 18 (5.5) 27 (3.6) CYP2F1, cytochrome P450 family 2 subfamily F member 1 rs58285195 CC 2 (0.6) 1 (0.1) 0.62 CT 29 (8.9) 51 (6.8) TT 295 (90.5) 696 (93.0) GSTP1, glutathione S-transferase pi 1 rs1695 AA 132 (40.5) 321 (42.9) 0.52 AG 157 (48.2) 332 (44.4) GG 37 (11.3) 95 (12.7) GSTA2, glutathione S-transferase alpha 2 rs2180314 CC 50 (15.3) 104 (13.9) 0.13 CG 139 (42.6) 318 (42.5) GG 129 (39.6) 309 (41.3) GSTA1, glutathione S-transferase alpha 1 rs4715332 AA 104 (31.9) 259 (34.6) 0.29 AC 159 (48.8) 374 (50.0) CC 63 (19.3) 114 (15.2) GSTA5, glutathione S-transferase alpha 5 rs4715354 AA 55 (16.9) 143 (19.1) 0.71 AG 160 (49.1) 362 (48.4) GG 110 (33.7) 243 (32.5) NAT2, N-acetyltransferase 1 rs1799931 AA 1 (0.3) 1 (0.1) 0.45 AG 16 (4.9) 40 (5.3) GG 309 (94.8) 707 (94.5) CHST2, carbohydrate sulfotransferase 2 rs6664 CC 177 (54.3) 419 (56.0) 0.34 CT 128 (39.3) 275 (36.8) TT 21 (6.4) 54 (7.2) ALB_c, albumin rs3756067 AA 37 (11.3) 91 (12.2) 0.33 AG 136 (41.7) 320 (42.8) GG 145 (44.5) 332 (44.4) SLCO3A1, solute carrier organic anion transporter rs2283458 AA 33 (10.1) 100 (13.4) 1.00 family member 3A1 AG 169 (51.8) 348 (46.5) GG 124 (38.0) 300 (40.1) by the interaction estimates which are on the bor- variation were at higher odds of UGIH than carriers of derline of statistical significance: any NSAID the wild-type genotype. A moderate non-statistically [S ¼ 2.11 (95%CI: 0.9–4.97); RERI ¼ 3.46 (95%CI: significant excess risk was observed for the presence 0.40–7.31)], non-aspirin NSAIDs [S ¼ 2.03 (95%CI: of these genetic variants: rs4715332:C>A [any NSAID 0.81–5.08); RERI ¼ 3.11 (95%CI: 0.82–7.05)]. The (S ¼ 1.64; RERI ¼ 2.34), non-aspirin NSAIDs (S ¼ 1.75; interaction estimates for aspirin exposure— RERI ¼ 2.25) and aspirin (S ¼ 1.61; RERI ¼ 1.99)] and rs4809957:A>G are inconclusive due to the limited rs4715354:G>A [any NSAID (S ¼ 1.37; RERI ¼ 1.53), number of observations (Table 4). non-aspirin NSAIDs (S ¼ 1.30; RERI ¼ 1.16) and aspirin (S ¼ 1.26; RERI ¼ 0.88)] (Table 4). 3.3.2. Genotypes associated with moderate excess Similar observations were observed for aspirin users of risk of UGIH carrying rs6664:C>T. Aspirin users carrying this genetic An increased odds of UGIH was observed from any variant had substantially higher odds of UGIH in com- NSAID, non-aspirin NSAIDs, or aspirin intake by both parison with patients carrying the wild-type genotype the carriers of the genetic variants (rs4715332:C>A [ORwild-type: 2.22 (95%CI: 0.69–7.17) vs. ORgenetic-variation: and rs4715354:G>A) or their corresponding wild-type 7.72 (95%CI: 2.75–21.68)]. Nonetheless the number of genotype (Table 4). However, carriers of the genetic aspirin users in this subgroup was limited which Table 4. Odds ratios (OR) for UGIH stratified by patients’ genotype and NSAID (any NSAID, aspirin, non-aspirin) exposure and their interaction represented by synergism index 386 (S) and relative excess risk due to interaction (RERI). Wildtype genotype Genetic variation SNP (reference number) N (%) (cases/controls) OR† (95% CI); p-value N (%) (cases/controls) OR† (95% CI); p-value RERI (95% CI) S (95% CI) rs2180314:C > G Any NSAID (No) 91 (26.1)/258 (73.9) 1 99 (21.4)/363 (78.6) 0.74 (0.50, 1.09); p ¼ .1229 4.39 (0.70, 8.07) 3.3 (1.24, 8.8) Any NSAID (Yes) 48 (44.4)/60 (55.6) 3.17 (1.79, 5.63); p ¼ .0001 80 (61.5)/50 (38.5) 7.30 (4.27, 12.48); p < .0001 Non-aspirin NSAID (No) 100 (27.2)/267 (72.8) 1 110 (22.9)/370 (77.1) 0.75 (0.52, 1.09); p ¼ .1343 3.97 (0.44, 7.50) 3.42 (1.12, 10.47) N. MALLAH ET AL. Non-aspirin NSAID (Yes) 39 (43.3)/51 (56.7) 2.89 (1.57, 5.31); p ¼ .0006 69 (61.6)/43 (38.4) 6.61 (3.82, 11.46); p < .0001 Aspirin intake (No) 115 (30.4)/263 (69.6) 1.0 142 (28.4)/358 (71.6) 0.92 (0.66–1.30); p ¼ .6481 8.39 (4.20, 20.99) 7.65 (0.81–72.33) Aspirin intake (Yes) 10 (47.6)/11(52.4) 2.34 (0.84–6.49); p ¼ .1031 14 (70.0)/6 (30.0) 10.65 (3.27–34.71); p ¼ .0001 rs4809957:A > G Any NSAID (No) 68 (23.4)/222 (76.6) 1 127 (23.5)/414 (76.5) 0.99 (0.66, 1.47); p ¼ .9515 3.46 (0.40, 7.31) 2.11 (0.9, 4.97) Any NSAID (Yes) 40 (44.9)/49 (55.1) 4.12 (2.18, 7.79); p < .0001 91 (59.1)/63 (40.9) 7.57 (4.43, 12.93); p < .0001 Non-aspirin NSAID (No) 74 (24.3)/230 (75.7) 1 141 (25.0)/422 (75.0) 1.04 (0.71, 1.53); p ¼ .8433 3.11 (0.82, 7.05) 2.03 (0.81, 5.08) Non-aspirin NSAID (Yes) 34 (45.3)/41 (54.7) 3.99 (2.06, 7.75); p < .0001 77 (58.3)/55 (41.7) 7.15 (4.10, 12.46); p < .0001 Aspirin intake (No) 92 (28.8)/227 (71.2) 1.0 170 (29.4)/409 (70.6) 1.09 (0.76–1.54); p ¼ .6474 6.04 (1.89, 13.98) 7.72 (0.38–158.17) Aspirin intake (Yes) 6 (40.0)/9 (60.0) 1.81 (0.45–7.37); p ¼ .4045 18 (69.2)/8 (30.8) 7.94 (2.99–21.13); p < .0001 rs6664:C > T Any NSAID (No) 75 (24.4)/233 (75.6) 1 120 (22.9)/403 (77.1) 1.13 (0.76, 1.68); p ¼ .5438 1.45 (6.05, 3.15) 0.78 (0.37, 1.65) Any NSAID (Yes) 53 (55.8)/42 (44.2) 7.47 (4.02, 13.88); p < .0001 78 (52.7)/70 (47.3) 6.15 (3.60, 10.50); p < .0001 Non-aspirin NSAID (No) 83 (25.6)/241 (74.4) 1 132 (24.3)/411 (75.7) 1.16 (0.79, 1.70); p ¼ .4486 3.37 (8.77, 2.03) 0.55 (0.24, 1.25) Non-aspirin NSAID (Yes) 45 (57.0)/34 (43.0) 8.38 (4.33, 16.18); p < .0001 66 (51.6)/62 (48.4) 5.16 (3.00, 8.90); p < .0001 Aspirin intake (No) 104 (31.0)/231 (69.0) 1.0 158 (28.1)/405 (71.9) 0.95 (0.67–1.34); p ¼ .7793 5.55 (2.60, 13.70) 5.74 (0.49–67.83) Aspirin intake (Yes) 8 (47.1)/9 (52.9) 2.22 (0.69–7.17); p ¼ .1829 16 (66.7)/8 (33.3) 7.72 (2.75–21.68); p ¼ .0001 rs2283458:A > G Any NSAID (No) 107 (26.7)/294 (73.3) 1 88 (20.5)/342 (79.5) 0.69 (0.47, 1.01); p ¼ .0532 0.56 (2.62, 3.73) 1.15 (0.50, 2.64) Any NSAID (Yes) 62 (53.4)/54 (46.6) 4.92 (2.83, 8.58); p < .0001 69 (54.3)/58 (45.7) 5.17 (3.07, 8.70); p < .0001 Non-aspirin NSAID (No) 117 (28.1)/300 (71.9) 1 98 (21.8)/352 (78.2) 0.71 (0.49, 1.03); p ¼ .068 0.77 (2.41, 3.96) 1.24 (0.5, 3.09) Non-aspirin NSAID (Yes) 52 (52.0)/48 (48.0) 4.46 (2.51, 7.93); p < .0001 59 (55.1)/48 (44.9) 4.95 (2.86, 8.57); p < .0001 Aspirin intake (No) 134 (31.7)/289 (68.3) 1.0 128 (26.9)/347 (73.1) 0.78 (0.56–1.08); p ¼ .1369 0.02 (6.07, 6.10) 1.01 (0.14–7.55) Aspirin intake (Yes) 13 (65.0)/7 (35.0) 4.24 (1.38–13.06); p ¼ .0118 11 (52.4)/10 (47.6) 4.03 (1.46–11.15); p ¼ .0072 rs1060463:C > G/C > T Any NSAID (No) 98 (22.5)/337 (77.5) 1 97 (24.5)/299 (75.5) 1.36 (0.93, 1.99); p ¼ .1143 0.73 (3.88, 5.33) 1.12 (0.55, 2.29) Any NSAID (Yes) 67 (52.3)/61 (47.7) 6.67 (3.94, 11.28); p < .0001 64 (55.7)/51 (44.3) 7.75 (4.41, 13.62); p < .0001 Non-aspirin NSAID (No) 103 (22.9)/346 (77.1) 1 112 (26.8)/306 (73.2) 1.42 (0.98, 2.05); p ¼ .0603 0.019 (4.65, 4.69) 1 (0.46, 2.19) Non-aspirin NSAID (Yes) 62 (54.4)/52 (45.6) 6.53 (3.81, 11.19); p < .0001 49 (52.7)/44 (47.3) 6.97 (3.81, 12.74); p < .0001 Aspirin intake (No) 138 (28.8)/342 (71.3) 1.0 124 (29.7)/294 (70.3) 1.28 (0.91–1.79); p ¼ .1542 2.07 (5.66, 9.79) 1.62 (0.27–9.83) Aspirin intake (Yes) 8 (50.0)/8 (50.0) 4.05 (1.26–12.98); p ¼ .0186 16 (64.0)/9 (36.0) 6.39 (2.35–17.38); p ¼ .0003 rs1695:A > G Any NSAID (No) 94 (24.5)/289 (75.5) 1 101 (22.5)/347 (77.5) 0.82 (0.56, 1.20); p ¼ .3084 0.11 (3.67, 3.46) 0.98 (0.44, 2.18) Any NSAID (Yes) 63 (59.4)/43 (40.6) 5.70 (3.27, 9.92); p < .0001 68 (49.6)/69 (50.4) 5.41 (3.19, 9.18); p < .0001 Non-aspirin NSAID (No) 101 (25.4)/297 (74.6) 1 114 (24.3)/355 (75.7) 0.90 (0.62, 1.30); p ¼ .5776 1.24 (5.08, 2.60) 0.75 (0.32, 1.77) Non-aspirin NSAID (Yes) 56 (61.5)/35 (38.5) 6.08 (3.40, 10.88); p < .0001 55 (47.4)/61 (52.6) 4.74 (2.72, 8.27); p < .0001 Aspirin intake (No) 133 (31.4)/290 (68.6) 1.0 129 (27.2)/346 (72.8) 0.83 (0.59–1.17); p ¼ .2858 1.14 (5.04, 7.31) 1.44 (0.18–11.37) Aspirin intake (Yes) 9 (56.3)/7 (43.8) 3.73 (1.14–12.21); p ¼ .0295 15 (60.0)/10 (40.0) 4.70 (1.76–12.52); p ¼ .0020 rs3756067:G > A (continued) Table 4. Continued. Wildtype genotype Genetic variation SNP (reference number) N (%) (cases/controls) OR† (95% CI); p-value N (%) (cases/controls) OR† (95% CI); p-value RERI (95% CI) S (95% CI) Any NSAID (No) 79 (23.0)/265 (77.0) 1 114 (23.8)/366 (76.3) 0.93 (0.63, 1.38); p ¼ .7199 0.03 (3.53, 3.46) 0.99 (0.45, 2.18) Any NSAID (Yes) 57 (50.9)/55 (49.1) 5.52 (3.15, 9.68); p < .0001 68 (54.4)/57 (45.6) 5.42 (3.10, 9.46); p < .0001 Non-aspirin NSAID (No) 88 (24.3)/274 (75.7) 1 124 (24.9)/373 (75.1) 0.87 (0.60, 1.27); p ¼ .483 0.22 (3.11, 3.56) 1.06 (0.44, 2.57) Non-aspirin NSAID (Yes) 48 (51.1)/46 (48.9) 4.79 (2.66, 8.64); p < .0001 58 (53.7)/50 (46.3) 4.89 (2.77, 8.64); p < .0001 Aspirin intake (No) 105 (28.2)/267 (71.8) 1.0 150 (29.2)/364 (70.8) 0.94 (0.67–1.32); p ¼ .7272 0.08 (6.60, 6.76) 1.02 (0.15–7.02) Aspirin intake (Yes) 13 (56.5)/10 (43.5) 4.48 (1.62–12.35); p ¼ .0038 10 (58.8)/7 (41.2) 4.50 (1.41–14.40); p ¼ .0113 rs3765070:A > G/A > T Any NSAID (No) 99 (22.7)/337 (77.3) 1 96 (24.3)/299 (75.7) 1.32 (0.90, 1.93); p ¼ .1534 0.99 (3.57, 5.54) 1.17 (0.57, 2.40) Any NSAID (Yes) 66 (52.0)/61 (48.0) 6.46 (3.82, 10.93); p < .0001 65 (56.0)/51 (44.0) 7.77 (4.43, 13.61); p < .0001 Non-aspirin NSAID (No) 104 (23.1)/346 (76.9) 1 111 (26.6)/306 (73.4) 1.38 (0.96, 2.00); p ¼ .0822 0.31 (4.32, 4.94) 1.05 (0.48, 2.30) Non-aspirin NSAID (Yes) 61 (54.0)/52 (46.0) 6.33 (3.69, 10.86); p < .0001 50 (53.2)/44 (46.8) 7.02 (3.85, 12.80); p < .0001 Aspirin intake (No) 138 (28.8)/342 (71.3) 1.0 124 (29.7)/294 (70.3) 1.26 (0.9–1.76); p ¼ .1772 2.07 (5.60, 9.75) 1.63 (0.27–9.96) Aspirin intake (Yes) 8 (50.0)/8 (50.0) 4.02 (1.26–12.88); p ¼ .0191 16 (64.0)/9 (36.0) 6.35 (2.34–17.27); p ¼ .0003 rs3869579:G > A/G > C Any NSAID (No) 89 (23.1)/296 (76.9) 1 106 (23.8)/340 (76.2) 1.14 (0.78, 1.67); p ¼ .5035 0.33 (4.53, 3.87) 0.94 (0.45, 1.95) Any NSAID (Yes) 58 (53.7)/50 (46.3) 6.68 (3.78, 11.79); p < .0001 73 (54.1)/62 (45.9) 6.49 (3.82, 11.03); p < .0001 Non-aspirin NSAID (No) 99 (24.6)/303 (75.4) 1 116 (24.9)/349 (75.1) 1.15 (0.80, 1.66); p ¼ .4474 0.63 (4.86, 3.59) 0.89 (0.4, 1.99) Non-aspirin NSAID (Yes) 48 (52.7)/43 (47.3) 6.35 (3.50, 11.50); p < .0001 63 (54.3)/53 (45.7) 5.87 (3.38, 10.19); p < .0001 Aspirin intake (No) 116 (28.4)/293 (71.6) 1.0 146 (29.9)/343 (70.1) 1.09 (0.78–1.52); p ¼ .6258 0.34 (6.81, 7.50) 1.09 (0.18–6.72) Aspirin intake (Yes) 10 (58.8)/7 (41.2) 4.69 (1.53–14.36); p ¼ .0068 14 (58.3)/10 (41.7) 5.12 (1.85–14.20); p ¼ .0017 rs4715332:C > A Any NSAID (No) 98 (23.8)/314 (76.2) 1 97 (23.2)/322 (76.8) 0.88 (0.60, 1.28); p ¼ .507 2.34 (1.49, 6.16) 1.64 (0.74, 3.65) Any NSAID (Yes) 61 (50.4)/60 (49.6) 4.76 (2.76, 8.22); p < .0001 70 (57.9)/51 (42.1) 6.98 (4.09, 11.92); p < .0001 Non-aspirin NSAID (No) 110 (25.3)/324 (74.7) 1 105 (24.3)/327 (75.7) 0.85 (0.59, 1.23); p ¼ .3941 2.25 (1.36, 5.86) 1.75 Non-aspirin NSAID (Yes) 49 (49.5)/50 (50.5) 4.15 (2.31, 7.43); p < .0001 62 (57.4)/46 (42.6) 6.25 (3.62, 10.77); p < .0001 (0.71, 4.32) Aspirin intake (No) 125 (28.8)/318 (71.8) 1.0 137 (30.1)/318 (69.9) 0.98 (0.70–1.38); p ¼ .9270 1.99 (6.68, 10.66) 1.61 (0.24–10.9) Aspirin intake (Yes) 14 (58.3)/10 (41.7) 4.27 (1.62–11.24); p ¼ .0033 10 (62.5)/6 (37.5) 6.24 (1.76–22.12); p ¼ .0046 rs4715354:G > A Any NSAID (No) 99 (24.7)/302 (75.3) 1 95 (22.1)/334 (77.9) 0.95 (0.65, 1.40); p ¼ .8103 1.53 (2.25, 5.32) 1.37 (0.63, 2.96) Any NSAID (Yes) 61 (50.4)/60 (49.6) 5.24 (3.03, 9.08); p < .0001 70 (57.4)/52 (42.6) 6.73 (3.95, 11.46); p < .0001 Non-aspirin NSAID (No) 108 (25.7)/313 (74.3) 1 106 (23.8)/339 (76.2) 0.95 (0.66, 1.37); p ¼ .7886 1.16 (2.54, 4.85) 1.30 (0.56, 3.04) Non-aspirin NSAID (Yes) 52 (51.5)/49 (48.5) 4.90 (2.73, 8.79); p < .0001 59 (55.7)/47 (44.3) 6.01 (3.48, 10.36); p < .0001 Aspirin intake (No) 127 (29.4)/305 (70.6) 1.0 135 (29.0)/331 (71.0) 1.02 (0.73–1.42); p ¼ .9198 0.88 (6.37, 8.13) 1.26 (0.20–8.05) Aspirin intake (Yes) 14 (58.3)/10 (41.7) 4.39 (1.61–11.97); p ¼ .0039 10 (58.8)/7 (41.2) 5.28 (1.68–16.61); p ¼ .0044 rs1799931:G > A Any NSAID (No) 186 (23.6)/601 (76.4) 1 9 (20.5)/35 (79.5) 0.98 (0.42, 2.28); p ¼ .9634 2.49 (8.06, 3.07) 0.53 (0.08, 3.4) Any NSAID (Yes) 123 (53.7)/106 (46.3) 6.30 (4.20, 9.46); p < .0001 8 (57.1)/6 (42.9) 3.79 (0.98, 14.63) Non-aspirin NSAID (No) 203 (24.8)/615 (75.2) 1 12 (24.5)/37 (75.5) 0.99 (0.45, 2.21); p ¼ .9854 2.07 (8.31, 4.17) 0.56 (0.06, 5.18) Non-aspirin NSAID (Yes) 106 (53.5)/92 (46.5) 5.74 (3.77, 8.75); p < .0001 5 (55.6)/4 (44.4) 3.67 (0.74, 18.11); p ¼ .1107 Aspirin intake (No) 280 (28.9)/690 (71.1) 1 14 (26.4)/39 (73.6) 0.91 (0.43, 1.94); p ¼ .8103 1.63 (10.30, 7.03) 0.57 (0.01, 24.24) Aspirin intake (Yes) 29 (63.0)/17 (37.0) 4.87 (2.20, 10.81); p ¼ .0001 3 (60.0)/2 (40.0) 3.15 (0.26, 37.77); p ¼ .3644 ANNALS OF MEDICINE rs28399433:A > C (continued) 387 Table 4. Continued. 388 Wildtype genotype Genetic variation SNP (reference number) N (%) (cases/controls) OR† (95% CI); p-value N (%) (cases/controls) OR† (95% CI); p-value RERI (95% CI) S (95% CI) Any NSAID (No) 172 (23.4)/563 (56.6) 1 22 (24.7)/67 (75.3) 0.93 (0.50, 1.72); p ¼ .8125 0.23 (5.51, 5.96) 1.05 (0.34, 3.2) Any NSAID (Yes) 116 (54.0)/99 (46.0) 6.02 (3.96, 9.15); p < .0001 14 (51.9)/13 (48.1) 6.18 (2.50, 15.23); p ¼ .0001 Non-aspirin NSAID (No) 190 (24.8)/577 (75.2) 1 24 (25.8)/69 (74.2) 1.01 (0.56, 1.81); p ¼ .9841 0.86 (5.92, 4.19) 0.82 (0.23, 2.92) Non-aspirin NSAID (Yes) 98 (53.6)/85 (46.4) 5.71 (3.69, 8.83); p < .0001 12 (52.2)/11 (47.8) 4.85 (1.84, 12.81); p ¼ .0014 Aspirin intake (No) 258 (28.5)/646 (71.5) 1 34 (30.6)/77 (69.4) 0.97 (0.57, 1.64); p ¼ .9016 0.025 (10.43, 10.48) 1.01 (0.06, 17.32) Aspirin intake (Yes) 30 (65.2)/16 (34.8) 4.69 (2.09, 10.50); p ¼ .0002 3 (60.0)/2 (40.0) 4.68 (0.57, 38.56); p ¼ .1514 N. MALLAH ET AL. rs4536:C > T Any NSAID (No) 191 (23.6)/618 (76.4) 1 4 (19.0)/17 (81.0) 0.32 (0.06, 1.66); p ¼ .1743 2.80 (8.08, 2.47) 0.37 (0.02, 7.43) Any NSAID (Yes) 127 (54.3)/107 (45.7) 6.10 (4.08, 9.12); p < .0001 3 (37.5)/5 (62.5) 2.61 (0.41, 16.49); p ¼ .3070 Non-aspirin NSAID (No) 210 (24.9)/633 (75.1) 1 5 (21.7)/18 (78.3) 0.29 (0.06, 1.53); p ¼ .1450 2.38 (7.41, 2.66) 0.39 (0.02, 8.2) Non-aspirin NSAID (Yes) 108 (54.0)/92 (46.0) 5.60 (3.69, 8.52); p < .0001 2 (33.3)/4 (66.7) 2.52 (0.41, 15.62); p ¼ .3213 Aspirin intake (No) 287 (28.9)/707 (71.1) 1 6 (22.2)/21 (77.8) 0.37 (0.11, 1.27); p ¼ .1139 Not applicable Not applicable Aspirin intake (Yes) 31 (63.3)/18 (36.7) 4.63 (2.16, 9.91); p ¼ .0001 1 (50.0)/1 (50.0) Not applicable rs58285195:T > C Any NSAID (No) 173 (22.7)/588 (77.3) 1 22 (31.4)/48 (68.6) 1.57 (0.84, 2.92); p ¼ .1553 7.68 (12.96, 28.31) 2.35 (0.48, 11.4) Any NSAID (Yes) 122 (53.0)/108 (47.0) 6.10 (4.05, 9.18); p < .0001 9 (69.2)/4 (30.8) 14.35 (3.38, 60.98); p ¼ .0003 Non-aspirin NSAID (No) 193 (24.2)/604 (75.8) 1 22 (31.4)/48 (68.6) 5.50 (3.60, 8.42); p < .0001 7.23 (11.67, 26.14) 2.46 (0.5, 12.11) Non-aspirin NSAID (Yes) 102 (52.6)/92 (47.4) 5.50 (3.60, 8.42); p ¼ .2383 9 (69.2)/4 (30.8) 13.18 (3.12, 55.72); p ¼ .0005 Aspirin intake (No) 264 (28.0)/678 (72.0) 1 30 (37.0)/51 (63.0) 1.70 (0.96, 3.02); p ¼ .0679 Not applicable Not applicable Aspirin intake (Yes) 31 (63.3)/18 (36.7) 5.36 (2.45, 11.73); p < .0001 1 (50.0)/1 (50.0) Not applicable SNP: single nucleotide polymorphism. † Odds Ratio adjusted for: period of patients’ recruitment, previous history of arthrosis, infection with Helicobacter pylori, gastrointestinal disorders (ulcer and bleeding), 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). ANNALS OF MEDICINE 389 yielded a non-statistically significant measure of inter- tested in the present work and NSAID (non-aspirin action [S ¼ 5.74 (95%CI: 0.49–67.83); RERI ¼ 5.55 and aspirin) on UGIH. Consequently, it was not pos- (95%CI: 2.60–13.70)] sible to compare our findings to that of other studies. Few studies examined the associations between the 3.3.3. Genotypes not associated with modification genetic variants assessed in the current study of the risk of UGIH and other gastrointestinal disorders (ulcers or small Carriers of the wild-type genotypes and carriers of the bowel bleeding). Shiotani and colleagues suggested a following genetic variants are associated with a similar possible relation between the genetic variants magnitude of risk of UGIH: rs2283458:A>G, rs1060463: rs2180314:G>C, rs4809957:A>G, rs1060463:C>G/C>T, C>G/C>T, rs1695:A>G, rs3756067:G>A, and rs1695:A>G and the risk of small bowel bleeding rs3765070:A>G/A>T, rs3869579:G>A/G>C, in Japanese patients on aspirin therapy, however, no rs28399433:A>C (Table 4). significant association was demonstrated [25]. Another The risk associated with rs36079186 could not be study in Japan also reported a significant association determined because it was monovariant (the same of rs6664:C>T genotype with the risk of ulcer bleeding genotype was observed in all the study population). in aspirin users [26]. Shiotani and colleagues also sug- Inconclusive results were also obtained for rs4536:C>T gested an association between rs3765070:A>G/A>T, and rs58285195:T>C due to the limited number of rs4715354:G>A, rs2283458:A>G, and rs3756067:G>A cases and controls who are on NSAIDs treatment and and small bowel bleeding in a genome-wide analysis carriers for these genetic variations. in the Japanese population; nevertheless, the associ- ation disappeared upon the validation of those SNPs [25]. 4. Discussion The explanation of the increased odds of UGIH in To the best of our knowledge, this is the first study NSAID consumers who are carriers of the genetic var- that finds a statistically significant additive synergism iations rs2180314:C>G or rs4809957:A>G is limited. interaction between certain genetic polymorphisms in However, we hypothesize that this excess in risk could genes involved in drug metabolism (rs2180314:C>G be a consequence of an altered function of the corre- and rs4809957:A>G) and NSAIDs on UGIH. Our results sponding genes due to polymorphisms. indicate that the joint effect of these SNPs with rs2180314:C>G belongs to the glutathione S-transfer- NSAIDs is more than three times higher ase (GSTA) gene family which plays an important role [rs2180314:C>G (S ¼ 3.30; 95%CI: 1.24, 8.80), in the detoxification of electrophilic compounds, rs4809957:A>G (S ¼ 2.11; 95%CI: 0.90, 4.97)] than the including therapeutic drugs, and protects the cells sum of their individual effects. Since (1) UGIH contrib- against damage. GSTA genes are highly polymorphic, utes to high mortality and morbidity rates [28], (2) and their genetic variation may alter the toxicity and NSAIDs are among the most commonly used medi- efficacy of some drugs [56,57]. rs4809957:A>G belongs cines worldwide [4], (3) a large fraction of the to gene members of the P450 family. Enzymes European population carries the genetic variants encoded by P450 are monooxygenases that catalyze rs2180314:C>G (58%) and rs4809957:A>G (21%) [53], many drug metabolism reactions [58]. and (4) genotyping is a low-cost test, our findings In general, testing a large number of SNPs increases could enable identifying better the individuals at risk type 1 error, and therefore increments the chance of and those who are not at risk of UGIH from obtaining false-positive conclusions [59]. Nevertheless, NSAID exposure. in this study, we attempted to minimize type 1 error The interindividual variations in therapeutic by performing a pre-hoc SNP selection whereby we responses to NSAIDs were associated earlier with sev- chose specifically those SNPs that belong to genes eral demographic and clinical factors. Variations in involved in drug metabolism, and which were sug- patients’ response to aspirin NSAIDs were also sug- gested to be associated with gastrointestinal disorders gested to be related to the metabolic rate of this drug [25,26]. Furthermore, we analyzed each SNP in an and the excretion of aspirin metabolites [54]. Patients independent model and reported all the implemented are classified as fast and slow acetylators, and these analyses. Our strategy of SNP selection and data metabolic differences were associated with genetic reporting exempts from adjusting for multiple testing factors [55]. and leads to fewer errors of interpretation [60,61]. We are not aware of any previous study that eval- Another strength of this study is the control for all uated the interaction between the polymorphisms possible biases. The memory bias was reduced by 390 N. MALLAH ET AL. showing prompt cards of the most frequent NSAID Acknowledgements commercial boxes to the patients during the interview The authors would like to thank Professor Bahi Takkouche and by reviewing the medical records. The exclusion for reviewing the earlier version of this article and for his of non-white patients also allowed to prevent bias helpful comments. The authors are also thankful for the per- due to racial differences between populations. sonnel who helped in recruiting and interviewing Moreover, performing the study in biologically unre- the patients. lated patients, exclusively, avoided the over-represen- tation of the bias of genotype within families. Finally, Author contributions the measures of effect reported in this study were all adjusted to baseline risk factors that were known to C.A., L.I., X.V., and A.F. formulated the research idea, designed, and supervised the study. N.M. carried out the lit- increase the risk of gastrointestinal bleeding. erature review and conceptualized and wrote the manu- The main limitation of our study was the sample script. N.M. and M.P. analyzed the data. A.F. supervised data size. In fact, upon stratification by genotype and types analysis. M.Z.C., E.I.-G., I.P.-Z., F.M.G., J.I.G., L.V., L.M.-A., M.S.G., of NSAID, a limited number of observations were left V.V.G., A.S., and A.E.-G. recruited patients and registered the in the subgroups mainly for aspirin, which conse- data. All authors approved the manuscript for submission. quently decreased the statistical power for the associa- tions of many SNPs. Low statistical power is a Disclosure statement frequent limitation in candidate gene studies [62,63]. Another consequence imposed by the modest sample The authors declare they have no conflict of interest. size is the curse of dimensionality; i.e. the number of observations was very small when various SNPs were Funding combined [64]. Therefore, it was not feasible to ana- This study was supported by grants from: Carlos III Health lyze the combined effect of different genetic varia- Institute (P I12/02414, of the P E I+D+I 2012-2016); Fondo tions. In addition, it was not possible to undertake a Europeo de Desarrollo Regional (FEDER); the Novartis, Pfizer dose-response analysis. Therefore, we believe that fur- and Dr Esteve pharmaceutical companies; the Health ther studies with a larger sample size are needed to Research Fund/Fondo de Investigacion n Sanitaria (P I021512, confirm these results before their implantation in clin- P I021364, P I020661, and P I021572); Ministry of Health & ical settings. Another limitation of our study is the Consumer Affairs, Spain (SAF2002-04057); Galician Regional Authority, Spain (P GIDIT03P XIC20806P N); Department of potential presence of false H. pylori test results. Health of the Basque Country (03/11092 and 11/111103); Though we intended to minimize the false positive Fundacion Vasca de innovacion e investigacion sanitarias rate by inquiring about treatment for H. pylori infec- (OSIBG19/002 and OSIBG18/105). The genotyping service tion in the past, we cannot rule out the possibility of was carried out at CEGEN-P RB3-ISCIII; Carlos III Health having false negative results since the rate of eradica- Institute and ERDF (P T17/0019, of the P E I+D+I 2013-2016). tion of H. pylori from a single treatment varies The funding sources do not have any role in the study design; data collection, analysis and interpretation; writing between 60% and 90% [65]. the manuscript; and in the decision to submit the article for In conclusion, this study revealed that genetic varia- publication. tions might alter the pharmacological and clinical response to NSAIDs. The risk of UGIH in NSAID users with the wild type genotypes of rs2180314 and rs4809957 is significantly lower than that in those Data availability statement users who carry the genetic variants. 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