**1. Introduction**

Conventional open-heart surgery is performed annually in more than one million patients worldwide, and the incidence of postoperative sequelae including acute organ dysfunction remains high [1,2]. A more precise identification of patients' risk for postoperative complications is desirable both for prognostic guidance and for the application of earlier and more effective interventions. While clinical scoring systems such as the well-established EuroSCORE were primarily developed for the preoperative risk stratification of mortality in cardiac surgery patients, only limited evidence exists about its value for postoperative organ dysfunction and other complications [3]. The identification of genomic risk alleles could be especially helpful to more accurately predict outcomes and to enable personalized medicine approaches.

Oxidative stress and a systemic inflammatory response contribution to the pathogenesis of postoperative organ dysfunctions following cardiac surgery [4]. Macrophage migration inhibitory factor (MIF) is a stress-regulating cytokine that increases in the circulation after cardiac surgery [5]. Within the *MIF* gene promoter, two polymorphisms, a G>C single nucleotide polymorphism 270 bases before *MIF* transcription start (−270) (originally described as −173; HGVS nomenclature: NM\_002415.2 c.−270G>C, rs755622) and a CATT tetranucleotide repeat CATT5-7 (rs3063368), have been associated with disease severity of multiple chronic inflammatory diseases, including osteoporosis, ankylosing spondylitis, and multiple sclerosis [6–8]. A higher number of CATT repeats has been reported to increase *MIF* promotor activity and to be associated with higher circulating MIF concentrations in different autoimmune and chronic inflammatory conditions [9–12]. The clinical significance of functional polymorphisms in *MIF* for postoperative outcome after cardiac surgery is unknown. In this study, we analyzed 1116 patients who underwent elective cardiac surgery with a cardiopulmonary bypass. We analyzed whether *MIF* promoter polymorphisms impact the risk of postoperative organ dysfunction and mortality in patients undergoing cardiac surgery. In a subset of patients, we also examined the correlation between *MIF* genotypes and circulating MIF levels.

#### **2. Materials and Methods**

#### *2.1. Study Design and Patients*

The present study is a predefined sub-study performed in cardiac surgery patients of the Remote Ischemic Preconditioning Heart (RIPHeart) study (January 2011–May 2014), which investigated whether upper limb remote ischemic conditioning reduced mortality and the incidence of myocardial infarction, stroke, and acute kidney injury (AKI) in adults scheduled for elective cardiac surgery requiring a cardiopulmonary bypass [13]. As the initial intervention study did not show group differences, this *MIF* polymorphism study includes all patients irrespective of the initial group assignment [13]. The trial was undertaken in compliance with International Conference on Harmonisation Good Clinical Practice guidelines, the Declaration of Helsinki (2008), and European Directive 2001/20/CE regarding the

conduct of clinical trials (4 April 2001). The study was registered at ClinicalTrials.gov (NCT01067703). The study protocol was approved by the ethics committees of the University of Kiel, Aachen, and all participating centers of this prospective multicenter study.

Patients scheduled for elective cardiac surgery with use of cardiopulmonary bypass (e.g., coronary artery bypass graft (CABG), valve surgery, ascending aorta replacement) were eligible for this study. Of the 1403 patients screened for the study, blood samples were available from 1196 patients. Seven patients were excluded due to missing outcome data and 70 patients were excluded due to missing genotype information (Supplemental Figure S1). Thus, data from 1119 patients were included in the current study. The CATT8 genotype was excluded from analysis because of its low frequency (*N* = 3), which is in accord with prior reports [14].

Blood samples were collected before surgery, at 45 min after cardiopulmonary bypass (CPB) initiation, at 2 min after opening of the cross-clamp (reperfusion), at 15 min after weaning from CPB, and at 1, 6, and 24 h after admission to ICU. Blood samples were processed no later than 2 h after collection and were stored at −70 ◦C or −20 ◦C until further transfer. The final study visit took place either before hospital discharge, or at the latest 30 days after ICU admission.

#### *2.2. Outcome Measures*

The primary exposure was the *MIF* genotype, which we analyzed according to the following groups: alleles (e.g., carriers of at least one C allele), genotypes (e.g., GC), and individual genotype combinations (e.g., CATT5,7-GC). The endpoints were the association between the *MIF* genotype and the incidence of postoperative organ dysfunctions, including AKI, myocardial infarction, new onset of atrial fibrillation, stroke, delirium, and death. Each of these outcome parameters were analyzed as single events, and in the composite outcome "multiple organ dysfunction", when patients su ffered from more than one organ dysfunction.

According to the KDIGO Clinical Practice Guideline 2012, AKI was defined as a ≥ 5-fold increase of serum creatinine from baseline, and a urine output of ≤0.5 mL/kg/h for more than 6 h, or the use of renal replacement therapy within 72 h [15]. However, a total creatinine increase of ≥ 0.3 mg/dL was not considered a diagnostic criterion for AKI, as this criterion had not ye<sup>t</sup> been established in 2011 when the data collection started. Non-fatal myocardial infarction was defined by biomarker values more than five times higher than the 99th percentile of the normal reference range combined with new pathological Q-waves or new left bundle branch block within the first 72 h, standard clinical criteria for myocardial infarction from 72 h on, new ischemic finding by echocardiography or angiography, or myocardial infarction diagnosed at autopsy [16]. New onset of atrial fibrillation was defined as a new onset within the first four days after surgery [13]. The occurrence of postoperative delirium was assessed with the CAM-ICU score (preoperative, 24, 48, 72, and 96 h after surgery) [17]. Stroke was defined by any new, temporary or permanent, focal or global neurological deficit, or evidence of stroke on autopsy, and was evaluated according to the National Institutes of Health Stroke Scale (≥4 points) [18].

Myocardial infarction, atrial fibrillation, and stroke were analyzed until hospital discharge with a maximum of 14 days after surgery.
