*2.3. ELISA*

Serum was available from 100 patients in the RIPheart Study. Serum MIF levels were quantified by ELISA in duplicates as previously described and according to the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA) ([5]). Samples were diluted 1:10 before analysis to obtain measures in the valid assay range.

#### *2.4. Nomenclature and Genotyping of the Tetranucleotide Repeat Polymorphism CATTn (rs3063368)*

In adherence to the U.S. National Library of Medicine dbSNP database (ncbi.nlm.nih.gov/snp/), the tetranucleotide repeat polymorphism formerly described as rs5844572, referring to the CATT6

allele, will be referred to with the reference SNP number rs3063368, which comprises the multiallelic repeat polymorphism CATTn present at this site. The tetranucleotide repeat polymorphism is located at position chr22:23893566-23893569: (GRCh38.p12) (ncbi.nlm.nih.gov/snp/rs3063368), and based on older transcript annotations (NM\_002415.2) -794 nucleotides, or based on more accurate transcript annotations (NM\_002415.2) -909 nucleotides upstream of the start codon. The repeat polymorphism is a deletion, or respectively a duplication, of TTCA tetranucleotide repeats. In parallel with former publications, in this study this SNP will be referred to as a tetranucleotide 5-, 6-, 7-, or 8- fold repeat of CATT. The DNA sequence of this SNP is illustrated in Supplemental Figure S2 (UCSC Genome Browser, genome.ucsc.edu).

EDTA-anticoagulated whole blood was used for genotyping. DNA was extracted with the Autopure LS automated system according to the manufacturer's recommendations (Qiagen, Hilden, Germany).

For the analysis of the tetranucleotide repeat (rs3063368), as formerly described, a fragment length polymorphism PCR with fluorescently labeled primers and fragment length analysis via capillary electrophoresis was applied [10]. Of note, with this technique a phase analysis with the rs755622 single nucleotide polymorphism (SNP) is not possible. DNA was amplified in a Mastercycler gradient (Eppendorf AG, Germany). The PCR reaction mix contained 2.5 μL 10× PCR bu ffer, 3 μL (25 mM) MgCl2, 2 μL dNTP Mix, 0.8 μL of each primer, 0.15 μL (1 U) Taq polymerase, and 13.75 μL of purified DNA, in a total end volume of 25 μL. The PCR consisted of the following steps: an initial denaturation step (95 ◦C, 12 min), 35 amplification cycle (95 ◦C, 30 s; X (primer annealing temperature see Supplemental Table S1, 30 s; 72 ◦C, 30 s) and a final elongation step (72 ◦C, 10 min). The reverse primer was labeled with 6-Carboxyfluorescein (6-FAM) [19]. The PCR was performed in a SimpliAmp Thermal Cycler (Life Technologies, Carlsbad, CA, USA). The PCR products were subjected to capillary electrophoresis on an ABI 3730XL Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Data collection was performed with Data Collection v3.0 software (Applied Biosystems, Foster City, CA, USA) and the results were analyzed by GeneMapper ID v5 software (Applied Biosystems, Foster City, CA, USA).

#### *2.5. Nomenclature and Genotyping of the SNP G*>*C Substitution (rs755622)*

The SNP rs755622 is a substitution of G>C in the non-coding region at position chr22:23,894,205 (GRCh38.p12). Based on the older transcript annotations (NM\_002415.1), this SNP has been formerly described with the position NM\_002415.1:m.-173. According to the more accurate transcript annotation (NM\_002415.2), the G>C substitution is located at position NM\_002415.2:m.-178 and NM\_002415.2:c.-270 G>C. (The start codon is located at position chr22:23,894,475.)

DNA was extracted from EDTA-anticoagulated whole blood with Autopure (Qiagen, Hilden, Germany). Genotyping of the SNP rs755622 G>C was performed using an Assays-on-Demand ® allelic discrimination on a TaqMan platform according to the manufacturer's instructions (ThermoFisher Scientific, Waltham, MA, USA). The polymerase chain reaction (PCR) contained 10 ng of genomic DNA, 10 μL TaqMan master mix, and 0.125 μL of 40× assay mix. PCR was performed using 96-well plates on an ABI 9700 thermal cycler (Applied Biosystems, Foster City, CA, USA) (reaction conditions 50 ◦C for 2 min, 95 ◦C for 10 min, followed by 40 cycles of 95 ◦C for 15 s and 60 ◦C for 1 min). The TaqMan 7700 platform was used to perform an end-plate reading using the allelic discrimination option.

Sample and marker quality control (QC) was performed with PLINK (v1.9; https://www.coggenomics.org/plink/1.9)(PMID:25722852).
