Comprehensive Biomarker Profiling in Disease Phenotypes

It was our objective to unravel and to validate laboratory biomarkers significantly associated with disease phenotypes and risks. An extensive panel of laboratory tests covering 83 analytes and biomarkers (clinical chemistry, hematology, immunology, endocrinology and metabolism) was performed on fresh biospecimen directly on the day of sample collection in a highly standardized manner (Table 4). It is a major goal to investigate and to identify novel genetic modifiers of phenotypes and disease risk. To this end, we aimed to genotype all participants using the Affymetrix AXIOM-CEU genome-wide SNP array, addressing a total of 587,352 variants. Genotyping was accompanied by genome-wide gene expression analyses for the whole blood, which was collected in Tempus Blood RNA Tubes (Life Technologies) and transferred to -80 °C prior to further processing. Isolated RNA was processed and hybridised to Illumina HT-12 v4 Expression BeadChips (Illumina, San Diego, CA, USA) and scanned on the Illumina HiScan. We investigated the association of metabolic biomarkers (quantitiative tandem mass spectrometry: amino acidy, fatty acid oxidation, steroids, sterols, eicosanoids, phospholipids, tri- and diacylglycerols, apolipoproteins and others) with major disease phenotypes, the genome and other biomarkers. For the whole blood analysis of 26 amino acids, free carnitine and 34 acylcarnitines, 40 μl of native EDTA whole blood were spotted on filter paper WS 903 (GE Healthcare, Germany). Dried blood spots were stored at -80 °C after 3 h of drying until batch analysis. Sample pretreatment and measurement are described in detail elsewhere [74 (link), 75 (link)]. Analyses were performed on an API 2000 and API 4000 tandem mass spectrometer (Applied Biosystems, Germany). Quantification was performed using ChemoView™ 1.4.2 software (Applied Biosystems, Germany).
In a subcohort of over 900 participants comprising all age groups, a detailed leukocyte subtype phenotyping with an extensive antibody panel was performed from EDTA whole blood samples using 10 colour flow cytometry (Navios flow cytometer, Beckman Coulter, Pasadena, CA, USA) [76 (link), 77 ].