Genome-wide association studies (GWAS) with intermediate phenotypes, like changes in metabolite and protein levels, provide functional evidence to map disease associations and translate them into clinical applications. However, although hundreds of genetic variants have been associated with complex disorders, the underlying molecular pathways often remain elusive. Associations with intermediate traits are key in establishing functional links between GWAS-identified risk-variants and disease end points. Here we describe a GWAS using a highly multiplexed aptamer-based affinity proteomics platform. We quantify 539 associations between protein levels and gene variants (pQTLs) in a German cohort and replicate over half of them in an Arab and Asian cohort. Fifty-five of the replicated pQTLs are located in trans. Our associations overlap with 57 genetic risk loci for 42 unique disease end points. We integrate this information into a genome-proteome network and provide an interactive web-tool for interrogations. Our results provide a basis for novel approaches to pharmaceutical and diagnostic applications.

Author contributions In an academic-industry partnership, SomaLogic, Inc. and the academic collaborators worked together on study design, interpretation of the data and preparation of the manuscript. S.A.W., P.G. and N.W. were responsible for designing, writing and final editing of the manuscript and responses to reviewer comments. In addition to all authors being generally involved in the program, specific contributions were as follows: M.K. and M.J.S. were accountable for the data from the Whitehall II study and advised on the study design for the CV and diabetes models. C.L. and N.W. were accountable for the data from the Fenland study and advising on diabetes risk and behavioral models. C.B. and M.A.S. were accountable for the data from the Heritage Family study. C.J. was accountable for the data from the HUNT3 study. R.O. was accountable for the data from the Covance study.

SignificanceDuchenne muscular dystrophy (DMD) is a rare and devastating muscle disease caused by mutations in the X-linked DMD gene (which encodes the dystrophin protein). Serum biomarkers hold significant potential as objective phenotypic measures of DMD disease state, as well as potential measures of pharmacological effects of and response to therapeutic interventions. Here we describe a proteomics approach to determine serum levels of 1,125 proteins in 93 DMD patients and 45 controls. The study identified 44 biomarkers that differed significantly between patients and controls. These data are being made available to DMD researchers and clinicians to accelerate the search for new diagnostic, prognostic, and therapeutic approaches.

The vitamin D receptor (VDR) heterodimerizes with retinoid X receptors (RXR) on many vitamin D-responsive promoter elements, suggesting that this complex is the active factor in vitamin D-mediated transcription. However, the mechanism of transcriptional regulation following VDR-RXR binding to DNA is not well characterized. Using a yeast two-hybrid protein interaction assay, we demonstrate that VDR forms specific protein: protein contacts with the basal transcription factor TFIIB. Deletion analysis indicated that the carboxyl-terminal ligand binding domain of VDR interacted with a 43-residue amino-terminal domain in TFIIB. The interaction with TFIIB showed selectivity for the ligand binding domain of VDR as similar regions of RXR alpha or of retinoic acid receptor alpha did not couple with TFIIB. Binding assays with purified proteins showed a direct interaction between VDR and TFIIB in vitro. These data suggest a mechanism for VDR-dependent transcription in which protein contacts between VDR and TFIIB may impart regulatory information to the transcription preinitiation complex.

Gemfibrozil-1-O-beta-glucuronide (GEM-1-O-gluc), a major metabolite of the antihyperlipidemic drug gemfibrozil, is a mechanism-based inhibitor of P450 2C8 in vitro, and this irreversible inactivation may lead to clinical drug-drug interactions between gemfibrozil and other P450 2C8 substrates. In light of this in vitro finding and the observation that the glucuronide conjugate does not contain any obvious structural alerts, the current study was conducted to determine the potential site of GEM-1-O-gluc bioactivation and the subsequent mechanism of P450 2C8 inhibition (i.e., modification of apoprotein or heme). LC/MS analysis of a reaction mixture containing recombinant P450 2C8 and GEM-1-O-gluc revealed that the substrate was covalently linked to the heme prosthetic heme group during catalysis. A combination of mass spectrometry and deuterium isotope effects revealed that a benzylic carbon on the 2',5'-dimethylphenoxy group of GEM-1-O-gluc was covalently bound to the heme of P450 2C8. The regiospecificity of substrate addition to the heme group was not confirmed experimentally, but computational modeling experiments indicated that the gamma-meso position was the most likely site of modification. The metabolite profile, which consisted of two benzyl alcohol metabolites and a 4'-hydroxy-GEM-1-O-gluc metabolite, indicated that oxidation of GEM-1-O-gluc was limited to the 2',5'-dimethylphenoxy group. These results are consistent with an inactivation mechanism wherein GEM-1-O-gluc is oxidized to a benzyl radical intermediate, which evades oxygen rebound, and adds to the gamma-meso position of heme. Mechanism-based inhibition of P450 2C8 can be rationalized by the formation of the GEM-1-O-gluc-heme adduct and the consequential restriction of additional substrate access to the catalytic iron center.