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Factor VII (FVII) is an important component of the coagulation cascade. Few genetic loci regulating FVII activity and/or levels have been discovered to date. We conducted a meta-analysis of 9 genome-wide association studies of plasma FVII levels (7 FVII activity and 2 FVII antigen) among 27 495 participants of European and African ancestry. Each study performed ancestry-specific association analyses. Inverse variance weighted meta-analysis was performed within each ancestry group and then combined for a trans-ancestry meta-analysis. Our primary analysis included the 7 studies that measured FVII activity, and a secondary analysis included all 9 studies. We provided functional genomic validation for newly identified significant loci by silencing candidate genes in a human liver cell line (HuH7) using small-interfering RNA and then measuring F7 messenger RNA and FVII protein expression. Lastly, we used meta-analysis results to perform Mendelian randomization analysis to estimate the causal effect of FVII activity on coronary artery disease, ischemic stroke (IS), and venous thromboembolism. We identified 2 novel (REEP3 and JAZF1-AS1) and 6 known loci associated with FVII activity, explaining 19.0% of the phenotypic variance. Adding FVII antigen data to the meta-analysis did not result in the discovery of further loci. Silencing REEP3 in HuH7 cells upregulated FVII, whereas silencing JAZF1 downregulated FVII. Mendelian randomization analyses suggest that FVII activity has a positive causal effect on the risk of IS. Variants at REEP3 and JAZF1 contribute to FVII activity by regulating F7 expression levels. FVII activity appears to contribute to the etiology of IS in the general population.

Coagulation factor XI (FXI) has become increasingly interesting for its role in pathogenesis of thrombosis. While elevated plasma levels of FXI have been associated with venous thromboembolism and ischemic stroke, its deficiency is associated with mild bleeding. We aimed to determine novel genetic and post-transcriptional plasma FXI regulators. We performed a genome-wide association study (GWAS) for plasma FXI levels, using novel data imputed to the 1000 Genomes reference panel. Individual GWAS analyses, including a total of 16,169 European individuals from the ARIC, GHS, MARTHA and PROCARDIS studies, were meta-analysed and further replicated in 2,045 individuals from the F5L family, GAIT2 and MEGA studies. Additional association with activated partial thromboplastin time (aPTT) was tested for the top SNPs. In addition, a study on the effect of miRNA on FXI regulation was performed using in silico prediction tools and in vitro luciferase assays. Three loci showed robust, replicating association with circulating FXI levels: KNG1 (rs710446, P-value = 2.07 x 10(-302)), F11 (rs4253417, P-value = 2.86 x 10(-193)), and a novel association in GCKR (rs780094, P-value = 3.56 x 10(-09)), here for the first time implicated in FXI regulation. The two first SNPs (rs710446 and rs4253417) also associated with aPTT. Conditional and haplotype analyses demonstrated a complex association signal, with additional novel SNPs modulating plasma FXI levels in both the F11 and KNG1 loci. Finally, eight miRNAs were predicted to bind F11 mRNA. Over-expression of either miR-145 or miR-181 significantly reduced the luciferase activity in cells transfected with a plasmid containing FXI-3'UTR. These results should open the door to new therapeutic targets for thrombosis prevention.

Venous thromboembolism is a complex disease with a high heritability. There are significant associations among Factor XI (FXI) levels and SNPs in the KNG1 and F11 loci. Our aim was to identify the genetic variation of KNG1 and F11 that might account for the variability of FXI levels. The KNG1 and F11 loci were sequenced completely in 110 unrelated individuals from the GAIT-2 (Genetic Analysis of Idiopathic Thrombophilia 2) Project using Next Generation Sequencing on an Illumina MiSeq. The GAIT-2 Project is a study of 935 individuals in 35 extended Spanish families selected through a proband with idiopathic thrombophilia. Among the 110 individuals, a subset of 40 individuals was chosen as a discovery sample for identifying variants. A total of 762 genetic variants were detected. Several significant associations were established among common variants and low-frequency variants sets in KNG1 and F11 with FXI levels using the PLINK and SKAT packages. Among these associations, those of rs710446 and five low-frequency variant sets in KNG1 with FXI level variation were significant after multiple testing correction and permutation. Also, two putative pathogenic mutations related to high and low FXI levels were identified by data filtering and in silico predictions. This study of KNG1 and F11 loci should help to understand the connection between genotypic variation and variation in FXI levels. The functional genetic variants should be useful as markers of thromboembolic risk., Peer Reviewed, Postprint (published version)

There is a clear clinical need for high-specificity plasma biomarkers for predicting risk of venous thromboembolism (VTE), but thus far, such markers have remained elusive. Utilizing affinity reagents from the Human Protein Atlas project and multiplexed immuoassays, we extensively analyzed plasma samples from 2 individual studies to identify candidate protein markers associated with VTE risk. We screened plasma samples from 88 VTE cases and 85 matched controls, collected as part of the Swedish Venous Thromboembolism Biomarker Study, using suspension bead arrays composed of 755 antibodies targeting 408 candidate proteins. We identified significant associations between VTE occurrence and plasma levels of human immunodeficiency virus type I enhancer binding protein 1 (HIVEP1), von Willebrand factor (VWF), glutathione peroxidase 3 (GPX3), and platelet-derived growth factor beta (PDGFB). For replication, we profiled plasma samples of 580 cases and 589 controls from the French FARIVE study. These results confirmed the association of VWF and PDGFB with VTE after correction for multiple testing, whereas only weak trends were observed for HIVEP1 and GPX3. Although plasma levels of VWF and PDGFB correlated modestly (rho similar to 0.30) with each other, they were independently associated with VTE risk in a joint model in FARIVE (VWF P < .001; PDGFB P = .002). PDGF. was verified as the target of the capture antibody by immunocapture mass spectrometry and sandwich enzyme-linked immunosorbent assay. In conclusion, we demonstrate that high-throughput affinity plasma proteomic profiling is a valuable research strategy to identify potential candidate biomarkers for thrombosis-related disorders, and our study suggests a novel association of PDGFB plasma levels with VTE.