Antithrombin, Protein C, and Protein S: Genome and Transcriptome-Wide Association Studies Identify 7 Novel Loci Regulating Plasma Levels.

BACKGROUND: Antithrombin, PC (protein C), and PS (protein S) are circulating natural anticoagulant proteins that regulate hemostasis and of which partial deficiencies are causes of venous thromboembolism. Previous genetic association studies involving antithrombin, PC, and PS were limited by modest sample sizes or by being restricted to candidate genes. In the setting of the Cohorts for Heart and Aging Research in Genomic Epidemiology consortium, we meta-analyzed across ancestries the results from 10 genome-wide association studies of plasma levels of antithrombin, PC, PS free, and PS total. METHODS: Study participants were of European and African ancestries, and genotype data were imputed to TOPMed, a dense multiancestry reference panel. Each of the 10 studies conducted a genome-wide association studies for each phenotype and summary results were meta-analyzed, stratified by ancestry. Analysis of antithrombin included 25 243 European ancestry and 2688 African ancestry participants, PC analysis included 16 597 European ancestry and 2688 African ancestry participants, PSF and PST analysis included 4113 and 6409 European ancestry participants. We also conducted transcriptome-wide association analyses and multiphenotype analysis to discover additional associations. Novel genome-wide association studies and transcriptome-wide association analyses findings were validated by in vitro functional experiments. Mendelian randomization was performed to assess the causal relationship between these proteins and cardiovascular outcomes. RESULTS: Genome-wide association studies meta-analyses identified 4 newly associated loci: 3 with antithrombin levels (GCKR, BAZ1B, and HP-TXNL4B) and 1 with PS levels (ORM1-ORM2). transcriptome-wide association analyses identified 3 newly associated genes: 1 with antithrombin level (FCGRT), 1 with PC (GOLM2), and 1 with PS (MYL7). In addition, we replicated 7 independent loci reported in previous studies. Functional experiments provided evidence for the involvement of GCKR, SNX17, and HP genes in antithrombin regulation. CONCLUSIONS: The use of larger sample sizes, diverse populations, and a denser imputation reference panel allowed the detection of 7 novel genomic loci associated with plasma antithrombin, PC, and PS levels.

Pseudothrombocytopenia, beyond a laboratory phenomenon: study of 192 cases.

The platelet antibodies that cause pseudothrombocytopenia (PTCP) act only in vitro and do not produce clinical bleeding. Most studies on PTCP have focused on improving differential diagnosis with true thrombocytopenia but studies on the characteristics of patients with PTCP are limited. In this study, we aimed to evaluate the clinical and biological characteristics of 192 patients with PTCP. In addition to general variables, we evaluated automated and microscopic platelet counts, platelet clumps, platelet diameters, immature platelet fraction (IPF), and platelet antibodies. Adult women accounted for the largest subgroup of patients (n=82; 42.7%) and 67 patients (34.9%) were grouped into families. Forty-four patients (22.9%) had one or more associated autoimmune disorders (ADs); 39 relatives of these patients (19.8%) had ADs and 45 relatives (23.4%) had immune thrombocytopenia (ITP) or unspecified thrombocytopenia. Platelet cryptantibodies and/or autoantibodies were positive in 56 patients (30.1%). Most patients (n=169; 80%) had automated platelet counts >80×109/L. In all patients, microscopic platelet counts were ≥150×109/L. The platelet clump index (% increase in microscopic platelet count compared to automatic count) ranged from 30 to >7000%. Platelet diameters and IPF parameters were significantly greater in the PTCP versus healthy controls (p<0.001). A total of 17 patients (8.8%) had had previous ITP or the PTCP evolved into ITP. Our data suggest that PTCP should be considered a situation of autoimmunity; the assessment of platelet clumps has a high diagnostic value; the close association between ITP and PTCP suggests that these conditions could be different phases of the same process.

A genome-wide association study identifies new loci for factor VII and implicates factor VII in ischemic stroke etiology.

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.

Identification of a Plasma MicroRNA Profile Associated With Venous Thrombosis.

OBJECTIVE: Venous thrombosis (VT) is a complex condition with a highly heritable genetic component that predisposes one to its development. Certain microRNAs (miRNAs) might be used as biomarkers of VT, but few studies have examined miRNA expression in this respect. The aim of the present work was to identify a plasma miRNA profile associated with VT. Approach and Results: miRNAs were analyzed by quantitative polymerase chain reaction in plasma samples from members of the GAIT-2 (Genetic Analysis of Idiopathic Thrombophilia 2) population (n=935). A discovery phase involving the screening of 752 miRNAs from a subset of 104 GAIT-2 subjects was followed by an internal validation phase in which the selected miRNAs were quantified in the whole GAIT-2 population. In the discovery phase, 16 miRNAs were selected, including 9 associated with VT and 7 that correlated with an intermediate phenotype of VT. In the next phase, 4 miRNAs were validated as differentially expressed (false discovery rate, <0.1) in VT: hsa-miR-126-3p, hsa-miR-885-5p, hsa-miR-194-5p, and hsa-miR-192-5p. The 4 miRNAs each returned a significant (P<0.05) odds ratio for VT (range of 1.3-1.8). A risk model including the 4 miRNAs, age, and sex returned an area under the receiver operating characteristic curve of 0.77. Moreover, all 4 miRNAs showed significant correlations with intermediate phenotypes of VT (eg, protein S and factor VII). The targets of the miRNAs in the blood coagulation pathway and their interactions are also discussed. CONCLUSIONS: The present results suggest a 4-miRNA plasma profile associated with VT is of potential use in predicting the risk of this condition.

Genome-wide association study with additional genetic and post-transcriptional analyses reveals novel regulators of plasma factor XI levels.

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 × 10-302), F11 (rs4253417, P-value = 2.86 × 10-193), and a novel association in GCKR (rs780094, P-value = 3.56 ×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.

Genome-wide association analysis of self-reported events in 6135 individuals and 252 827 controls identifies 8 loci associated with thrombosis.

Thrombotic diseases are among the leading causes of morbidity and mortality in the world. To add insights into the genetic regulation of thrombotic disease, we conducted a genome-wide association study (GWAS) of 6135 self-reported blood clots events and 252 827 controls of European ancestry belonging to the 23andMe cohort of research participants. Eight loci exceeded genome-wide significance. Among the genome-wide significant results, our study replicated previously known venous thromboembolism (VTE) loci near the F5, FGA-FGG, F11, F2, PROCR and ABO genes, and the more recently discovered locus near SLC44A2 In addition, our study reports for the first time a genome-wide significant association between rs114209171, located upstream of the F8 structural gene, and thrombosis risk. Analyses of expression profiles and expression quantitative trait loci across different tissues suggested SLC44A2, ILF3 and AP1M2 as the three most plausible candidate genes for the chromosome 19 locus, our only genome-wide significant thrombosis-related locus that does not harbor likely coagulation-related genes. In addition, we present data showing that this locus also acts as a novel risk factor for stroke and coronary artery disease (CAD). In conclusion, our study reveals novel common genetic risk factors for VTE, stroke and CAD and provides evidence that self-reported data on blood clots used in a GWAS yield results that are comparable with those obtained using clinically diagnosed VTE. This observation opens up the potential for larger meta-analyses, which will enable elucidation of the genetics of thrombotic diseases, and serves as an example for the genetic study of other diseases.

PDGFB, a new candidate plasma biomarker for venous thromboembolism: results from the VEREMA affinity proteomics study.

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 ß (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 (ρ ∼ 0.30) with each other, they were independently associated with VTE risk in a joint model in FARIVE (VWF P < .001; PDGFB P = .002). PDGFΒ 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.

Cost-effectiveness analysis of dabigatran and anticoagulation monitoring strategies of vitamin K antagonist.

BACKGROUND: Vitamin K antagonists are commonly used for the prevention of thromboembolic events. Patient self-monitoring of vitamin K antagonists has proved superior to usual care. Dabigatran has been shown, relative to warfarin, to reduce thromboembolic events without increasing bleeding. METHODS: We constructed a Markov model to compare vitamin K self-monitoring strategies to dabigatran including effectiveness and costs of monitoring and complications (thromboembolism and major bleeding). The model was used to project the incidence of these complications, life years, quality-adjusted life years, and health system costs with anticoagulant treatment throughout life. The analysis was conducted from the health system perspective and from the societal perspective. RESULTS: Low quality evidence suggests that self-monitoring is at least as effective as dabigatran for the outcomes of thrombosis, bleeding and death. Moderate quality evidence that patient self-monitoring is more effective than other forms of monitoring degree of anticoagulation with vitamin K antagonists, reducing the relative risk of thromboembolism by 41% and death by 34%. The cost per quality adjusted year gained relative to other warfarin monitoring strategies is well below 30,000 € in the short term, and is a dominant alternative from the fourth year. In comparison with dabigatran, the lower annual cost and its equivalence in terms of effectiveness made self-monitoring the dominant option. These results were confirmed in the probabilistic sensitivity analysis. CONCLUSIONS: We have moderate quality evidence that self-monitoring of vitamin K antagonists is a cost-effective alternative compared with hospital and primary care monitoring, and low quality evidence, compared with dabigatran. Our analyses contrast with the available cost analysis of dabigatran and usual care of anticoagulated patients.

Predicting individual risk of venous thrombosis.

de Haan et al have developed a risk score algorithm that is able to improve the individual prediction of venous thrombosis, taking into account information from multiple risk SNPs in addition to clinical data.

Self-monitoring of oral anticoagulation: systematic review and meta-analysis of individual patient data.

BACKGROUND: Uptake of self-testing and self-management of oral anticoagulation [corrected] has remained inconsistent, despite good evidence of their effectiveness. To clarify the value of self-monitoring of oral anticoagulation, we did a meta-analysis of individual patient data addressing several important gaps in the evidence, including an estimate of the effect on time to death, first major haemorrhage, and thromboembolism. METHODS: We searched Ovid versions of Embase (1980-2009) and Medline (1966-2009), limiting searches to randomised trials with a maximally sensitive strategy. We approached all authors of included trials and requested individual patient data: primary outcomes were time to death, first major haemorrhage, and first thromboembolic event. We did prespecified subgroup analyses according to age, type of control-group care (anticoagulation-clinic care vs primary care), self-testing alone versus self-management, and sex. We analysed patients with mechanical heart valves or atrial fibrillation separately. We used a random-effect model method to calculate pooled hazard ratios and did tests for interaction and heterogeneity, and calculated a time-specific number needed to treat. FINDINGS: Of 1357 abstracts, we included 11 trials with data for 6417 participants and 12,800 person-years of follow-up. We reported a significant reduction in thromboembolic events in the self-monitoring group (hazard ratio 0·51; 95% CI 0·31-0·85) but not for major haemorrhagic events (0·88, 0·74-1·06) or death (0·82, 0·62-1·09). Participants younger than 55 years showed a striking reduction in thrombotic events (hazard ratio 0·33, 95% CI 0·17-0·66), as did participants with mechanical heart valve (0·52, 0·35-0·77). Analysis of major outcomes in the very elderly (age ≥85 years, n=99) showed no significant adverse effects of the intervention for all outcomes. INTERPRETATION: Our analysis showed that self-monitoring and self-management of oral coagulation is a safe option for suitable patients of all ages. Patients should also be offered the option to self-manage their disease with suitable health-care support as back-up. FUNDING: UK National Institute for Health Research (NIHR) Technology Assessment Programme, UK NIHR National School for Primary Care Research.

A genome-wide association study identifies KNG1 as a genetic determinant of plasma factor XI Level and activated partial thromboplastin time.

OBJECTIVE: Elevated plasma levels of coagulation factor XI (FXI) are implicated in the pathogenesis of venous thromboembolism and ischemic stroke, and polymorphisms in the F11 gene are associated both with risk of venous thromboembolism and an elevated plasma FXI level. METHODS AND RESULTS: Here, we report the first hypothesis-free genome-wide genetic analysis of plasma FXI levels. Two genome-wide significant loci were detected in the family-based Genetic Analysis of Idiopathic Thrombophilia 1 cohort: one located in the kininogen 1 gene (KNG1) (rs710446; P=7.98 × 10(-10)) and one located in the structural F11 gene (rs4241824; P=1.16 × 10(-8)). Both associations were replicated in a second population-based Swedish cohort. A significant effect on KNG1 mRNA expression was also seen for the 2 most robustly FXI-associated single nucleotide polymorphisms located in KNG1. Furthermore, both KNG1 single nucleotide polymorphisms were associated with activated partial thromboplastin time, suggesting that FXI may be the main mechanistic pathway by which KNG1 and F11 influence activated partial thromboplastin time and risk of thrombosis. CONCLUSIONS: These findings contribute to the emerging molecular basis of venous thromboembolism and, more importantly, help in understanding the biological regulation of a phenotype that has proved to have promising therapeutic properties in relation to thrombosis.

Clinical outcomes in patients with atrial fibrillation treated with DOACs in a specialized anticoagulation center: Critical appraisal of real-world data.

AIMS: Direct oral anticoagulants (DOAC) are progressively replacing vitamin K antagonists in the prevention of thromboembolism in patients with atrial fibrillation. However, their real-world clinical outcomes appear to be contradictory, with some studies reporting fewer and others reporting higher complications than the pivotal randomized controlled trials. We present the results of a clinical model for the management of DOACs in real clinical practice and provide a review of the literature. METHODS: The MACACOD project is an ongoing, observational, prospective, single-center study with unselected patients that focuses on rigorous DOAC selection, an educational visit, laboratory measurements, and strict follow-up. RESULTS: A total of 1,259 patients were included. The composite incidence of major complications was 4.93% py in the whole cohort vs 4.49% py in the edoxaban cohort. The rate of all-cause mortality was 6.11% py for all DOACs vs 5.12% py for edoxaban. There weren't differences across sex or between Edoxaban reduced or standard doses. However, there were differences across ages, with a higher incidence of major bleeding complications in patients >85 years (5.13% py vs 1.69% py in <75 years). CONCLUSIONS: We observed an incidence of serious complications of 4.93% py, in which severe bleeding predominated (3.65% py). Considering our results, more specialized attention seems necessary to reduce the incidence of severe complications and also a more critical view of the literature. Considering our results, and our indirect comparison with many real-world studies, more specialized attention seems necessary to reduce the incidence of severe complications in AF patients receiving DOACs.

Development of a predictive model of venous thromboembolism recurrence in anticoagulated cancer patients using machine learning.

INTRODUCTION: Patients with cancer and venous thromboembolism (VTE) show a high risk of VTE recurrence during anticoagulant treatment. This study aimed to develop a predictive model to assess the risk of VTE recurrence within 6 months at the moment of primary VTE diagnosis in these patients. MATERIALS AND METHODS: Using the EHRead® technology, based on Natural Language Processing (NLP) and machine learning (ML), the unstructured data in electronic health records from 9 Spanish hospitals between 2014 and 2018 were extracted. Both clinically- and ML-driven feature selection were performed to identify predictors for VTE recurrence. Logistic regression (LR), decision tree (DT), and random forest (RF) algorithms were used to train different prediction models, which were subsequently validated in a hold-out data set. RESULTS: A total of 16,407 anticoagulated cancer patients with diagnosis of VTE were identified (54.4 % male and median age 70). Deep vein thrombosis, pulmonary embolism and metastases were observed in 67.2 %, 26.6 %, and 47.7 % of the patients, respectively. During the study follow-up, 11.4 % of the patients developed a recurrent VTE, being more frequent in patients with lung cancer. Feature selection and model training based on ML identified primary pulmonary embolism, deep vein thrombosis, metastasis, adenocarcinoma, hemoglobin and serum creatinine levels, platelet and leukocyte count, family history of VTE, and patients' age as predictors of VTE recurrence within 6 months of VTE diagnosis. The LR model had an AUC-ROC (95 % CI) of 0.66 (0.61, 0.70), the DT of 0.69 (0.65, 0.72) and the RF of 0.68 (0.63, 0.72). CONCLUSIONS: This is the first ML-based predictive model designed to predict 6-months VTE recurrence in patients with cancer. These results hold great potential to assist clinicians to identify the high-risk patients and improve their clinical management.

Multiallelic Copy Number Variation in ORM1 is Associated with Plasma Cell-Free DNA Levels as an Intermediate Phenotype for Venous Thromboembolism.

Venous thromboembolism (VTE) is a common disease with high heritability. However, only a small portion of the genetic variance of VTE can be explained by known genetic risk factors. Neutrophil extracellular traps (NETs) have been associated with prothrombotic activity. Therefore, the genetic basis of NETs could reveal novel risk factors for VTE. A recent genome-wide association study of plasma cell-free DNA (cfDNA) levels in the Genetic Analysis of Idiopathic Thrombophilia 2 (GAIT-2) Project showed a significant associated locus near ORM1. We aimed to further explore this candidate region by next-generation sequencing, copy number variation (CNV) quantification, and expression analysis using an extreme phenotype sampling design involving 80 individuals from the GAIT-2 Project. The RETROVE study with 400 VTE cases and 400 controls was used to replicate the results. A total of 105 genetic variants and a multiallelic CNV (mCNV) spanning ORM1 were identified in GAIT-2. Of these, 17 independent common variants, a region of 22 rare variants, and the mCNV were significantly associated with cfDNA levels. In addition, eight of these common variants and the mCNV influenced ORM1 expression. The association of the mCNV and cfDNA levels was replicated in RETROVE (p-value = 1.19 × 10-6). Additional associations between the mCNV and thrombin generation parameters were identified. Our results reveal that increased mCNV dosages in ORM1 decreased gene expression and upregulated cfDNA levels. Therefore, the mCNV in ORM1 appears to be a novel marker for cfDNA levels, which could contribute to VTE risk.

Polygenic risk of major depressive disorder as a risk factor for venous thromboembolism.

Major depressive disorder (MDD), bipolar disorder (BD), and schizophrenia (SCZ) are associated with an increased risk of cardiovascular diseases, including venous thromboembolism (VTE). The reasons for this are complex and include obesity, smoking, and use of hormones and psychotropic medications. Genetic studies have increasingly provided evidence of the shared genetic risk of psychiatric and cardiometabolic illnesses. This study aimed to determine whether a genetic predisposition to MDD, BD, or SCZ is associated with an increased risk of VTE. Genetic correlations using the largest genome-wide genetic meta-analyses summary statistics for MDD, BD, and SCZ (Psychiatric Genetics Consortium) and a recent genome-wide genetic meta-analysis of VTE (INVENT Consortium) demonstrated a positive association between VTE and MDD but not BD or SCZ. The same summary statistics were used to construct polygenic risk scores for MDD, BD, and SCZ in UK Biobank participants of self-reported White British ancestry. These were assessed for impact on self-reported VTE risk (10 786 cases, 285 124 controls), using logistic regression, in sex-specific and sex-combined analyses. We identified significant positive associations between polygenic risk for MDD and the risk of VTE in men, women, and sex-combined analyses, independent of the known risk factors. Secondary analyses demonstrated that this association was not driven by those with lifetime experience of mental illness. Meta-analyses of individual data from 6 additional independent cohorts replicated the sex-combined association. This report provides evidence for shared biological mechanisms leading to MDD and VTE and suggests that, in the absence of genetic data, a family history of MDD might be considered when assessing the risk of VTE.

Sequence variation and genetic evolution at the human F12 locus: mapping quantitative trait nucleotides that influence FXII plasma levels.

The level of Factor XII (FXII) is an important phenotype that exhibits a high genetic component and is associated with thrombotic disease. In a genome-wide linkage scan, we demonstrated that the F12 gene represents a quantitative trait locus (QTL) that influences FXII levels. The current study investigated the genetic architecture of the F12 gene to locate polymorphism(s) responsible for the variation of FXII levels. Re-sequencing of the F12 gene in 40 unrelated individuals (selected from the tails of normal distribution of FXII levels) identified 26 polymorphisms which were genotyped in 398 individuals belonging to 21 families from the GAIT Project. By a measured genotype association analysis, eight of 26 SNPs showed significant P-values less than 10(-5) (after multiple test correction) with FXII levels. In addition, the Bayesian Quantitative Trait Nucleotide method, which infers those polymorphisms most likely to have a direct influence on the trait under study, provided evidence that only rs1801020 variation accounted for the variance attributed to this QTL. Moreover, we have analyzed the evolutionary processes that produced the variation in F12 gene and concluded that is evolutionarily neutral and that the T allele of the rs1801020 appeared approximately 100 000 years ago and spread to most human populations rising to high frequencies by genetic drift. Our study provides a template for future genetic studies of human quantitative traits, as we move beyond QTL localization to the polymorphisms responsible for the variation of important biomedical phenotypes.

C4BPB/C4BPA is a new susceptibility locus for venous thrombosis with unknown protein S-independent mechanism: results from genome-wide association and gene expression analyses followed by case-control studies.

Through its binding with protein S (PS), a key element of the coagulation/fibrinolysis cascade, the C4b-binding protein (C4BP) has been hypothesized to be involved in the susceptibility to venous thrombosis (VT). To identify genetic factors that may influence the plasma levels of the 3 C4BP existing isoforms, alpha(7)beta(1), alpha(6)beta(1), and alpha(7)beta(0), we conducted a genome-wide association study by analyzing 283 437 single nucleotide polymorphisms (SNPs) in the Genetic Analysis of Idiopathic Thrombophilia (GAIT) study composed of 352 persons. Three SNPs at the C4BPB/C4BPA locus were found genome-wide significantly associated with alpha(7)beta(0) levels. One of these SNPs was further found to explain approximately 11% of the variability of mRNA C4BPA expression in the Gutenberg Heart Study composed of 1490 persons, with no effect on C4BPB mRNA expression. The allele associated with increased alpha(7)beta(0) plasma levels and increased C4BPA expression was further found associated with increased risk of VT (odds ratio [OR] = 1.24 [1.03-1.53]) in 2 independent case-control studies (MARseille THrombosis Association study [MARTHA] and FActeurs de RIsque et de récidives de la maladie thromboembolique VEineuse [FARIVE]) gathering 1706 cases and 1379 controls. This SNP was not associated with free PS or total PS. In conclusion, we observed strong evidence that the C4BPB/C4BPA locus is a new susceptibility locus for VT through a PS-independent mechanism that remains to be elucidated.

Integrated GWAS and Gene Expression Suggest ORM1 as a Potential Regulator of Plasma Levels of Cell-Free DNA and Thrombosis Risk.

Plasma cell-free DNA (cfDNA) is a surrogate marker of neutrophil extracellular traps (NETs) that contribute to immunothrombosis. There is growing interest about the mechanisms underlying NET formation and elevated cfDNA, but little is known about the factors involved. We aimed to identify genes involved in the regulation of cfDNA levels using data from the Genetic Analysis of Idiopathic Thrombophilia (GAIT-2) Project.Imputed genotypes, whole blood RNA-Seq data, and plasma cfDNA quantification were available for 935 of the GAIT-2 participants from 35 families with idiopathic thrombophilia. We performed heritability and GWAS analysis for cfDNA. The heritability of cfDNA was 0.26 (p = 3.7 × 10-6), while the GWAS identified a significant association (rs1687391, p = 3.55 × 10-10) near the ORM1 gene, on chromosome 9. An eQTL (expression quantitative trait loci) analysis revealed a significant association between the lead GWAS variant and the expression of ORM1 in whole blood (p = 6.14 × 10-9). Additionally, ORM1 expression correlated with levels of cfDNA (p = 4.38 × 10-4). Finally, genetic correlation analysis between cfDNA and thrombosis identified a suggestive association (ρ g = 0.43, p = 0.089).All in all, we show evidence of the role of ORM1 in regulating cfDNA levels in plasma, which might contribute to the susceptibility to thrombosis through mechanisms of immunothrombosis.

Polymorphonuclear neutrophils and cancer: intense and sustained neutrophilia as a treatment against solid tumors.

Polymorphonuclear neutrophils (PMN) are the most abundant circulating immune cells and represent the first line of immune defense against infection. This review of the biomedical literature of the last 40 years shows that they also have a powerful antitumoral effect under certain circumstances. Typically, the microenvironment surrounding a solid tumor possesses many of the characteristics of chronic inflammation, a condition considered very favorable for tumor growth and spread. However, there are many circumstances that shift the chronic inflammatory state toward an acute inflammatory response around a tumor. This shift seems to convert PMN into very efficient anticancer effector cells. Clinical reports of unexpected antitumoral effects linked to the prolonged use of granulocyte colony-stimulating factor, which stimulates an intense and sustained neutrophilia, suggest that an easy way to fight solid tumors would be to encourage the development of intense peritumoral PMN infiltrates. Specifically designed clinical trials are urgently needed to evaluate the safety and efficacy of such drug-induced neutrophilia in patients with solid tumors. This antitumoral role of neutrophils may provide new avenues for the clinical treatment of cancer.