Plasma levels of complement components C5 and C9 are associated with thrombin generation.

BACKGROUND: The thrombin generation assay (TGA) evaluates the potential of plasma to generate thrombin over time, providing a global picture of an individual's hemostatic balance. OBJECTIVES: This study aimed to identify novel biological determinants of thrombin generation using a multiomics approach. METHODS: Associations between TGA parameters and plasma levels of 377 antibodies targeting 236 candidate proteins for cardiovascular risk were tested using multiple linear regression analysis in 770 individuals with venous thrombosis from the Marseille Thrombosis Association (MARTHA) study. Proteins associated with at least 3 TGA parameters were selected for validation in an independent population of 536 healthy individuals (Etablissement Français du Sang Alpes-Méditerranée [EFS-AM]). Proteins with strongest associations in both groups underwent additional genetic analyses and in vitro experiments. RESULTS: Eighteen proteins were associated (P < 1.33 × 10⁻4) with at least 3 TGA parameters in MARTHA, among which 13 demonstrated a similar pattern of associations in EFS-AM. Complement proteins C5 and C9 had the strongest associations in both groups. Ex vivo supplementation of platelet-poor plasma with purified C9 protein had a significant dose-dependent effect on TGA parameters. No effect was observed with purified C5. Several single nucleotide polymorphisms associated with C5 and C9 plasma levels were identified, with the strongest association for the C5 missense variant rs17611, which was associated with a decrease in C5 levels, endogenous thrombin potential, and peak in MARTHA. No association of this variant with TGA parameters was observed in EFS-AM. CONCLUSION: This study identified complement proteins C5 and C9 as potential determinants of thrombin generation. Further studies are warranted to establish causality and elucidate the underlying mechanisms.

Genome-wide investigation of exogenous female hormones, genetic variation, and venous thromboembolism risk.

BACKGROUND: Increased risk of venous thromboembolism (VTE) is a life-threatening side effect for users of oral contraceptives (OCs) or hormone therapy (HT). OBJECTIVES: To investigate the potential for genetic predisposition to VTE in OC or HT users, we conducted a gene-by-environment case-only meta-analysis of genome-wide association studies (GWAS). METHODS: Use or nonuse of OCs (7 studies) or HT (8 studies) at the time of the VTE event was determined by pharmacy records or self-report. A synergy index (SI) was modeled for each variant in each study and submultiplicative/supramultiplicative gene-by-environment interactions were estimated. The SI parameters were first meta-analyzed across OC and HT studies and subsequently meta-analyzed to obtain an overall estimate. The primary analysis was agnostic GWAS and interrogated all imputed genotypes using a P value threshold of <5.0 × 10-8; secondary analyses were candidate-based. RESULTS: The VTE case-only OC meta-analysis included 2895 OC users and 6607 nonusers; the case-only HT meta-analysis included 2434 HT users and 12 793 nonusers. In primary GWAS meta-analyses, no variant reached genome-wide significance, but the smallest P value approached statistical significance: rs9386463 (P = 5.03 × 10-8). We tested associations for 138 candidate variants and identified 2 that exceeded statistical significance (0.05/138 = 3.62 × 10-4): F5 rs6025 (P = 1.87 × 10-5; SI, 1.29; previously observed) and F11 rs2036914 (P = 2.0 × 10-4; SI, 0.91; new observation). CONCLUSION: The candidate variant approach to identify submultiplictive/supramultiplicative associations between genetic variation and OC and HT use identified a new association with common genetic variation in F11, while the agnostic interrogations did not yield new discoveries.

Genome-wide association study of a semicontinuous trait: illustration of the impact of the modeling strategy through the study of Neutrophil Extracellular Traps levels.

Over the last years, there has been a considerable expansion of genome-wide association studies (GWAS) for discovering biological pathways underlying pathological conditions or disease biomarkers. These GWAS are often limited to binary or quantitative traits analyzed through linear or logistic models, respectively. In some situations, the distribution of the outcome may require more complex modeling, such as when the outcome exhibits a semicontinuous distribution characterized by an excess of zero values followed by a non-negative and right-skewed distribution. We here investigate three different modeling for semicontinuous data: Tobit, Negative Binomial and Compound Poisson-Gamma. Using both simulated data and a real GWAS on Neutrophil Extracellular Traps (NETs), an emerging biomarker in immuno-thrombosis, we demonstrate that Compound Poisson-Gamma was the most robust model with respect to low allele frequencies and outliers. This model further identified the MIR155HG locus as significantly (P = 1.4 × 10-8) associated with NETs plasma levels in a sample of 657 participants, a locus recently highlighted to be involved in NETs formation in mice. This work highlights the importance of the modeling strategy for GWAS of a semicontinuous outcome and suggests Compound Poisson-Gamma as an elegant but neglected alternative to Negative Binomial for modeling semicontinuous outcome in the context of genomic investigations.

Elevated plasma complement factor H related 5 protein is associated with venous thromboembolism.

Venous thromboembolism (VTE) is a common, multi-causal disease with potentially serious short- and long-term complications. In clinical practice, there is a need for improved plasma biomarker-based tools for VTE diagnosis and risk prediction. Here we show, using proteomics profiling to screen plasma from patients with suspected acute VTE, and several case-control studies for VTE, how Complement Factor H Related 5 protein (CFHR5), a regulator of the alternative pathway of complement activation, is a VTE-associated plasma biomarker. In plasma, higher CFHR5 levels are associated with increased thrombin generation potential and recombinant CFHR5 enhanced platelet activation in vitro. GWAS analysis of ~52,000 participants identifies six loci associated with CFHR5 plasma levels, but Mendelian randomization do not demonstrate causality between CFHR5 and VTE. Our results indicate an important role for the regulation of the alternative pathway of complement activation in VTE and that CFHR5 represents a potential diagnostic and/or risk predictive plasma biomarker.

Association of ABO blood groups with venous thrombosis recurrence in middle-aged patients: insights from a weighted Cox analysis dedicated to ambispective design.

BACKGROUND: In studies of time-to-events, it is common to collect information about events that occurred before the inclusion in a prospective cohort. When the studied risk factors are independent of time, including both pre- and post-inclusion events in the analyses, generally referred to as relying on an ambispective design, increases the statistical power but may lead to a selection bias. In the field of venous thromboembolism (VT), ABO blood groups have been the subject of extensive research due to their substantial effect on VT risk. However, few studies have investigated their effect on the risk of VT recurrence. Motivated by the study of the association of genetically determined ABO blood groups with VT recurrence, we propose a methodology to include pre-inclusion events in the analysis of ambispective studies while avoiding the selection bias due to mortality. METHODS: This work relies on two independent cohorts of VT patients, the French MARTHA study built on an ambispective design and the Dutch MEGA study built on a standard prospective design. For the analysis of the MARTHA study, a weighted Cox model was developed where weights were defined by the inverse of the survival probability at the time of data collection about the events. Thanks to the collection of information on the vital status of patients, we could estimate the survival probabilities using a delayed-entry Cox model on the death risk. Finally, results obtained in both studies were then meta-analysed. RESULTS: In the combined sample totalling 2,752 patients including 993 recurrences, the A1 blood group has an increased risk (Hazard Ratio (HR) of 1.18, p = 4.2 × 10-3) compared with the O1 group, homogeneously in MARTHA and in MEGA. The same trend (HR = 1.19, p = 0.06) was observed for the less frequent A2 group. CONCLUSION: The proposed methodology increases the power of studies relying on an ambispective design which is frequent in epidemiologic studies about recurrent events. This approach allowed to clarify the association of ABO blood groups with the risk of VT recurrence. Besides, this methodology has an immediate field of application in the context of genome wide association studies.

Bayesian network analysis of plasma microRNA sequencing data in patients with venous thrombosis.

MicroRNAs (miRNAs) are small regulatory RNAs participating to several biological processes and known to be involved in various pathologies. Measurable in body fluids, miRNAs have been proposed to serve as efficient biomarkers for diseases and/or associated traits. Here, we performed a next-generation-sequencing based profiling of plasma miRNAs in 344 patients with venous thrombosis (VT) and assessed the association of plasma miRNA levels with several haemostatic traits and the risk of VT recurrence. Among the most significant findings, we detected an association between hsa-miR-199b-3p and haematocrit levels (P = 0.0016), these two markers having both been independently reported to associate with VT risk. We also observed suggestive evidence for association of hsa-miR-370-3p (P = 0.019), hsa-miR-27b-3p (P = 0.016) and hsa-miR-222-3p (P = 0.049) with VT recurrence, the observations at the latter two miRNAs confirming the recent findings of Wang et al. Besides, by conducting Genome-Wide Association Studies on miRNA levels and meta-analyzing our results with some publicly available, we identified 21 new associations of single nucleotide polymorphisms with plasma miRNA levels at the statistical significance threshold of P < 5 × 10-8, some of these associations pertaining to thrombosis associated mechanisms. In conclusion, this study provides novel data about the impact of miRNAs' variability in haemostasis and new arguments supporting the association of few miRNAs with the risk of recurrence in patients with venous thrombosis.

Los micro-ARN (miARN) son pequeñas moléculas de ARN reguladoras que participan en varios procesos biológicos y están implicados en diversas patologías. Mensurables en los líquidos corporales, se ha planteado que los miARN pueden ser biomarcadores eficaces para el diagnóstico de enfermedades y/o características asociadas. Aquí hemos llevado a cabo un análisis de miARN plasmático con tecnología de secuenciación de última generación en 344 pacientes con trombosis venosa (TV) y hemos evaluado la asociación de los niveles de miARN con distintas características hemostáticas y el riesgo de recidiva de TV. Entre los hallazgos más significativos, hemos detectado una asociación entre hsa-miR-199b-3p y los niveles de hematocritos (p = 0,0016); dos marcadores que se habían asociado de forma independiente con el riesgo de sufrir TV. Asimismo, hemos observado una evidencia indicativa de asociación entre hsa-miR-370-3p (p = 0,019), hsa-miR-27b-3p (p = 0,016) y hsa-miR-222-3p (p = 0,049) y la recidiva de TV; los resultados los dos últimos miARN confirman los hallazgos recientes de Wang et al. (Clin Epigenetics, 2019). Además, al efectuar estudios de asociación del genoma completo sobre los niveles de miARN y al metaanalizar nuestros resultados con otros disponibles públicamente, hemos identificado 21 asociaciones nuevas de polimorfismos de un solo nucleótido (PSN) con niveles de miARN plasmático con un umbral de significación estadística de p < 5 × 10−8; algunas de estas asociaciones pertenecen a los mecanismos patogénicos de la trombosis.Como conclusión, en este estudio se proporcionan nuevos datos sobre el impacto de la variabilidad de miARN en la hemostasia y nuevos argumentos que apoyan la asociación de algunas secuencias de miARN con el riesgo de recidiva en pacientes con trombosis venosa.