Multi-ancestry polygenic risk scores for venous thromboembolism.

Venous thromboembolism (VTE) is a significant contributor to morbidity and mortality, with large disparities in incidence rates between Black and White Americans. Polygenic risk scores (PRSs) limited to variants discovered in genome-wide association studies in European-ancestry samples can identify European-ancestry individuals at high risk of VTE. However, there is limited evidence on whether high-dimensional PRS constructed using more sophisticated methods and more diverse training data can enhance the predictive ability and their utility across diverse populations. We developed PRSs for VTE using summary statistics from the International Network against Venous Thrombosis (INVENT) consortium genome-wide association studies meta-analyses of European- (71 771 cases and 1 059 740 controls) and African-ancestry samples (7482 cases and 129 975 controls). We used LDpred2 and PRS-CSx to construct ancestry-specific and multi-ancestry PRSs and evaluated their performance in an independent European- (6781 cases and 103 016 controls) and African-ancestry sample (1385 cases and 12 569 controls). Multi-ancestry PRSs with weights tuned in European-ancestry samples slightly outperformed ancestry-specific PRSs in European-ancestry test samples (e.g. the area under the receiver operating curve [AUC] was 0.609 for PRS-CSx_combinedEUR and 0.608 for PRS-CSxEUR [P = 0.00029]). Multi-ancestry PRSs with weights tuned in African-ancestry samples also outperformed ancestry-specific PRSs in African-ancestry test samples (PRS-CSxAFR: AUC = 0.58, PRS-CSx_combined AFR: AUC = 0.59), although this difference was not statistically significant (P = 0.34). The highest fifth percentile of the best-performing PRS was associated with 1.9-fold and 1.68-fold increased risk for VTE among European- and African-ancestry subjects, respectively, relative to those in the middle stratum. These findings suggest that the multi-ancestry PRS might be used to improve performance across diverse populations to identify individuals at highest risk for VTE.

Testing for association with rare variants in the coding and non-coding genome: RAVA-FIRST, a new approach based on CADD deleteriousness score.

Rare variant association tests (RVAT) have been developed to study the contribution of rare variants widely accessible through high-throughput sequencing technologies. RVAT require to aggregate rare variants in testing units and to filter variants to retain only the most likely causal ones. In the exome, genes are natural testing units and variants are usually filtered based on their functional consequences. However, when dealing with whole-genome sequence (WGS) data, both steps are challenging. No natural biological unit is available for aggregating rare variants. Sliding windows procedures have been proposed to circumvent this difficulty, however they are blind to biological information and result in a large number of tests. We propose a new strategy to perform RVAT on WGS data: "RAVA-FIRST" (RAre Variant Association using Functionally-InfoRmed STeps) comprising three steps. (1) New testing units are defined genome-wide based on functionally-adjusted Combined Annotation Dependent Depletion (CADD) scores of variants observed in the gnomAD populations, which are referred to as "CADD regions". (2) A region-dependent filtering of rare variants is applied in each CADD region. (3) A functionally-informed burden test is performed with sub-scores computed for each genomic category within each CADD region. Both on simulations and real data, RAVA-FIRST was found to outperform other WGS-based RVAT. Applied to a WGS dataset of venous thromboembolism patients, we identified an intergenic region on chromosome 18 enriched for rare variants in early-onset patients. This region that was missed by standard sliding windows procedures is included in a TAD region that contains a strong candidate gene. RAVA-FIRST enables new investigations of rare non-coding variants in complex diseases, facilitated by its implementation in the R package Ravages.

Two SERPINC1 variants affecting N-glycosylation of Asn224 cause severe thrombophilia not detected by functional assays.

Antithrombin deficiency, the most severe congenital thrombophilia, might be underestimated, as some pathogenic variants are not detected by routine functional methods. We have identified 2 new SERPINC1 variants, p.Glu227Lys and p.Asn224His, in 4 unrelated thrombophilic patients with early and recurrent thrombosis that had normal antithrombin activity. In one case, the mutation was identified by whole genome sequencing, while in the 3 remaining cases, the mutation was identified by sequencing SERPINC1 based on a single functional positive finding supporting deficiency. The 2 variants shared a common functional defect, an impaired or null N-glycosylation of Asn224 according to a eukaryotic expression model. Carriers had normal anti-FXa or anti-FIIa activities but impaired anti-FVIIa activity and a detectable loss of inhibitory function when incubating the plasma for 1 hour at 41°C. Moreover, the ß glycoform of the variants, lacking 2 N-glycans, had reduced secretion, increased heparin affinity, no inhibitory activity, and a potential dominant-negative effect. These results explain the increased thrombin generation observed in carriers. Mutation experiments reflected the role that Lysine residues close to the N-glycosylation sequon have in impairing the efficacy of N-glycosylation. Our study shows new elements involved in the regulation of N-glycosylation, a key posttranslational modification that, according to our results, affects folding, secretion, and function, providing new evidence of the pathogenic consequence of an incorrect N-glycosylation of antithrombin. This study supports that antithrombin deficiency is underestimated and encourages the development of new functional and genetic tests to diagnose this severe thrombophilia.

Relationship between plasma tissue Factor Pathway Inhibitor (TFPI) levels, thrombin generation and clinical risk of bleeding in patients with severe haemophilia A or B.

INTRODUCTION: Bleeding severity in severe haemophilic patients, with low thrombin generation (TG) capacity, can vary widely between patients, possibly reflecting differences in tissue factor pathway inhibitor (TFPI) level. AIM: To compare free TFPI (fTFPI) levels in patients with severe haemophilia A (sHA) and severe haemophilia B (sHB) and to investigate in these patients as a whole the relationships between bleeding and TG potential, between TG potential and fTFPI level and between fTFPI level and bleeding tendency. METHODS: Data on bleeding episodes retrospectively recorded during follow-up visits over 5-10 years were collected and used to calculate the annualised joint bleeding rate (AJBR). fTFPI levels and basal TG parameters were determined in platelet-poor plasma (PPP) and platelet-rich plasma (PRP) using calibrated automated tomography (CAT). RESULTS: Mean fTFPI levels did not differ significantly between sHA (n = 34) and sHB (n = 19) patients. Mean values of endogenous thrombin potential (ETP) and thrombin peak (peak) in PPP and PRP were two-fold higher when fTFPI levels < 9.4 versus > 14.3 ng/mL. In patients treated on demand, ETP and peak in PRP were doubled when AJBR was ≤ 4.9 $ \le 4.9$ , AJBR being halved in patients with a low fTFPI level (9.4 ng/mL). In patients on factor prophylaxis, no association was found between TG parameters and either fTFPI level or AJBR. CONCLUSION: In patients treated on demand, bleeding tendency was influenced by fTFPI levels, which in turn affected basal TG potential. In patients on prophylaxis, bleeding tendency is probably determined primarily by the intensity of this treatment.

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.

A rare coding mutation in the MAST2 gene causes venous thrombosis in a French family with unexplained thrombophilia: The Breizh MAST2 Arg89Gln variant.

Rare variants outside the classical coagulation cascade might cause inherited thrombosis. We aimed to identify the variant(s) causing venous thromboembolism (VTE) in a family with multiple relatives affected with unprovoked VTE and no thrombophilia defects. We identified by whole exome sequencing an extremely rare Arg to Gln variant (Arg89Gln) in the Microtubule Associated Serine/Threonine Kinase 2 (MAST2) gene that segregates with VTE in the family. Free-tissue factor pathway inhibitor (f-TFPI) plasma levels were significantly decreased in affected family members compared to healthy relatives. Conversely, plasminogen activator inhibitor-1 (PAI-1) levels were significantly higher in affected members than in healthy relatives. RNA sequencing analysis of RNA interference experimental data conducted in endothelial cells revealed that, of the 13,387 detected expressed genes, 2,354 have their level of expression modified by MAST2 knockdown, including SERPINE1 coding for PAI-1 and TFPI. In HEK293 cells overexpressing the MAST2 Gln89 variant, TFPI and SERPINE1 promoter activities were respectively lower and higher than in cells overexpressing the MAST2 wild type. This study identifies a novel thrombophilia-causing Arg89Gln variant in the MAST2 gene that is here proposed as a new molecular player in the etiology of VTE by interfering with hemostatic balance of endothelial cells.

A Platelet Function Modulator of Thrombin Activation Is Causally Linked to Cardiovascular Disease and Affects PAR4 Receptor Signaling.

Dual antiplatelet therapy reduces ischemic events in cardiovascular disease, but it increases bleeding risk. Thrombin receptors PAR1 and PAR4 are drug targets, but the role of thrombin in platelet aggregation remains largely unexplored in large populations. We performed a genome-wide association study (GWAS) of platelet aggregation in response to full-length thrombin, followed by clinical association analyses, Mendelian randomization, and functional characterization including iPSC-derived megakaryocyte and platelet experiments. We identified a single sentinel variant in the GRK5 locus (rs10886430-G, p = 3.0 × 10-42) associated with increased thrombin-induced platelet aggregation (ß = 0.70, SE = 0.05). We show that disruption of platelet GRK5 expression by rs10886430-G is associated with enhanced platelet reactivity. The proposed mechanism of a GATA1-driven megakaryocyte enhancer is confirmed in allele-specific experiments. Utilizing further data, we demonstrate that the allelic effect is highly platelet- and thrombin-specific and not likely due to effects on thrombin levels. The variant is associated with increased risk of cardiovascular disease outcomes in UK BioBank, most strongly with pulmonary embolism. The variant associates with increased risk of stroke in the MEGASTROKE, UK BioBank, and FinnGen studies. Mendelian randomization analyses in independent samples support a causal role for rs10886430-G in increasing risk for stroke, pulmonary embolism, and venous thromboembolism through its effect on thrombin-induced platelet reactivity. We demonstrate that G protein-coupled receptor kinase 5 (GRK5) promotes platelet activation specifically via PAR4 receptor signaling. GRK5 inhibitors in development for the treatment of heart failure and cancer could have platelet off-target deleterious effects. Common variants in GRK5 may modify clinical outcomes with PAR4 inhibitors, and upregulation of GRK5 activity or signaling in platelets may have therapeutic benefits.

Impaired adhesion of neutrophils expressing Slc44a2/HNA-3b to VWF protects against NETosis under venous shear rates.

Genome-wide association studies linked expression of the human neutrophil antigen 3b (HNA-3b) epitope on the Slc44a2 protein with a 30% decreased risk of venous thrombosis (VT) in humans. Slc44a2 is a ubiquitous transmembrane protein identified as a receptor for von Willebrand factor (VWF). To explain the link between Slc44a2 and VT, we wanted to determine how Slc44a2 expressing either HNA-3a or HNA-3b on neutrophils could modulate their adhesion and activation on VWF under flow. Transfected HEK293T cells or neutrophils homozygous for the HNA-3a- or HNA-3b-coding allele were purified from healthy donors and perfused in flow chambers coated with VWF at venous shear rates (100 s-1). HNA-3a expression was required for Slc44a2-mediated neutrophil adhesion to VWF at 100 s-1. This adhesion could occur independently of ß2 integrin and was enhanced when neutrophils were preactivated with lipopolysaccharide. Moreover, specific shear conditions with high neutrophil concentration could act as a "second hit," inducing the formation of neutrophil extracellular traps. Neutrophil mobilization was also measured by intravital microscopy in venules from SLC44A2-knockout and wild-type mice after histamine-induced endothelial degranulation. Mice lacking Slc44a2 showed a massive reduction in neutrophil recruitment in inflamed mesenteric venules. Our results show that Slc44a2/HNA-3a is important for the adhesion and activation of neutrophils in veins under inflammation and when submitted to specific shears. The fact that neutrophils expressing Slc44a2/HNA-3b have a different response on VWF in the conditions tested could thus explain the association between HNA-3b and a reduced risk for VT in humans.

Association between ABO haplotypes and the risk of venous thrombosis: impact on disease risk estimation.

Genetic risk score (GRS) analysis is a popular approach to derive individual risk prediction models for complex diseases. In venous thrombosis (VT), such type of analysis shall integrate information at the ABO blood group locus, which is one of the major susceptibility loci. However, there is no consensus about which single nucleotide polymorphisms (SNPs) must be investigated when properly assessing association between ABO locus and VT risk. Using comprehensive haplotype analyses of ABO blood group tagging SNPs in 5425 cases and 8445 controls from 6 studies, we demonstrate that using only rs8176719 (tagging O1) to correctly assess the impact of ABO locus on VT risk is suboptimal, because 5% of rs8176719-delG carriers do not have an increased risk of developing VT. Instead, we recommend the use of 4 SNPs, rs2519093 (tagging A1), rs1053878 (A2), rs8176743 (B), and rs41302905 (O2), when assessing the impact of ABO locus on VT risk to avoid any risk misestimation. Compared with the O1 haplotype, the A2 haplotype is associated with a modest increase in VT risk (odds ratio, âˆ¼1.2), the A1 and B haplotypes are associated with an âˆ¼1.8-fold increased risk, whereas the O2 haplotype tends to be slightly protective (odds ratio, âˆ¼0.80). In addition, although the A1 and B blood groups are associated with increased von Willebrand factor and factor VIII plasma levels, only the A1 blood group is associated with ICAM levels, but in an opposite direction, leaving additional avenues to be explored to fully understand the spectrum of biological effects mediated by ABO locus on cardiovascular traits.

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.

OPTIMIR, a novel algorithm for integrating available genome-wide genotype data into miRNA sequence alignment analysis.

Next-generation sequencing is an increasingly popular and efficient approach to characterize the full set of microRNAs (miRNAs) present in human biosamples. MiRNAs' detection and quantification still remain a challenge as they can undergo different posttranscriptional modifications and might harbor genetic variations (polymiRs) that may impact on the alignment step. We present a novel algorithm, OPTIMIR, that incorporates biological knowledge on miRNA editing and genome-wide genotype data available in the processed samples to improve alignment accuracy. OPTIMIR was applied to 391 human plasma samples that had been typed with genome-wide genotyping arrays. OPTIMIR was able to detect genotyping errors, suggested the existence of novel miRNAs and highlighted the allelic imbalance expression of polymiRs in heterozygous carriers. OPTIMIR is written in python, and freely available on the GENMED website (http://www.genmed.fr/index.php/fr/) and on Github (github.com/FlorianThibord/OptimiR).

CD169 and CD64 could help differentiate bacterial from CoVID-19 or other viral infections in the Emergency Department.

The identification of a bacterial, viral, or even noninfectious cause is essential in the management of febrile syndrome in the emergency department (ED), especially in epidemic contexts such as flu or CoVID-19. The aim was to assess discriminative performances of two biomarkers, CD64 on neutrophils (nCD64) and CD169 on monocytes (mCD169), using a new flow cytometry procedure, in patients presenting with fever to the ED during epidemics. Eighty five adult patients presenting with potential infection were included during the 2019 flu season in the ED of La Timone Hospital. They were divided into four diagnostic outcomes according to their clinical records: no-infection, bacterial infection, viral infection and co-infection. Seventy six patients with confirmed SARS-CoV-2 infection were also compared to 48 healthy volunteers. For the first cohort, 38 (45%) patients were diagnosed with bacterial infections, 11 (13%) with viral infections and 29 (34%) with co-infections. mCD169 was elevated in patients with viral infections, with a majority of Flu A virus or Respiratory Syncytial Virus, while nCD64 was elevated in subjects with bacterial infections, with a majority of Streptococcus pneumoniae and Escherichia coli. nCD64 and mCD169 showed 90% and 80% sensitivity, and 78% and 91% specificity, respectively, for identifying patients with bacterial or viral infections. When studied in a second cohort, mCD169 was elevated in 95% of patients with SARS-CoV-2 infections and remained at normal level in 100% of healthy volunteers. nCD64 and mCD169 have potential for accurately distinguishing bacterial and acute viral infections. Combined in an easy and rapid flow cytometry procedure, they constitute a potential improvement for infection management in the ED, and could even help for triage of patients during emerging epidemics.

Plasma Biomarkers and Identification of Resilient Metabolic Disruptions in Patients With Venous Thromboembolism Using a Metabolic Systems Approach.

OBJECTIVE: Deep vein thrombosis and pulmonary embolism referred as venous thromboembolism (VTE) are a common cause of morbidity and mortality. Plasma from healthy controls or individuals who have experienced a VTE were analyzed using metabolomics to characterize biomarkers and metabolic systems of patients with VTE. Approach and Results: Polar metabolite and lipidomic profiles from plasma collected 3 months after an incident VTE were obtained using liquid chromatography mass spectrometry. Fasting-state plasma samples from 42 patients with VTE and 42 healthy controls were measured. Plasma metabolomic profiling identified 512 metabolites forming 62 biological clusters. Multivariate analysis revealed a panel of 21 metabolites altogether capable of predicting VTE status with an area under the curve of 0.92 (P=0.00174, selectivity=0.857, sensitivity=0.971). Multiblock systems analysis revealed 25 of the 62 functional biological groups as significantly affected in the VTE group (P<0.05 to control). Complementary correlation network analysis of the dysregulated functions highlighted a subset of the lipidome composed mainly of n-3 long-chain polyunsaturated fatty acids within the predominant triglycerides as a potential regulator of the post-VTE event biological response, possibly controlling oxidative and inflammatory defence systems, and metabolic disorder associated dysregulations. Of interest was microbiota metabolites including trimethylamine N-oxide that remained associated to post incident VTE patients, highlighting a possible involvement of gut microbiota on VTE risk and relapse. CONCLUSIONS: These findings show promise for the elucidation of underlying mechanisms and the design of a diagnostic test to assess the likely efficacy of clinical care in patients with VTE.

Clinical validation of immunoassay HemosIL® AcuStar HIT-IgG (PF4-H) in the diagnosis of Heparin-induced thrombocytopenia.

Heparin induced thrombocytopenia (HIT) is a life and limb-threatening complication of heparin exposure. The misdiagnosis of this disease can have major consequences on the patients. The objective of this study was to evaluate a diagnostic strategy that combines the 4Ts score with the result of HemosIL® AcuStar HIT-IgG (PF4-H) to confirm the diagnosis of HIT. Citrated plasmas from 1300 patients with suspicion of HIT were analyzed with a fully automated quantitative chemiluminescent immunoassay (HemosIL® AcuStar HIT-IgG (PF4/H)). If the IgG anti-PF4/H antibodies were positive (cut-off, 1 U/mL), HIT diagnosis was confirmed using functional tests. In total, 1300 samples of consecutive patients were enrolled, 94 (7.2%) of which gave positive results in HemosIL® AcuStar-IgG. HIT was diagnosed in 65 out of these patients, corresponding to a prevalence of 5%. Using ROC curve analysis, patients were divided into three groups according to their titer of antibodies. Higher values of the IgG (PF4-H) were associated with increased probability of HIT, and the diagnostic specificity was greatly increased using the combination of a 4Ts score > 3 and a positive titer ≥ 3.25 U/mL. Importantly, the diagnostic specificity is 100% when the titer is > 12.40 U/mL. We demonstrated that higher values of Anti PF4/H Antibodies were associated with a high probability of having HIT. A titer of HemosIL® IgG (PF4-H) > 12.40 U/mL has a specificity of 100% which should no require a functional test to confirm the diagnosis of HIT.

A Granulocytic Signature Identifies COVID-19 and Its Severity.

BACKGROUND: An unbiased approach to SARS-CoV-2-induced immune dysregulation has not been undertaken so far. We aimed to identify previously unreported immune markers able to discriminate COVID-19 patients from healthy controls and to predict mild and severe disease. METHODS: An observational, prospective, multicentric study was conducted in patients with confirmed mild/moderate (n = 7) and severe (n = 19) COVID-19. Immunophenotyping of whole-blood leukocytes was performed in patients upon hospital ward or intensive care unit admission and in healthy controls (n = 25). Clinically relevant associations were identified through unsupervised analysis. RESULTS: Granulocytic (neutrophil, eosinophil, and basophil) markers were enriched during COVID-19 and discriminated between patients with mild and severe disease. Increased counts of CD15+CD16+ neutrophils, decreased granulocytic expression of integrin CD11b, and Th2-related CRTH2 downregulation in eosinophils and basophils established a COVID-19 signature. Severity was associated with emergence of PD-L1 checkpoint expression in basophils and eosinophils. This granulocytic signature was accompanied by monocyte and lymphocyte immunoparalysis. Correlation with validated clinical scores supported pathophysiological relevance. CONCLUSIONS: Phenotypic markers of circulating granulocytes are strong discriminators between infected and uninfected individuals as well as between severity stages. COVID-19 alters the frequency and functional phenotypes of granulocyte subsets with emergence of CRTH2 as a disease biomarker.

A large-scale exome array analysis of venous thromboembolism.

Although recent Genome-Wide Association Studies have identified novel associations for common variants, there has been no comprehensive exome-wide search for low-frequency variants that affect the risk of venous thromboembolism (VTE). We conducted a meta-analysis of 11 studies comprising 8,332 cases and 16,087 controls of European ancestry and 382 cases and 1,476 controls of African American ancestry genotyped with the Illumina HumanExome BeadChip. We used the seqMeta package in R to conduct single variant and gene-based rare variant tests. In the single variant analysis, we limited our analysis to the 64,794 variants with at least 40 minor alleles across studies (minor allele frequency [MAF] ~0.08%). We confirmed associations with previously identified VTE loci, including ABO, F5, F11, and FGA. After adjusting for multiple testing, we observed no novel significant findings in single variant or gene-based analysis. Given our sample size, we had greater than 80% power to detect minimum odds ratios greater than 1.5 and 1.8 for a single variant with MAF of 0.01 and 0.005, respectively. Larger studies and sequence data may be needed to identify novel low-frequency and rare variants associated with VTE risk.

Interest of IgG and IgM antiprothrombin autoantibodies in the exploration of antiphospholipid syndrome: a 5-year retrospective study.

OBJECTIVES: Non-conventional aPL have been described in patients presenting clinical manifestations of antiphospholipid syndrome but negative for conventional markers. Among them, detection of autoantibodies against prothrombin has been proposed to improve diagnosis and management of these patients. However autoantibodies against prothrombin are heterogeneous and their use in clinical practice still remains unclear. The aim of this study was to evaluate the interest of IgG and IgM autoantibodies directed against the prothrombin only (aPT). METHODS: We retrospectively studied IgM and IgG aPT results, conventional antiphospholipid syndrome markers and clinical data of a large cohort of 441 patients referred for antiphospholipid syndrome exploration with aPT detection over a period of 5 years. RESULTS: We observed a total prevalence of 17% of aPT-positive patients (75/441). A significant association was found between aPT and thrombosis (P = 0.035), with 70% of patients having unexplained thrombosis, aPT representing the sole aPL detected. aPT positivity was significantly more frequent in venous thrombosis than in arterial thrombosis (P = 0.004). Interestingly, we demonstrated for the first time that aPT IgG levels were higher in recurrent thrombosis than in isolated thrombosis (P = 0.013), leading us to propose a predictive level of recurrence for thrombosis. CONCLUSION: Our results show that aPT are associated with thrombosis and demonstrate the interest of assessing both IgG and IgM aPT, in particular in venous thrombosis when conventional markers are negative. Quantification of aPT could predict recurrence of thrombosis and influence subsequent treatment strategy. Prospective clinical studies are now required to confirm these results.

DNA methylation age is associated with an altered hemostatic profile in a multiethnic meta-analysis.

Many hemostatic factors are associated with age and age-related diseases; however, much remains unknown about the biological mechanisms linking aging and hemostatic factors. DNA methylation is a novel means by which to assess epigenetic aging, which is a measure of age and the aging processes as determined by altered epigenetic states. We used a meta-analysis approach to examine the association between measures of epigenetic aging and hemostatic factors, as well as a clotting time measure. For fibrinogen, we performed European and African ancestry-specific meta-analyses which were then combined via a random effects meta-analysis. For all other measures we could not estimate ancestry-specific effects and used a single fixed effects meta-analysis. We found that 1-year higher extrinsic epigenetic age as compared with chronological age was associated with higher fibrinogen (0.004 g/L/y; 95% confidence interval, 0.001-0.007; P = .01) and plasminogen activator inhibitor 1 (PAI-1; 0.13 U/mL/y; 95% confidence interval, 0.07-0.20; P = 6.6 × 10-5) concentrations, as well as lower activated partial thromboplastin time, a measure of clotting time. We replicated PAI-1 associations using an independent cohort. To further elucidate potential functional mechanisms, we associated epigenetic aging with expression levels of the PAI-1 protein encoding gene (SERPINE1) and the 3 fibrinogen subunit-encoding genes (FGA, FGG, and FGB) in both peripheral blood and aorta intima-media samples. We observed associations between accelerated epigenetic aging and transcription of FGG in both tissues. Collectively, our results indicate that accelerated epigenetic aging is associated with a procoagulation hemostatic profile, and that epigenetic aging may regulate hemostasis in part via gene transcription.

A novel rare c.-39C>T mutation in the PROS1 5'UTR causing PS deficiency by creating a new upstream translation initiation codon.

Autosomal dominant inherited Protein S deficiency (PSD) (MIM 612336) is a rare disorder caused by rare mutations, mainly located in the coding sequence of the structural PROS1 gene, and associated with an increased risk of venous thromboembolism. To identify the molecular defect underlying PSD observed in an extended French pedigree with seven PSD affected members in whom no candidate deleterious PROS1 mutation was detected by Sanger sequencing of PROS1 exons and their flanking intronic regions or via an multiplex ligation-dependent probe amplification (MLPA) approach, a whole genome sequencing strategy was adopted. This led to the identification of a never reported C to T substitution at c.-39 from the natural ATG codon of the PROS1 gene that completely segregates with PSD in the whole family. This substitution ACG→ATG creates a new start codon upstream of the main ATG. We experimentally demonstrated in HeLa cells that the variant generates a novel overlapping upstream open reading frame (uORF) and inhibits the translation of the wild-type PS. This work describes the first example of 5'UTR PROS1 mutation causing PSD through the creation of an uORF, a mutation that is not predicted to be deleterious by standard annotation softwares, and emphasizes the need for better exploration of such type of non-coding variations in clinical genomics.

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.