Integrative Multi-omics Analysis of Childhood Aggressive Behavior.

This study introduces and illustrates the potential of an integrated multi-omics approach in investigating the underlying biology of complex traits such as childhood aggressive behavior. In 645 twins (cases = 42%), we trained single- and integrative multi-omics models to identify biomarkers for subclinical aggression and investigated the connections among these biomarkers. Our data comprised transmitted and two non-transmitted polygenic scores (PGSs) for 15 traits, 78,772 CpGs, and 90 metabolites. The single-omics models selected 31 PGSs, 1614 CpGs, and 90 metabolites, and the multi-omics model comprised 44 PGSs, 746 CpGs, and 90 metabolites. The predictive accuracy for these models in the test (N = 277, cases = 42%) and independent clinical data (N = 142, cases = 45%) ranged from 43 to 57%. We observed strong connections between DNA methylation, amino acids, and parental non-transmitted PGSs for ADHD, Autism Spectrum Disorder, intelligence, smoking initiation, and self-reported health. Aggression-related omics traits link to known and novel risk factors, including inflammation, carcinogens, and smoking.

The mode of action of sugammadex on coagulation.

OBJECTIVE: To explore the mode of action (MoA) by which sugammadex interferes with coagulation. MATERIALS AND METHODS: The effect of sugammadex on various steps in the coagulation cascade including thrombin generation, factor Xa activity, and factor Xa generation was explored in human plasma. RESULTS: Sugammadex did not affect a conventional thrombin generation test (TGT), while it prolongs activated partial thromboplastin time (APTT) and prothrombin time (PT). However, a customized TGT with PT reagent revealed sugammadex effects. In addition, sugammadex prolonged a one-step prothrombinase-induced clotting time (PiCT) using human factor Xa. Furthermore, sugammadex interfered with factor Xa generation induced by an intrinsic and not by an extrinsic activator, nor by Russell's viper venom factor X (RVV-X). CONCLUSION: Adapted, rather than standard, experiments show that sugammadex is likely to decrease factor Xa activity in the common pathway and activation of factor X specifically in the intrinsic pathway.

Interactions of sugammadex with various anticoagulants
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OBJECTIVE: To investigate in vitro the effect of sugammadex on activated partial thromboplastin time (APTT) and prothrombin time (PT) prolongations with various anticoagulants as well as the neutralizing effect of rocuronium and vecuronium on sugammadex effects on APTT and PT. MATERIALS AND METHODS: We investigated in vitro the effect of sugammadex on APTT and/or PT in plasma of patients on a vitamin K antagonist and with elevated international normalized ratios (INRs), in plasma of healthy subjects spiked with either a low or high concentration of enoxaparin, fondaparinux, rivaroxaban, and dabigatran, and in perioperatively collected patient plasma. In addition, we explored whether the effects of sugammadex persisted in the presence of rocuronium or vecuronium, or whether they were counteracted by these compounds. RESULTS: Sugammadex concentration-dependently increased APTT and PT(INR) in all anticoagulant conditions, mainly in a proportional manner, with no differences between perioperatively collected patient and control plasma. Rocuronium and vecuronium both neutralized the effects of sugammadex on APTT and PT. CONCLUSION: Sugammadex has a transient effect on coagulation and is unlikely to increase bleeding risk, this possibility cannot be excluded for scenarios not clinically studied.

Thrombin Generation in Vitreous and Subretinal Fluid of Patients with Retinal Detachment.

PURPOSE: To measure prothrombin fragments (F1+2) and thrombin-antithrombin complex (TAT) in vitreous and subretinal fluid (SRF) of rhegmatogenous retinal detachment (RRD) patients and to validate and further specify our earlier finding of increased thrombin activity in patients with proliferative vitreoretinopathy (PVR). METHODS: F1+2 and TAT were measured in 31 vitreous and 16 SRF samples using the Enzygnost® immunoassays. RESULTS: We found significant levels of F1+2 and TAT in the vitreous of all patients with RRD compared to patients with macular hole or macular pucker. However, there was no significant difference between patients who would develop PVR in the future, had established PVR, and patients with uncomplicated RRD both in vitreous concentrations of F1+2 (Kruskal-Wallis p = 0.963) and TAT (p = 0.516). CONCLUSION: The analysis of F1+2 and TAT confirmed significant thrombin generation in both vitreous and SRF of patients with RRD. An imbalance between the thrombin regulation mechanisms TAT and α2-macroglobulin possibly explains the difference from our previous findings.

Assessing Safety of Thrombolytic Therapy.

Thrombolytic therapy involves thrombolytic agents administered to patients suffering from venous or arterial thrombosis. The therapy induces systemic effects interrelated with the thrombolytic agent used. Bleeding is a prominent complication of thrombolytic therapy. Exhaustion of coagulation factors, generation of excessive amounts of fibrin degradation products (FDPs), therapy-induced activation of coagulation, therapy-induced anticoagulation, and formation of new fibrin all illustrate the complexity of effects of the treatment and challenges the hemostatic balance in the patients. The therapy-induced effects can be modulated by parallel administration of anticoagulants. Risk assessment is mandatory prior to thrombolytic therapy. Anticoagulated and unconscious patients represent particular safety concerns, and should be fully evaluated. Several guidelines describe the choice of tests and their safety limits in relation to pretreatment evaluation of anticoagulated patients. Fibrinogen depletion and FDPs during treatment may be promising markers for the evaluation of bleeding risk posttreatment. Future risk assessment measures should focus on the dynamics of the hemostatic balance. Here, thromboelastography may be considered a tool addressing clot formation, fibrin structure, and fibrinolytic resistance in parallel. Suitable laboratory analysis performed shortly after treatment may help to recognize severe treatment-induced systemic effects that can be counteracted by rational treatment, thereby reducing bleeding risk.

2SNP heritability and effects of genetic variants for neutrophil-to-lymphocyte and platelet-to-lymphocyte ratio.

Neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) are important biomarkers for disease development and progression. To gain insight into the genetic causes of variance in NLR and PLR in the general population, we conducted genome-wide association (GWA) analyses and estimated SNP heritability in a sample of 5901 related healthy Dutch individuals. GWA analyses identified a new genome-wide significant locus on the HBS1L-MYB intergenic region for PLR, which replicated in a sample of 2538 British twins. For platelet count, we replicated three known genome-wide significant loci in our cohort (at CCDC71L-PIK3CG, BAK1 and ARHGEF3). For neutrophil count, we replicated the PSMD3 locus. For the identified top SNPs, we found significant cis and trans expression quantitative trait loci effects for several loci involved in hematological and immunological pathways. Linkage Disequilibrium score (LD) regression analyses for PLR and NLR confirmed that both traits are heritable, with a polygenetic SNP heritability for PLR of 14.1%, and for NLR of 2.4%. Genetic correlations were present between ratios and the constituent counts, with the genetic correlation (r=0.45) of PLR with platelet count reaching statistical significance. In conclusion, we established that two important biomarkers have a significant heritable SNP component, and identified the first genome-wide locus for PLR.

Biological Variation of Hemostasis Variables in Thrombosis and Bleeding: Consequences for Performance Specifications.

BACKGROUND: Levels of hemostasis factors vary between and within individuals as a result of genetic and environmental factors and analytical variation of the assays. The current state of the art for defining analytical precision requirements for analytical testing is based on this between- and within-individual (biological) variation. However, information on biological variation in hemostasis variables is still limited.The aim of this study was to determine the biological variation of coagulation variables involved in thrombosis and bleeding to provide a recommendation for performance specifications and to assess whether hemostasis assays fulfill the recommendation. METHODS: We performed a longitudinal study by repeated blood sampling (in total 13 times over a 1-year period) in 40 healthy individuals and measured prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen, antithrombin, factor VIII, factor IX, von Willebrand factor (VWF), protein C, and protein S. We evaluated the effect of the biological variation on parameters of analytical variation and propose required performance specifications. RESULTS: Biological variation was highly different for various hemostasis variables: the within-subject variation ranged from 2.6% (PT) to 25.6% [VWF collagen binding (CB) activity], the between-subject variation varied from 4.1% (PT) to 31.2% (VWF:ristocetin cofactor acitivity), and the assay variation from 1.3% (PT) to 12.9% (VWF:CB). CONCLUSIONS: With the reagents and analyzers used in this study, most of the hemostasis tests variables fulfill the current quality criteria for diagnosis and monitoring of routine hemostasis assays.

Discovery of biochemical biomarkers for aggression: A role for metabolomics in psychiatry.

Human aggression encompasses a wide range of behaviors and is related to many psychiatric disorders. We introduce the different classification systems of aggression and related disorders as a basis for discussing biochemical biomarkers and then present an overview of studies in humans (published between 1990 and 2015) that reported statistically significant associations of biochemical biomarkers with aggression, DSM-IV disorders involving aggression, and their subtypes. The markers are of different types, including inflammation markers, neurotransmitters, lipoproteins, and hormones from various classes. Most studies focused on only a limited portfolio of biomarkers, frequently a specific class only. When integrating the data, it is clear that compounds from several biological pathways have been found to be associated with aggressive behavior, indicating complexity and the need for a broad approach. In the second part of the paper, using examples from the aggression literature and psychiatric metabolomics studies, we argue that a better understanding of aggression would benefit from a more holistic approach such as provided by metabolomics. © 2016 Wiley Periodicals, Inc.

The contribution of the functional IL6R polymorphism rs2228145, eQTLs and other genome-wide SNPs to the heritability of plasma sIL-6R levels.

The non-synonymous SNP rs2228145 in the IL6R gene on chromosome 1q21.3 is associated with a wide range of common diseases, including asthma, rheumatoid arthritis, type 1 diabetes and coronary heart disease. We examined the contribution of this functional IL6R gene polymorphism rs2228145 versus other genome-wide SNPs to the variance of sIL-6R levels in blood plasma in a large population-based sample (N ~5,000), and conducted an expression QTL analysis to identify SNPs associated with IL6R gene expression. Based on data from 2,360 twin families, the broad heritability of sIL-6R was estimated at 72 and 51% of the total variance was explained by the functional SNP rs2228145. Converging findings from GWAS, linkage, and GCTA analyses indicate that additional variance of sIL-6R levels can be explained by other variants in the IL6R region, including variants at the 3'-end of IL6R tagged by rs60760897 that are associated with IL6R RNA expression.

Effects of sugammadex on activated partial thromboplastin time and prothrombin time in healthy subjects.

OBJECTIVES: To assess the impact of sugammadex on activated partial thromboplastin time (APTT) and international normalized ratio for prothrombin time (PT(INR)) in healthy subjects and characterize the concentration-dependency of sugammadex effects on APTT and prothrombin time (PT) in normal human plasma in vitro. METHODS: Eight healthy subjects (18 - 45 years of age) were administered intravenous doses of 4 mg/kg sugammadex, 16 mg/kg sugammadex, or placebo in a randomized, placebo-controlled, three period cross-over trial. The primary endpoint was area under the curve from 2 to 60 minutes post-dose (AUC2-60min) for APTT and PT(INR). In vitro, the effects of sugammadex on APTT and PT were assessed in pooled normal human citrate plasma. RESULTS: In subjects dosed with 4 and 16 mg/kg sugammadex, geometric mean ratios (treated vs. placebo) for AUC2-60min were 1.085 (95% confidence interval, 0.888 - 1.325) and 1.019 (0.868 - 1.195), respectively, for APTT, and 1.047 (0.904 - 1.213) and 1.096 (0.953 - 1.261), respectively, for PT(INR). At individual timepoints, mean APTT and PT(INR) increased by up to 22% after 16 mg/kg sugammadex compared with placebo. All such increases occurred within 30 minutes post-dose. Sugammadex was generally well tolerated. In the in vitro experiments, addition of sugammadex to plasma resulted in limited, concentration dependent increases in both APTT and PT. At 200 µg/mL (the mean maximum concentration reached therapeutically), the relative increases were 29% and 19%, respectively. CONCLUSIONS: Administration of sugammadex is associated with a dose-related, limited and transient prolongation of APTT and PT(INR) that is unlikely to be of clinical relevance.

Plasma cotinine levels in cigarette smokers: impact of mental health and other correlates.

BACKGROUND/AIMS: We examined in cigarette smokers whether cotinine was associated with depressive and/or anxiety disorders. METHODS: Data were derived from 1,026 smoking adults with and without depressive and/or anxiety disorders participating in the Netherlands Study of Depression and Anxiety (NESDA). Depressive and anxiety disorders were ascertained with the DSM-IV Composite International Diagnostic Interview. Cigarette consumption was inquired about during an interview. Cotinine was assessed in plasma. RESULTS: Currently depressed and/or anxious smokers (n=692) reported smoking a higher number of cigarettes per day (CPD) than smokers with a remitted disorder (n=190) and smokers with no lifetime disorder (n=144). After controlling for CPD and other covariates, depressed and/or anxious smokers had lower cotinine levels compared to smokers with no lifetime disorder (B=-56.0, p=0.001). In the full regression model, CPD was positively associated with cotinine levels, whereas current depression and/or anxiety and high body mass index were inversely associated with cotinine. CONCLUSION: After considering CPD, the presence of current depressive and/or anxiety disorders was associated with lower cotinine levels, which may point to a different smoking topography or a faster cotinine metabolism in individuals with affective disorders. The latter could help to explain the higher number of cigarettes smoked and poorer cessation rates among depressed or anxious patients.

Development of a novel non-invasive biomarker panel for hepatic fibrosis in MASLD.

Accurate non-invasive biomarkers to diagnose metabolic dysfunction-associated steatotic liver disease (MASLD)-related fibrosis are urgently needed. This study applies a translational approach to develop a blood-based biomarker panel for fibrosis detection in MASLD. A molecular gene expression signature identified from a diet-induced MASLD mouse model (LDLr-/-.Leiden) is translated into human blood-based biomarkers based on liver biopsy transcriptomic profiles and protein levels in MASLD patient serum samples. The resulting biomarker panel consists of IGFBP7, SSc5D and Sema4D. LightGBM modeling using this panel demonstrates high accuracy in predicting MASLD fibrosis stage (F0/F1: AUC = 0.82; F2: AUC = 0.89; F3/F4: AUC = 0.87), which is replicated in an independent validation cohort. The overall accuracy of the model outperforms predictions by the existing markers Fib-4, APRI and FibroScan. In conclusion, here we show a disease mechanism-related blood-based biomarker panel with three biomarkers which is able to identify MASLD patients with mild or advanced hepatic fibrosis with high accuracy.

Genetic architecture of the pro-inflammatory state in an extended twin-family design.

In this study we examined the genetic architecture of variation in the pro-inflammatory state, using an extended twin-family design. Within the Netherlands Twin Register Biobank, fasting Tumor Necrosis Factor-α (TNF-α), Interleukin-6 (IL-6), C-Reactive Protein (CRP), and fibrinogen levels were available for 3,534 twins, 1,568 of their non-twin siblings, and 2,227 parents from 3,095 families. Heritability analyses took into account the effects of current and recent illness, anti-inflammatory medication, female sex hormone status, age, sex, body mass index, smoking status, month of data collection, and batch processing. Moderate broad-sense heritability was found for all inflammatory parameters (39%, 21%, 45%, and 46% for TNF-α, IL-6, CRP and fibrinogen, respectively). For all parameters, the remaining variance was explained by unique environmental influences and not by environment shared by family members. There was no resemblance between spouses for any of the inflammatory parameters, except for fibrinogen. Also, there was no evidence for twin-specific effects. A considerable part of genetic variation was explained by non-additive genetic effects for TNF-α, CRP, and fibrinogen. For IL-6, all genetic variance was additive. This study may have implications for future genome-wide association studies by setting a clear numerical target for genome-wide screens that aim to find genetic variants regulating the levels of these pro-inflammatory markers.

The Adult Netherlands Twin Register: twenty-five years of survey and biological data collection.

Over the past 25 years, the Adult Netherlands Twin Register (ANTR) has collected a wealth of information on physical and mental health, lifestyle, and personality in adolescents and adults. This article provides an overview of the sources of information available, the main research findings, and an outlook for the future. Between 1991 and 2012, longitudinal surveys were completed by twins, their parents, siblings, spouses, and offspring. Data are available for 33,957 participants, with most individuals having completed two or more surveys. Smaller projects provided in-depth phenotyping, including measurements of the autonomic nervous system, neurocognitive function, and brain imaging. For 46% of the ANTR participants, DNA samples are available and whole genome scans have been obtained in more than 11,000 individuals. These data have resulted in numerous studies on heritability, gene x environment interactions, and causality, as well as gene finding studies. In the future, these studies will continue with collection of additional phenotypes, such as metabolomic and telomere length data, and detailed genetic information provided by DNA and RNA sequencing. Record linkage to national registers will allow the study of morbidity and mortality, thus providing insight into the development of health, lifestyle, and behavior across the lifespan.

Heritability of Urinary Amines, Organic Acids, and Steroid Hormones in Children.

Variation in metabolite levels reflects individual differences in genetic and environmental factors. Here, we investigated the role of these factors in urinary metabolomics data in children. We examined the effects of sex and age on 86 metabolites, as measured on three metabolomics platforms that target amines, organic acids, and steroid hormones. Next, we estimated their heritability in a twin cohort of 1300 twins (age range: 5.7-12.9 years). We observed associations between age and 50 metabolites and between sex and 21 metabolites. The monozygotic (MZ) and dizygotic (DZ) correlations for the urinary metabolites indicated a role for non-additive genetic factors for 50 amines, 13 organic acids, and 6 steroids. The average broad-sense heritability for these amines, organic acids, and steroids was 0.49 (range: 0.25-0.64), 0.50 (range: 0.33-0.62), and 0.64 (range: 0.43-0.81), respectively. For 6 amines, 7 organic acids, and 4 steroids the twin correlations indicated a role for shared environmental factors and the average narrow-sense heritability was 0.50 (range: 0.37-0.68), 0.50 (range; 0.23-0.61), and 0.47 (range: 0.32-0.70) for these amines, organic acids, and steroids. We conclude that urinary metabolites in children have substantial heritability, with similar estimates for amines and organic acids, and higher estimates for steroid hormones.

Introduction to haemostasis from a pharmacodynamic perspective.

Biochemical characterization of the haemostatic system has advanced significantly in the past decades. Sub-systems, such as coagulation, fibrinolysis, blood cells and platelets and the vessel wall have been studied by specialists, mostly separately and independently. The time has come to integrate the approaches, and, in particular, to develop tests to document the state of the whole system and to have available adequate pharmacodynamic tests to evaluate treatments. Many examples are available to show that traditional general methods of clotting and lysis do not provide the information that is desired. The present tendency is to use specific methods for specific factors or effects which are very limited in pharmacological information. There is also increasing awareness of the occurrence of rather broad interindividual variability in the haemostatic system. This suggests that individually tailored treatments are required. This is the more relevant since haemostasis is a balance and treatment should be positioned between efficacy and safety. The conclusion is reached that there is a need for integrated or global methods or sets of methods that reflect the complexity and individual status appropriately and allow the practitioner to judge the effects of interventions and their individual aspects.

A thrombin generation assay may reduce the need for compression ultrasonography for the exclusion of deep venous thrombosis in the elderly.

INTRODUCTION: The exclusion of deep venous thrombosis (DVT) by pre-test probability (PTP) and D-dimer in the elderly is safe, but has a low specificity. There is increasing interest in the diagnostic role of thrombin generation in patients with thrombosis. We evaluated the value of thrombin generation in the exclusion of DVT, especially in elderly patients. MATERIAL AND METHODS: All thrombin generation parameters of the Endogenous Thrombin Potential assay were tested retrospectively in a cohort of 443 patients suspected for DVT. The performance of the assay was calculated as AUC of the ROC curve and association with DVT as odds ratio (OR). The lag time value was combined with results of PTP and Innovance D-dimer assay. RESULTS: All thrombin generation parameters had a low AUC. In the 4th age quartile, the corrected OR of lag time increased to 16 and the AUC of lag time for the combination PTP < 2 and D-dimers ≥ 500 µg/L was 0.96 with a cut-off value of 23.0 sec. The exclusion of DVT in patients ≥ 75 years (n = 30), based on this combination and lag time values ≤ 23.0 sec had a NPV of 100% and a specificity of 96.0% in this subgroup of patients and decreased the number needed to test from 10 to 1.1. CONCLUSION: Thrombin generation parameters alone are inappropriate for the exclusion of DVT. However, in the elderly, the current algorithm of exclusion of DVT may improve markedly by the addition of the lag time results of the ETP.

Metabolic profile of a continuous versus a cyclic low-dose combined oral contraceptive after one year of use.

OBJECTIVES: To compare the effects of a combined oral contraceptive (COC) taken continuously with those of one of similar composition taken cyclically on 30 variables related to haemostasis, lipids, carbohydrates, bone metabolism, and sex hormone-binding globulin (SHBG). METHODS: Randomised, open-label, multicentre, comparative substudy of a larger phase 3 trial involving 147 healthy women (age 18-49 years). Participants received the COC either continuously (levonorgestrel [LNG] 90 µg/ethinylestradiol [EE] 20 µg) or cyclically (21/7 days pattern; LNG 100 µg/EE 20 µg). RESULTS: After 13 pill packs, changes in total cholesterol (+0.23 vs. -0.06 mmol/l), low-density lipoprotein cholesterol (+0.25 vs. -0.12 mmol/l), and high-density lipoprotein cholesterol(3) (-0.06 vs. -0.15 mmol/l) differed significantly (p<0.05) between the continuous and cyclic regimens, respectively. Increases were significantly greater (p <0.05) for protein C antigen (+11.8% vs. +6.1%) and SHBG (+791 vs. +565 nmol/l), and significantly smaller (p <0.05, ranks) for D-dimer (+19 vs. +37 µg FE/l). CONCLUSIONS: Overall, the continuous and cyclic regimens affected metabolic variables similarly. The larger increase in SHBG with the continuous COC is consistent with a higher net oestrogenic effect due to a lower daily dose of LNG. Prospective studies are required to determine the long-term effects of this continuous COC regimen.

External quality assessment for thrombin generation tests: an exploration.

External Quality Control of Diagnostic Assays and Tests (ECAT) surveys on thrombin generation rests (TGTs) have identified that various tests show a more than 30-fold difference in time to peak (TTP). The survey included pooled normal plasmas, microparticle (MP)-depleted plasmas, and factor (F)XII-deficient patient plasma. Between 4 and 11 laboratories participated per test; analyzed were a time (TTP) and quantity variable (AUC). MP depletion of plasma showed a progressive increase in TTP up to 29% with a decreasing tissue factor. The same was found for the AUC with the largest decrease of 38%. It was observed that MP depletion showed large individual differences. The FXII-deficient plasma showed no effect on TTP for rapid tests, but for slow tests it increased from 248 to 331%. The AUC declined gradually the slower the test, reaching a decline of 85%. The effects of FXII deficiency were not mimicked by the addition of corn trypsin inhibitor but were confirmed by inhibiting activated FXI. Interlaboratory variability was between 11% and 57% for all methods, showing differences mainly related to the used normal pooled plasma. The different sensitivities of TGTs to MPs and contact activation predict that they will associate differently with clinical situations, according to which of these aspects are important. Future ECAT surveys should include samples with variation in MPs and contact activation to match with features of the TGT variants.