In vitro and ex vivo rescue of a nonsense mutation responsible for severe coagulation factor V deficiency.

BACKGROUND: Coagulation factor V (FV) deficiency is a rare bleeding disorder that is usually managed with fresh-frozen plasma. Patients with nonsense mutations may respond to treatment with readthrough agents. OBJECTIVES: To investigate whether the F5 p.Arg1161Ter mutation, causing severe FV deficiency in several patients, would be amenable to readthrough therapy. METHODS: F5 mRNA and protein expression were evaluated in a F5 p.Arg1161Ter-homozygous patient. Five readthrough agents with different mechanisms of action, i.e. G418, ELX-02, PTC-124, 2,6-diaminopurine (2,6-DAP), and Amlexanox, were tested in in vitro and ex vivo models of the mutation. RESULTS: The F5 p.Arg1161Ter-homozygous patient showed residual F5 mRNA and functional platelet FV, indicating detectable levels of natural readthrough. COS-1 cells transfected with the FV-Arg1161Ter cDNA expressed 0.7% FV activity compared to wild-type. Treatment with 0-500 µM G418, ELX-02, and 2,6-DAP dose-dependently increased FV activity up to 7.0-fold, 3.1-fold, and 10.8-fold, respectively, whereas PTC-124 and Amlexanox (alone or in combination) were ineffective. These findings were confirmed by thrombin generation assays in FV-depleted plasma reconstituted with conditioned media of treated cells. All compounds except ELX-02 showed some degree of cytotoxicity. Ex vivo differentiated megakaryocytes of the F5 p.Arg1161Ter-homozygous patient, which were negative at FV immunostaining, turned positive after treatment with all 5 readthrough agents. Notably, they were also able to internalize mutant FV rescued with G418 or 2,6-DAP, which would be required to maintain the crucial platelet FV pool in vivo. CONCLUSION: These findings provide in vitro and ex vivo proof-of-principle for readthrough-mediated rescue of the F5 p.Arg1161Ter mutation.

Whole-Exome Sequencing in a Family with an Unexplained Tendency for Venous Thromboembolism: Multicomponent Prediction of Low-Frequency Variant Deleteriousness and of Individual Protein Interaction.

Whole-exome sequencing (WES) in families with an unexplained tendency for venous thromboembolism (VTE) may favor detection of low-frequency variants in genes with known contribution to hemostasis or associated with VTE-related phenotypes. WES analysis in six family members, three of whom affected by documented VTE, filtered for MAF < 0.04 in 192 candidate genes, revealed 22 heterozygous (16 missense and six synonymous) variants in patients. Functional prediction by multi-component bioinformatics tools, implemented by a database/literature search, including ClinVar annotation and QTL analysis, prioritized 12 missense variants, three of which (CRP Leu61Pro, F2 Asn514Lys and NQO1 Arg139Trp) were present in all patients, and the frequent functional variants FGB Arg478Lys and IL1A Ala114Ser. Combinations of prioritized variants in each patient were used to infer functional protein interactions. Different interaction patterns, supported by high-quality evidence, included eight proteins intertwined in the "acute phase" (CRP, F2, SERPINA1 and IL1A) and/or in the "fibrinogen complex" (CRP, F2, PLAT, THBS1, VWF and FGB) significantly enriched terms. In a wide group of candidate genes, this approach highlighted six low-frequency variants (CRP Leu61Pro, F2 Asn514Lys, SERPINA1 Arg63Cys, THBS1 Asp901Glu, VWF Arg1399His and PLAT Arg164Trp), five of which were top ranked for predicted deleteriousness, which in different combinations may contribute to disease susceptibility in members of this family.

Modulation of factor VIII pharmacokinetics by genetic components in factor VIII receptors.

INTRODUCTION: Gene variation in receptors for circulating factor VIII (FVIII) is candidate to explain the large inter-patient variability of infused FVIII pharmacokinetics (PK) in haemophilia A (HA). AIM: To compare in an Italian HA cohort (n = 26) the influence on FVIII PK of genetic components in four von Willebrand factor (VWF)/FVIII receptors. METHODS: Genotypes of low-density lipoprotein receptor (LDLR), asialoglycoprotein receptor minor subunit (ASGR2), family 4 member M (CLEC4M), stabilin2 (STAB2) and ABO blood-group, and VWF:Ag levels were included as independent variables in linear regression analyses of two-compartment model (TCM) - standard half-life (SHL) FVIII PK parameters. RESULTS: In the initial FVIII distribution phase, the STAB2 rs4981022 AA, ASGR2 rs2289645 TT and LDLR rs688 TT genotypes may contribute to increase Cmax , and prolong or shorten AlphaHL. In the elimination phase, a shorter BetaHL was associated with the CLEC4M rs868875 GG (beta-coefficient .366, p = .025) and ASGR2 rs2289645 TC (beta-coefficient .456, p = .006) genotypes, which also showed shorter mean residence time (MRT) than TT genotypes (p = .021). The alpha and beta phase effects were independent of ABO and VWF:Ag levels at baseline. The association of the LDLR rs2228671 genotypes with clearance was independent of ABO (beta-coefficient -.363, p = .035) but not of other receptors or VWF:Ag, which may point out multiple and competing interactions. CONCLUSIONS: With the limitation of the small number of HA patients, these observations highlight multiple genetic components acting in distinct phases of FVIII PK and contributing to explain FVIII PK variability. This analysis provides candidates for genotype-based, individual tailoring of FVIII substitutive treatment.

The p.P1127S pathogenic variant lowers von Willebrand factor levels through higher affinity for the macrophagic scavenger receptor LRP1: Clinical phenotype and pathogenic mechanisms.

BACKGROUND: The index case is a 21-year-old Italian woman with a mild hemorrhagic syndrome and von Willebrand factor antigen (VWF:Ag) = 34.3 U/dl, VWF recombinant glycoprotein Ib (VWF:GpIbR) = 32.8 U/dl, and factor VIII (FVIII) = 55.3 IU/dl. AIMS: The aim of this study is to characterize from a genetic and biochemical standpoint this low VWF phenotype. METHODS: Coagulation and biochemical methods were used to study the structural and functional pattern of VWF multimers in the index case's plasma. Recombinant wild-type and p.P1127S VWF variants were produced using human embryonic kidney (HEK)-293 cells. In addition, genetic screening was carried out to detect single nucleotide variants of some scavenger VWF/FVIII receptor genes such as CLEC4M, STAB2, and ASGR2. RESULTS: Genetic investigation revealed that the index case inherited from her mother the heterozygous missense mutation c.3379C > T (VWF exon 25), causing the p.P1127S substitution in the VWF D'D3 domain. The index case was also homozygous for the scavenger receptor ASGR2 c.-95 CC-genotype. Desmopressin normalized the VWF level of the patient, although its clearance was faster (t1/2  = 6.7 h) than in normal subjects (t1/2  = 12 ± 0.7 h). FVIII-VWF interaction, A Disintegrin And Metalloprotease with ThromboSpondin type 1 motif-13 levels, ristocetin-induced-platelet-aggregation, and VWF multimeric pattern were normal. The p.P1127S variant was normally synthesized and secreted by HEK-293 cells, and molecular modeling predicts a conformational change showing higher affinity for the macrophagic scavenger receptor lipoprotein receptor-related protein 1 (LRP1), as also experimentally verified. CONCLUSIONS: The p.P1127S variant may cause a low VWF phenotype, stemming from an increased VWF affinity for the scavenger receptor LRP1 and, consequently, an accelerated clearance of VWF.

Combination of CLEC4M rs868875 G-Carriership and ABO O Genotypes May Predict Faster Decay of FVIII Infused in Hemophilia A Patients.

The C-type lectin CLEC4M binds and internalizes factor VIII (FVIII). Common CLEC4M variants have been associated with FVIII pharmacokinetic (PK) profiles in hemophilia A (HA) patients. The two-compartment PK analysis of plasma-derived (pd-) and full length recombinant FVIII concentrates was conducted in twenty-six patients (FVIII:C ≤ 2 IU/dL). F8, ABO blood-groups, and the CLEC4M rs868875A/G polymorphism were genotyped. CLEC4M genotype groups differed for the elimination rate constant K 1-0 (p < 0.001), half-life (K 1-0 HL), and the Beta rate constant. Patients treated with pd-FVIII also differed in the Alpha phase. In linear regression models, the contribution of the CLEC4M genotypes to FVIII PK parameters remained significant after correction for ABO, age, and VWF antigen levels at PK. Combined CLEC4M rs868875A/G and ABO genotypes displayed significant interaction (K 1-0, p = 0.014). Compared to other combined genotypes, the G-carriers/O genotypes showed half-reduced K 1-0 HL (p = 0.008), and faster FVIII clearance (mean 7.1 ± 2.2 mL/h/kg SE) than in the G-carriers/non-O (mean 2.4 ± 0.3 mL/h/kg SE), (p = 0.038). Comparison in HA patients recruited in several countries suggests that CLEC4M genotypes coherently influence infused FVIII half-life and clearance. Our analysis supports substantially faster FVIII decay associated with the rs868875 G-carrier/ABO O genotypes, which has potential implications for genetically tailored substitutive HA treatment.

The Asialoglycoprotein Receptor Minor Subunit Gene Contributes to Pharmacokinetics of Factor VIII Concentrates in Hemophilia A.

BACKGROUND: The asialoglycoprotein receptor (ASGPR) binds with high affinity factor VIII (FVIII) through its N-linked oligosaccharides. However, its contribution to the wide inter-individual variation of infused FVIII pharmacokinetics (PK) in hemophilia A (HA) is unknown. OBJECTIVE: To investigate the variability in FVIII PK outcomes in relation to genetic variation in the ASGR2, encoding the ASGPR2 subunit. METHODS: Thirty-two HA patients with FVIII:C ≤2 IU/dL underwent 66 single-dose FVIII PK studies. PK parameters were evaluated in relation to ASGR2 5' untranslated region (5'UTR) polymorphisms, which were investigated by recombinant and white blood cell reverse transcription-polymerase chain reaction approaches. RESULTS: The 5'UTR polymorphisms determine a frequent and conserved haplotype (HT1) in a regulatory region. The HT1 homozygotes may differ in the amounts of alternatively spliced mRNA transcripts and thus ASGPR2 isoforms. Compared with the other ASGR2 genotypes, the c.-95TT homozygotes (n = 9), showed threefold longer Alpha HL (3.60 hours, 95% confidence interval: 1.44-5.76, p = 0.006), and the c.-95TC heterozygotes (n = 17) showed 25% shorter mean residence time (MRT; 18.5 hours, 15.0-22.0, p = 0.038) and 32% shorter Beta HL (13.5 hours, 10.9-16.0, p = 0.016). These differences were confirmed in patients (n = 27) undergoing PK studies (n = 54) with full-length FVIII only. In different linear regression models, the contribution of the ASGR2 genotypes remained significant after adjustment by ABO genotypes and von Willebrand factor (VWF) antigen levels, and explained 14% (MRT), 15 to 18% (Beta HL), and 22% (Alpha HL) of parameter variability. CONCLUSION: Infused FVIII distribution was modulated by frequent ASGR2 genotypes, independently from and together with ABO and VWF antigen levels, which has potential implications for genetically tailored substitutive treatment in HA.

F9 missense mutations impairing factor IX activation are associated with pleiotropic plasma phenotypes.

BACKGROUND: Circulating dysfunctional factor IX (FIX) might modulate distribution of infused FIX in hemophilia B (HB) patients. Recurrent substitutions at FIX activation sites (R191-R226, >300 patients) are associated with variable FIX activity and antigen (FIXag) levels. OBJECTIVES: To investigate the (1) expression of a complete panel of missense mutations at FIX activation sites and (2) contribution of F9 genotypes on the FIX pharmacokinetics (PK). METHODS: We checked FIX activity and antigen and activity assays in plasma and after recombinant expression of FIX variants and performed an analysis of infused FIX PK parameters in patients (n = 30), mostly enrolled in the F9 Genotype and PK HB Italian Study (GePKHIS; EudraCT ID2017-003902-42). RESULTS: The variable FIXag amounts and good relation between biosynthesis and activity of multiple R191 variants results in graded moderate-to-mild severity of the R191C>L>P>H substitutions. Recombinant expression may predict the absence in the HB mutation database of the benign R191Q/W/K and R226K substitutions. Equivalent changes at R191/R226 produced higher FIXag levels for R226Q/W/P substitutions, as also observed in p.R226W female carrier plasma. Pharmacokinetics analysis in patients suggested that infused FIX Alpha distribution and Beta elimination phases positively correlated with endogenous FIXag levels. Mean residence time was particularly prolonged (79.4 h, 95% confidence interval 44.3-114.5) in patients (n = 7) with the R191/R226 substitutions, which in regression analysis were independent predictors (ß coefficient 0.699, P = .004) of Beta half-life, potentially prolonged by the increasing over time ratio between endogenous and infused FIX. CONCLUSIONS: FIX activity and antigen levels and specific features of the dysfunctional R191/R226 variants may exert pleiotropic effects both on HB patients' phenotypes and substitutive treatment.

Cis-Segregation of c.1171C>T Stop Codon (p.R391*) in SERPINC1 Gene and c.1691G>A Transition (p.R506Q) in F5 Gene and Selected GWAS Multilocus Approach in Inherited Thrombophilia.

Inherited thrombophilia (e.g., venous thromboembolism, VTE) is due to rare loss-of-function mutations in anticoagulant factors genes (i.e., SERPINC1, PROC, PROS1), common gain-of-function mutations in procoagulant factors genes (i.e., F5, F2), and acquired risk conditions. Genome Wide Association Studies (GWAS) recently recognized several genes associated with VTE though gene defects may unpredictably remain asymptomatic, so calculating the individual genetic predisposition is a challenging task. We investigated a large family with severe, recurrent, early-onset VTE in which two sisters experienced VTE during pregnancies characterized by a perinatal in-utero thrombosis in the newborn and a life-saving pregnancy-interruption because of massive VTE, respectively. A nonsense mutation (CGA > TGA) generating a premature stop-codon (c.1171C>T; p.R391*) in the exon 6 of SERPINC1 gene (1q25.1) causing Antithrombin (AT) deficiency and the common missense mutation (c.1691G>A; p.R506Q) in the exon 10 of F5 gene (1q24.2) (i.e., FV Leiden; rs6025) were coinherited in all the symptomatic members investigated suspecting a cis-segregation further confirmed by STR-linkage-analyses [i.e., SERPINC1 IVS5 (ATT)5-18, F5 IVS2 (AT)6-33 and F5 IVS11 (GT)12-16] and SERPINC1 intragenic variants (i.e., rs5878 and rs677). A multilocus investigation of blood-coagulation balance genes detected the coexistence of FV Leiden (rs6025) in trans with FV HR2-haplotype (p.H1299R; rs1800595) in the aborted fetus, and F11 rs2289252, F12 rs1801020, F13A1 rs5985, and KNG1 rs710446 in the newborn and other members. Common selected gene variants may strongly synergize with less common mutations tuning potential life-threatening conditions when combined with rare severest mutations. Merging classic and newly GWAS-identified gene markers in at risk families is mandatory for VTE risk estimation in the clinical practice, avoiding partial risk score evaluation in unrecognized at risk patients.

Combination of Genomic and Transcriptomic Approaches Highlights Vascular and Circadian Clock Components in Multiple Sclerosis.

Aiming at exploring vascular components in multiple sclerosis (MS) with brain outflow disturbance, we combined transcriptome analysis in MS internal jugular vein (IJV) wall with WES in MS families with vertical transmission of disease. Main results were the differential expression in IJV wall of 16 MS-GWAS genes and of seven genes (GRIN2A, GRIN2B, IL20RB, IL26, PER3, PITX2, and PPARGC1A) not previously indicated by GWAS but encoding for proteins functionally interacting with MS candidate gene products. Strikingly, 22/23 genes have been previously associated with vascular or neuronal traits/diseases, nine encoded for transcriptional factors/regulators and six (CAMK2G, GRIN2A, GRIN2B, N1RD1, PER3, PPARGC1A) for circadian entrainment/rhythm components. Among the WES low-frequency (MAF ≤ 0.04) SNPs (n = 7) filtered in the 16 genes, the NR1D1 rs17616365 showed significantly different MAF in the Network for Italian Genomes affected cohort than in the 1000 Genome Project Tuscany samples. This pattern was also detected in five nonintronic variants (GRIN2B rs1805482, PER3 rs2640909, PPARGC1A rs2970847, rs8192678, and rs3755863) in genes coding for functional partners. Overall, the study proposes specific markers and low-frequency variants that might help (i) to understand perturbed biological processes in vascular tissues contributing to MS disease, and (ii) to characterize MS susceptibility genes for functional association with disease-pathways.

In vivo modulation of a dominant-negative variant in mouse models of von Willebrand disease type 2A.

Essentials Treatment options for von Willebrand disease (VWD) patients are limited. The p.P1127_C1948delinsR deletion/variant is a useful model to study VWD in vitro and in vivo. Counteracting dominant-negative effects restores von Willebrand factor multimerization in mice. This is the first siRNA-based treatment applied to a mouse model of VWD-type 2A. ABSTRACT: Background Treatment options for patients suffering from von Willebrand disease (VWD) are limited. Von Willebrand factor (VWF) is a polymeric protein that undergoes regulated dimerization and subsequent multimerization during its biosynthesis. Numerous heterozygous variants within the VWF gene display a dominant-negative effect and result in severe VWD. Previous studies have suggested that preventing the assembly of wild-type and mutant heteropolymers using siRNAs may have beneficial effects on VWF phenotypes in vitro. Objectives To study heterozygous dominant-negative variants in vivo, we developed a mouse model of VWD-type 2A and tested two independent strategies to modulate its detrimental effect. Methods The p.P1127_C1948delinsR deletion/variant, causing defective VWF multimerization, was expressed in mice as a model of VWD-type 2A variant. Two corrective strategies were applied. For the first time in a mouse model of VWD, we applied siRNAs selectively inhibiting translation of the mutant transcripts and we combined the VWD-type 2A deletion with the Cys to Arg substitution at position 2773, which is known to prevent dimerization. Results The RNA silencing approach induced a modest but consistent improvement of the VWF multimer profile. However, due to incomplete efficiency, the dominant-negative effect of the original variant could not be completely prevented. In contrast, the DNA approach resulted in increased antigen levels and restoration of a normal multimer profile. Conclusions Our data showed that preventing the detrimental impact of dominant-negative VWF variants by independent molecular mechanisms has beneficial consequences in vivo, in mouse models of dominant VWD.

Expression profiles of the internal jugular and saphenous veins: Focus on hemostasis genes.

INTRODUCTION: Venous bed specificity could contribute to differential vulnerability to thrombus formation, and is potentially reflected in mRNA profiles. MATERIALS AND METHODS: Microarray-based transcriptome analysis in wall and valve specimens from internal jugular (IJV) and saphenous (SV) veins collected during IJV surgical reconstruction in patients with impaired brain outflow. Multiplex antigenic assay in paired jugular and peripheral plasma samples. RESULTS: Most of the top differentially expressed transcripts have been previously associated with both vascular and neurological disorders. Large expression differences of HOX genes, organ patterning regulators, pinpointed the vein positional identity. The "complement and coagulation cascade" emerged among enriched pathways. In IJV, upregulation of genes for coagulation inhibitors (TFPI, PROS1), activated protein C pathway receptors (THBD, PROCR), fibrinolysis activators (PLAT, PLAUR), and downregulation of the fibrinolysis inhibitor (SERPINE1) and of contact/amplification pathway genes (F11, F12), would be compatible with a thromboprotective profile in respect to SV. Further, in SV valve the prothrombinase complex genes (F5, F2) were up-regulated and the VWF showed the highest expression. Differential expression of several VWF regulators (ABO, ST3GAL4, SCARA5, CLEC4M) was also observed. Among other differentially expressed hemostasis-related genes, heparanase (HPSE)/heparanase inhibitor (HPSE2) were up-/down-regulated in IJV, which might support procoagulant features and disease conditions. The jugular plasma levels of several proteins, encoded by differentially expressed genes, were lower and highly correlated with peripheral levels. CONCLUSIONS: The IJV and SV rely on differential expression of many hemostasis and hemostasis-related genes to balance local hemostasis, potentially related to differences in vulnerability to thrombosis.

Aptamer-modified FXa generation assays to investigate hypercoagulability in plasma from patients with ischemic heart disease.

BACKGROUND: High plasma levels of activated Factor VII-Antithrombin complex (FVIIa-AT) have been associated with an increased risk of cardiovascular mortality in patients with stable coronary artery disease (CAD). OBJECTIVES: To investigate if FVIIa-AT levels are associated with activated factor X generation (FXaG) in modified assays. PATIENTS/METHODS: Forty CAD patients were characterized for FVIIa-AT levels by ELISA and for FXaG in plasma. Novel fluorogenic FXaG assays, based on aptamers inhibiting thrombin and/or tissue factor pathway inhibitor (TFPI), were set up. RESULTS: FXaG correlated with FVIIa-AT levels (RAUC = 0.393, P = 0.012). The combination of thrombin inhibition and FXaG potentiation by using anti-thrombin and anti-TFPI aptamers, respectively, favors the study of time parameters. The progressive decrease in lag time from the lowest to the highest FVIIa-AT quartile was magnified by combining TFPI and thrombin inhibitory aptamers, thus supporting increased FXaG activity in the coagulation initiation phase. By exploring FXaG rates across FVIIa-AT quartiles, the largest relative differences were detectable at the early times (the highest versus the lowest quartile; 5.0-fold, P = 0.005 at 45 s; 3.5-fold, P = 0.001 at 55 s), and progressively decreased over time (2.3-fold, P = 0.002 at 75 s; 1.8-fold, P = 0.008 at 95 s; 1.6-fold, P = 0.022 at 115 s). Association between high FVIIa-AT levels and increased FXaG was independent of F7 -323 A1/A2 polymorphism influencing FVIIa-AT levels. CONCLUSIONS: High FVIIa-AT plasma levels were associated with increased FXaG. Hypercoagulability features were specifically detectable in the coagulation initiation phase, which may have implications for cardiovascular risk prediction by either FVIIa-AT complex measurement or modified FXaG assays.

C6orf10 Low-Frequency and Rare Variants in Italian Multiple Sclerosis Patients.

In light of the complex nature of multiple sclerosis (MS) and the recently estimated contribution of low-frequency variants into disease, decoding its genetic risk components requires novel variant prioritization strategies. We selected, by reviewing MS Genome Wide Association Studies (GWAS), 107 candidate loci marked by intragenic single nucleotide polymorphisms (SNPs) with a remarkable association (p-value ≤ 5 × 10-6). A whole exome sequencing (WES)-based pilot study of SNPs with minor allele frequency (MAF) ≤ 0.04, conducted in three Italian families, revealed 15 exonic low-frequency SNPs with affected parent-child transmission. These variants were detected in 65/120 Italian unrelated MS patients, also in combination (22 patients). Compared with databases (controls gnomAD, dbSNP150, ExAC, Tuscany-1000 Genome), the allelic frequencies of C6orf10 rs16870005 and IL2RA rs12722600 were significantly higher (i.e., controls gnomAD, p = 9.89 × 10-7 and p < 1 × 10-20). TET2 rs61744960 and TRAF3 rs138943371 frequencies were also significantly higher, except in Tuscany-1000 Genome. Interestingly, the association of C6orf10 rs16870005 (Ala431Thr) with MS did not depend on its linkage disequilibrium with the HLA-DRB1 locus. Sequencing in the MS cohort of the C6orf10 3' region revealed 14 rare mutations (10 not previously reported). Four variants were null, and significantly more frequent than in the databases. Further, the C6orf10 rare variants were observed in combinations, both intra-locus and with other low-frequency SNPs. The C6orf10 Ser389Xfr was found homozygous in a patient with early onset of the MS. Taking into account the potentially functional impact of the identified exonic variants, their expression in combination at the protein level could provide functional insights in the heterogeneous pathogenetic mechanisms contributing to MS.

Functional polymorphisms in the LDLR and pharmacokinetics of Factor VIII concentrates.

BACKGROUND: Optimization of factor VIII (FVIII) infusion in hemophilia A would benefit from identification of FVIII pharmacokinetics (PK) determinants. The low-density lipoprotein receptor (LDLR) contains an FVIII-binding site and might influence FVIII clearance. Consistently, LDLR polymorphisms have been associated with FVIII levels. OBJECTIVE: To investigate the relationships between individual FVIII PK and functional LDLR polymorphisms. PATIENTS/METHODS: Thirty-three hemophilia A patients (FVIII coagulant activity [FVIII:C] ≤2 IU/dL) without inhibitors underwent 85 FVIII single-dose (21.4-51.8 IU/kg) PKs with different FVIII concentrates. Twenty patients underwent repeated PKs (2-6). FVIII: C measured up to 72 hours was analyzed by two-compartment model. Parameters were evaluated in relation to F8 mutations, ABO blood-group and LDLR genotypes. RESULTS: F8 mutation types were not associated with PK parameters. ABO and LDLR c.1773C/T polymorphism were associated with Alpha, Alpha HL, CLD2, K1-2, and K2-1 parameters, suggesting an influence on the FVIII initial distribution phase. Regression analysis showed an independent association of both ABO and LDLR c.1773C/T with PK parameters (Alpha, ß-coefficient -0.311 vs 0.348; CLD2, ß-coefficient -0.335 vs 0.318), giving rise to an additive effect in subjects stratified by combined phenotypes. Differently, the LDLR c.81C/T was associated with FVIII clearance and volume of distribution at steady state, which could be related to distinct effects of polymorphisms, potentially linked to LDLR intracellular distribution and FVIII binding behavior. CONCLUSIONS: With the limitation of different FVIII concentrates and low number of patients, our data show plausible associations of LDLR polymorphisms with FVIII PK parameters, thus supporting their investigation as candidate functional determinants of FVIII PK.

Performance prediction models based on anthropometric, genetic and psychological traits of Croatian sprinters.

Elite athletes differ from each other in their characteristics according to their discipline. This study aimed to identify performance predictors in elite Croatian sprinters taking into consideration their anthropometric, psychological and genetic characteristics. One hundred and four elite Croatian sprinters (68 males and 36 females) participated in this study. Of them, 38 are currently competing in the 100-metre dash. The others are former sprinters. The participants underwent direct anthropometric assessment. Participants were also tested by means of the Competitive State Anxiety Inventory-2 and for ACE and ACTN3 polymorphisms. Multiple linear regression analysis was applied to identify the best model for performance prediction. Different models were developed for males and females. Anthropometric traits accounted for 44% of the variance in performance for males, 62% for females. Once other traits (psychological for females) were entered into the model, no additional contribution to the variance was observed. The most significant predictors of higher running velocity were bicristal diameter and foot dimensions in males, and leg length and clean one-repetition maximum in females. The findings suggest that performance in sprinters is associated with anthropometric characteristics, with biomechanical implications that may be used to provide a more complete evaluation of sprinters' performance.

Apolipoprotein C-III Strongly Correlates with Activated Factor VII-Anti-Thrombin Complex: An Additional Link between Plasma Lipids and Coagulation.

Activated factor VII-anti-thrombin (FVIIa-AT) complex is a potential biomarker of pro-thrombotic diathesis reflecting FVIIa-tissue factor (TF) interaction and has been associated with mortality in patients with coronary artery disease (CAD). Previous data indicated plasma lipids as predictors of FVIIa-AT variability, and plasma lipoproteins as potential stimulators of the coagulation cascade. Our aim was to evaluate the relationships between FVIIa-AT plasma concentration and a broad apolipoprotein profile (including ApoA-I, ApoB, ApoC-III and ApoE). Within the framework of the observational Verona Heart Study, we selected 666 subjects (131 CAD-free and 535 CAD, 75.4% males, mean age: 61.1 ± 10.9 years) not taking anticoagulant drugs and for whom plasma samples were available for both FVIIa-AT assay and a complete lipid profile. Plasma concentration of FVIIa-AT levels significantly and directly correlated with total and high-density lipoprotein cholesterol, triglycerides, ApoA-I, ApoC-III and ApoE levels. ApoC-III showed the strongest correlation (R = 0.235, p = 7.7 × 10-10), confirmed in all the sub-group analyses (males/females and CAD/CAD-free). Only ApoC-III remained associated with FVIIa-AT plasma concentration, even after adjustment for sex, age, CAD diagnosis, body mass index, renal function, smoking status, lipid-lowering therapies and FVIIa levels. The APOC3 gene locus-tagging polymorphism rs964184, previously linked with cardiovascular risk and plasma lipids by genome-wide association studies, was associated with both ApoC-III and FVIIa-AT plasma concentration. Our results indicate a strong association between ApoC-III and FVIIa-AT levels, thereby suggesting that an increased ApoC-III concentration may identify subjects with a pro-thrombotic diathesis characterized by an enhanced TF-FVIIa interaction and activity.

Changes in expression profiles of internal jugular vein wall and plasma protein levels in multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is an inflammatory, demyelinating and degenerative disorder of the central nervous system (CNS). Several observations support interactions between vascular and neurodegenerative mechanisms in multiple sclerosis (MS). To investigate the contribution of the extracranial venous compartment, we analysed expression profiles of internal jugular vein (IJV), which drains blood from CNS, and related plasma protein levels. METHODS: We studied a group of MS patients (n = 19), screened by echo-color Doppler and magnetic resonance venography, who underwent surgical reconstruction of IJV for chronic cerebrospinal venous insufficiency (CCSVI). Microarray-based transcriptome analysis was conducted on specimens of IJV wall from MS patients and from subjects undergoing carotid endarterectomy, as controls. Protein levels were determined by multiplex assay in: i) jugular and peripheral plasma from 17 MS/CCSVI patients; ii) peripheral plasma from 60 progressive MS patients, after repeated sampling and iii) healthy individuals. RESULTS: Of the differentially expressed genes (≥ 2 fold-change, multiple testing correction, P < 0.05), the immune-related CD86 (8.5 fold-change, P = 0.002) emerged among the up regulated genes (N = 409). Several genes encoding HOX transcription factors and histones potentially regulated by blood flow, were overexpressed. Smooth muscle contraction and cell adhesion processes emerged among down regulated genes (N = 515), including the neuronal cell adhesion L1CAM as top scorer (5 fold-change, P = 5 × 10- 4). Repeated measurements in jugular/peripheral plasma and overtime in peripheral plasma showed conserved individual plasma patterns for immune-inflammatory (CCL13, CCL18) and adhesion (NCAM1, VAP1, SELL) proteins, despite significant variations overtime (SELL P < 0.0001). Both age and MS disease phenotypes were determinants of VAP1 plasma levels. Data supported cerebral related-mechanisms regulating ANGPT1 levels, which were remarkably lower in jugular plasma and correlated in repeated assays but not between jugular/peripheral compartments. CONCLUSIONS: This study provides for the first time expression patterns of the IJV wall, suggesting signatures of altered vascular mRNA profiles in MS disease also independently from CCSVI. The combined transcriptome-protein analysis provides intriguing links between IJV wall transcript alteration and plasma protein expression, thus highlighting proteins of interest for MS pathophysiology.

An integrated genomic-transcriptomic approach supports a role for the proto-oncogene BCL3 in atherosclerosis.

Data with border-line statistical significance, copiously generated in genome-wide association studies of coronary artery disease (CAD), could include functionally relevant associations. We propose an integrated genomic and transcriptomic approach for unravelling new potential genetic signatures of atherosclerosis. Fifteen among 91 single nucleotide polymorphisms (SNPs) were first selected for association in a sex- and age-adjusted model by examining 510 patients with CAD and myocardial infarction and 388 subjects with normal coronary arteries (CAD-free) in the replication stages of a genome-wide association study. We investigated the expression of 71 genes proximal to the 15 tag-SNPs by two subsequent steps of microarray-based mRNA profiling, the former in vascular smooth muscle cell populations, isolated from non-atherosclerotic and atherosclerotic human carotid portions, and the latter in whole carotid specimens. BCL3 and PVRL2, contiguously located on chromosome 19, and ABCA1, extensively investigated before, were found to be differentially expressed. BCL3 and PVRL2 SNPs were genotyped within a second population of CAD patients (n=442) and compared with CAD-free subjects (n=393). The carriership of the BCL3 rs2965169 G allele was more represented among CAD patients and remained independently associated with CAD after adjustment for all the traditional cardiovascular risk factors (odds ratio=1.70 with 95% confidence interval 1.07-2.71), while the BCL3 rs8100239 A allele correlated with metabolic abnormalities. The up-regulation of BCL3 mRNA levels in atherosclerotic tissue samples was consistent with BCL3 protein expression, which was detected by immunostaining in the intima-media of atherosclerotic specimens, but not within non-atherosclerotic ones. Our integrated approach suggests a role for BCL3 in cardiovascular diseases.