Nuria García-Barberá

Pharmacogenetic relevance of CYP4F2 V433M polymorphism on acenocoumarol therapy

VKORC1 and CYP2C9 polymorphisms are used to predict the safe dose of oral anticoagulant therapy. A new variant of CYP4F2 (V433M) has recently been related to the required warfarin dose. We evaluated its influence in earliest response to acenocoumarol in 100 selected men who started anticoagulation (3 mg for 3 consecutive days). V433M genotype exerted a gene dosage-dependent effect on the decrease of factors II, VII, IX, and X in the earliest response to acenocoumarol, with homozygous 433V subjects being the most sensitive. Similarly, after the initiation of therapy, international normalized ratio also experienced a gene dosage-dependent effect (P = .015), and 433V subjects needed 4 mg/week less than 433M carriers to achieve a steady anticoagulation (P = .043). Multivariate linear regression analysis revealed a significant contribution of V433M polymorphism to variability of both early international normalized ratio value (R(2) = 0.14) and dose requirements (R(2) = 0.19). Our data underline the relevant role of CYP4F2 V433M polymorphism in the pharmacogenetics of coumarin anticoagulants.

Identification of miRNAs as potential modulators of tissue factor expression in patients with systemic lupus erythematosus and antiphospholipid syndrome

Summary.  Background: Tissue factor (TF) is the main initiator of the coagulation cascade and elements that may upregulate its expression might provoke thrombotic events. Systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS) are autoimmune diseases characterized by a high TF expression in monocytes. Objectives: To examine the role of microRNAs (miRNAs) in TF expression and to evaluate their levels in SLE and APS patients. Methods: An in silico search was performed to find potential putative binding sites of miRNAs in TF mRNA. In vitro validation was performed transfecting cells expressing TF (THP‐1 and MDA‐MB‐231) with oligonucleotide miRNA precursors and inhibitors. Additionally, reporter assays were performed to test for the binding of miR‐20a to TF mRNA. Levels of miRNAs and TF were measured by quantitative (qRT‐PCR) in patients with APS and SLE. Results: Overexpression of miRNA precursors, but not inhibitors, of two of the members of cluster miR‐17∼92, for example miR‐19b and miR‐20a, in cells expressing TF decreased TF mRNA, protein levels, and procoagulant activity between 30% and 60%. Reporter assays showed that miR‐20a binds to TF mRNA. Finally, we measured levels of miR‐19b and miR‐20a in monocytes from patients with APS and SLE and observed significantly lower miRNAs levels in comparison with healthy subjects inversely correlated with the levels of TF. Conclusions: Down‐regulation of miR‐19b and miR‐20a observed in patients with SLE and APS could contribute to increased TF expression and thus provoke the hypercoagulable state characteristic of these patients.

miR-133a regulates vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1), a key protein in the vitamin K cycle.

Regulation of key proteins by microRNAs (miRNAs) is an emergent field in biomedicine. Vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1) is a relevant molecule for cardiovascular diseases, since it is the target of oral anticoagulant drugs and plays a role in soft tissue calcification. The objective of this study was to determine the influence of miRNAs on the expression of VKORC1. Potential miRNAs targeting VKORC1 mRNA were searched by using online algorithms. Validation studies were carried out in HepG2 cells by using miRNA precursors; direct miRNA interaction was investigated with reporter assays. In silicostudies identified two putative conserved binding sites for miR-133a and miR-137 on VKORC1 mRNA. Ex vivo studies showed that only miR-133a was expressed in liver; transfection of miRNA precursors of miR-133a in HepG2 cells reduced VKORC1 mRNA expression in a dose-dependent manner, as assessed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) as well as protein expression. Reporter assays in HEK293T cells showed that miR-133a interacts with the 3′UTR of VKORC1. Additionally, miR-133a levels correlated inversely with VKORC1 mRNA levels in 23 liver samples from healthy subjects. In conclusion, miR-133a appears to have a direct regulatory effect on expression of VKORC1 in humans; this regulation may have potential importance for anticoagulant therapy or aortic calcification.

Creating a genotype-based dosing algorithm for acenocoumarol steady dose

Acenocoumarol is a commonly prescribed anticoagulant drug for the prophylaxis and treatment of venous and arterial thromboembolic disorders in several countries. In counterpart of warfarin, there is scarce information about pharmacogenetic algorithms for steady acenocoumarol dose estimation. The aim of this study was to develop an algorithm of prediction for acenocoumarol.The algorithm was created using the data from 973 retrospectively selected anticoagulated patients and was validated in a second independent cohort adding up to 2,683 patients. The best regression model to predict stable dosage in the Primary Cohort included clinical factors (age and body mass index, BSA) and genetic variants (VKORC1, CYP2C9* and CYP4F2 polymorphisms) and explained up to 50% of stable dose. In the validation study the clinical algorithm yielded an adjusted R²=0.15 (estimation´s standard error=4.5) and the genetic approach improved the dose forecast up to 30% (estimation´s standard error=4.6). Again, the best model combined clinical and genetic factors (R² = 0.48; estimation´s standard error=4) which provided the best results of doses estimates within 20% of the real dose in patients taking lower (≤ 7 mg/week) or higher (≥ 25 mg/week) acenocoumarol doses. In conclusion, we developed a prediction algorithm using clinical data and three polymorphisms in VKORC1, CYP2C9* and CYP4F2 genes that provided a steady acenocoumarol dose for about 50% of patients in the Validation Cohort. Such algorithm was especially useful to patients who need higher or lower acenocoumarol doses, those patients with higher time required until their stabilisation and are more prone to suffer a treatment derived complication.

Regulation of Coagulation Factor XI Expression by MicroRNAs in the Human Liver

High levels of factor XI (FXI) increase the risk of thromboembolic disease. However, the genetic and environmental factors regulating FXI expression are still largely unknown. The aim of our study was to evaluate the regulation of FXI by microRNAs (miRNAs) in the human liver. In silico prediction yielded four miRNA candidates that might regulate FXI expression. HepG2 cells were transfected with miR-181a-5p, miR-23a-3p, miR-16-5p and miR-195-5p. We used mir-494, which was not predicted to bind to F11, as a negative control. Only miR-181a-5p caused a significant decrease both in FXI protein and F11 mRNA levels. In addition, transfection with a miR-181a-5p inhibitor in PLC/PRF/5 hepatic cells increased both the levels of F11 mRNA and extracellular FXI. Luciferase assays in human colon cancer cells deficient for Dicer (HCT-DK) demonstrated a direct interaction between miR-181a-5p and 3′untranslated region of F11. Additionally, F11 mRNA levels were inversely and significantly correlated with miR-181a-5p levels in 114 healthy livers, but not with miR-494. This study demonstrates that FXI expression is directly regulated by a specific miRNA, miR-181a-5p, in the human liver. Future studies are necessary to further investigate the potential consequences of miRNA dysregulation in pathologies involving FXI.

Prognostic role of MIR146A polymorphisms for cardiovascular events in atrial fibrillation

There are few biomarkers able to forecast new thrombotic events in patients with AF. In this framework, microRNAs have emerged as critical players in cardiovascular biology. In particular, miR-146a-5p is recognised as an important negative regulator of inflammation. This study aims to evaluate the prognostic role and biological effect of functional MIR146A polymorphisms, rs2431697 and rs2910164, in non-valvular atrial fibrillation (AF) patients under oral anticoagulation.We studied 901 patients with permanent/paroxysmal AF stabilized for at least six months. Patients were followed-up for two years and adverse cardiovascular events (ACE) were recorded. In vitro studies were performed in monocytes from healthy homozygous for the two genotypes of rs2431697. Rs2910164 had no association with ACE. However, multivariate analysis (adjusted by CHA2DS2-VASc score) revealed that rs2431697TT was associated with adverse cardiovascular events [HR: 1.64 (1.09-2.47); p=0.017]. The predictive value of usefulness of the CHA2DS2-VASc+IL6+rs2431697 for predicting ACE, was statistically better than that predicted by CHA2DS2-VASc+IL6. Functional studies showed that after 24 hours incubation, monocytes from CC individuals showed a 65 % increase in miR-146a-5p levels, while TT individuals only showed a 28 % increase. Indeed, after 24 hours of LPS activation, TT monocytes showed a higher increase in IL6 mRNA expression than CC (52 % vs 26 %). Our study established MIR146A rs2431697 as a prognostic biomarker for ACE in anticoagulated AF patients. These data suggest that TT individuals, when submitted to an inflammatory stress, may be prone to a highest pro-inflammatory state due, in part, to lower levels of miR-146a-5p.

Pharmacogenetics of acenocoumarol in patients with extreme dose requirements.

Summary.  Background: There is currently intense debate as to whether pharmacogenetic algorithms for estimating the initial dose of coumarins provide a more accurate dose than the fixed‐dose approach. Recently, it has been suggested that the greatest benefit of pharmacogenetic algorithms is observed in patients with extreme dose requirements. Objectives: To identify clinical and genetic factors that better characterize patients who need extreme acenocoumarol doses for steady anticoagulation state. Patients/methods: We reviewed 9538 patients with a steady acenocoumarol dose from three Spanish hospitals, selecting 83 who took ≤ 5.00 mg week−1 (percentile 5, p5) and 203 taking ≥ 30.00 mg week−1 (p95). We also selected patients matched by gender and age taking 13.50–14.00 mg week−1 (p50). We genotyped VKORC1 (rs9923231), CALU (rs1043550), GGCX (rs699664), CYP2C9 (rs1799853; rs1057910), CYP4F2 (rs2108622) and F7 (rs5742910) single‐nucleotide polymorphisms (SNPs). Results: Comparison between p5 and p95 revealed five parameters with significant differences: body surface area (BSA) (P = 0.006), age, VKORC1, CYP2C9 and CYP4F2 genotypes (all P < 0.001). First VKORC1, and second, CYP2C9 SNPs played a strong effect by determining extreme doses, particularly in p95. Only one out of 203 p95 had the VKORC1 A‐1639A genotype, but this subject was CYP2C9*1/*1. In contrast, nine out of 83 p5 carried the VKORC1 G‐1639G genotype, although six of them were CYP2C9*3 homozygotes and another two were heterozygotes. Surprisingly, CYP4F2 V433M SNP displayed prevalences that suggest that its influence might only be evident when patients are treated with high doses. Conclusion: Two clinical data, age and BSA, and three SNPs in the VKORC1, CYP2C9 and CYP4F2 genes strongly predict outlier patients treated with acenocoumarol.

Chediak-Higashi syndrome: description of two novel homozygous missense mutations causing divergent clinical phenotype

Chediak–Higashi syndrome (CHS) is a rare autosomal recessive disease resulting from mutations in the LYST/CHS1 gene, which encodes for a 429 kDa protein, CHS1/LYST, that regulates vesicle trafficking and determines the size of lysosomes and other organelles. To date, 60 different mutations have been characterized, and a reasonably straightforward phenotype–genotype correlation has been suggested. We describe two patients on opposite ends of the CHS clinical spectrum with novel missense mutations. We characterized these patients in terms of their mutations, protein localization and expression, mRNA stability, and electrostatic potential. Patient 1 is the first report of a severe early‐onset CHS with a homozygous missense mutation (c.11362 G>A, p.G3725R) in the LYST/CHS1 gene. This molecular change results in a reduction at the CHS1 protein level, not due to an mRNA effect, but maybe a consequence of both, a change in the structure of the protein and most likely attributable to the remarkable serious perturbation in the electrostatic potential. Patient 2, who exhibited the adolescence form of the disease, was found to be homozygous for a novel missense mutation c.961 T>C, p.C258R, which seemed to have minor effect on the structure of the CHS1/LYST protein. Reexamining accepted premises of missense mutant alleles being reported among patients with clinically mild forms of the disorder should be carried out, and attempts to link genotype and clinical phenotype require identifying the actual molecular effect of the mutation. Early and accurate diagnosis of the severity of the disease is extremely important to early differentiate patients who would benefit from premature enrollment into a transplantation protocol.

Control of post-translational modifications in antithrombin during murine post-natal development by miR-200a

BackgroundDevelopmental haemostatic studies may help identifying new elements involved in the control of key haemostatic proteins like antithrombin, the most relevant endogenous anticoagulant.ResultsIn this study, we showed a significant reduction of sialic acid content in neonatal antithrombin compared with adult antithrombin in mice. mRNA levels of St3gal3 and St3gal4, two sialyltransferases potentially involved in antithrombin sialylation, were 85% lower in neonates in comparison with adults. In silico analysis of miRNAs overexpressed in neonates revealed that mir-200a might target these sialyltransferases. Moreover, in vitro studies in murine primary hepatocytes sustain this potential control.ConclusionsThese data suggest that in addition to the direct protein regulation, microRNAs may also modulate qualitative traits of selected proteins by an indirect control of post-translational processes.

JAK2 V617F, hemostatic polymorphisms, and clinical features as risk factors for arterial thrombotic events in essential thrombocythemia.

Dear Editor,ArecentmutationlocatedintheJAK2 gene, JAK2 V617F, hasbeen shown to play a transcendental role in the constitutiveactivation of its tyrosine kinase activity in an importantnumber of cases of Philadelphia negative myeloproliferativedisorders (Ph-MPDs). This mutation is a useful tool fordiagnosing this diseases because different studies show thatthis mutation is highly frequent in patients with polycythemiavera (PV) and is present in around 50% of the patients withessential thrombocythemia (ET) and idiopathic myelofibrosis(IMF) [1]. Among the clinical features and phenotypes thatcharacterize Ph-MPD patients, thrombotic and hemorrhagicepisodes have been shown to be frequent in these patients.Range values for thrombosis at diagnosis fluctuate between34% to 39% for PV and 10% to 29% for ET [ 2]. Forhemorrhage, the values are much more variable [ 3].The mechanisms accounting for the increased risk ofthrombosis and hemorrhage in Ph-MPD patients are notwell understood. It has been accepted that the occurrence ofprevious venous thrombotic events and age increase the riskof thrombosis [4, 5]. The contribution of cardiovasculardisease risk factors in the development of thrombosis inPh-MPD patients is still unclear. Risk factors for hemor-rhage are more elusive and few have been described [2, 4].Studies are limited concerning the impact of hereditarythrombophilia in the development of thrombotic episodes inPh-MPD patients [6, 7]. In addition, the effect of the JAK2F617 allele in the development of thrombotic events is stillcontroversial [2, 8, 9].Thus, we aimed to ascertain the role of JAK2 V617F andother genetic risk factors in the development of thrombosisin patients diagnosed with Ph-MPD. For this purpose, weevaluated the prevalence of JAK2 V617F mutation andnine additional genetic alterations [FV Leiden, PTG20210A, and ZPI R67Stop are polymorphisms associatedwith venous thrombosis; HPA-1 located in the αIIbβ3integrin, HPA-2 located in von Willebrand factor receptorGIb/IX/V, GPIa C807T, and PSGL-1 are polymorphismsassociated with arterial thrombosis; FVII -323 Del/Ins andTUBB1 Q43P located in the megakaryocyte-specifictubulin β1 are two polymorphisms that increase the riskof hemorrhage] in 212 patients [74 PV, 128 ET, and tenIMF] (94 males, 118 females, median age 60.5±16.1, range21–85 years) followed during a 4-year period in HospitalMorales Meseguer (Murcia) and Hospital del Mar (Barce-lona). Thrombotic complications included venous (N=13)and major arterial thromboses (N=42) as well as microvas-cular alterations such as erythromelalgia (N=17). Severalpatients had two episodes of vascular events: five patientshad both venous and major arterial thromboses and twopatients with erythromelalgia developed an episode ofarterial thrombosis. Fifteen patients had hemorrhage. Weconsidered asymptomatic patients those with no thromboticor hemorrhagic episodes. The analysis of the role of genetic