Genetic causes of resistance to vitamin K antagonists in Polish patients: a novel p.Ile123Met mutation in VKORC1 gene.

: Mutations in the genes encoding vitamin K epoxide reductase complex subunit 1 (VKORC1) and cytochrome P450 2C9 (CYP2C9) largely contribute to the inter-individual variations in vitamin K antagonists (VKAs) dose requirements. Up to 50% of the dosage variability can be explained by genetic polymorphisms in these genes. We sought to identify the mutations responsible for VKA resistance in a series of Polish patients. Of the 607 patients treated with VKA, 35 (6%) individuals with the VKA resistance defined as a daily dose of acenocoumarol more than 8 mg (n = 15, 43%) or warfarin more than 10 mg (n = 20, 57%) were selected for further mutational analysis using Sanger sequencing (VKORC1) or real-time PCR genotyping (CYP2C9). The indications for anticoagulant treatment were venous thromboembolism (n = 28, 80%), atrial fibrillation (n = 6, 17%), or artificial heart valve (n = 1, 3%). Patients taking medication interfering with VKA were ineligible. Almost all of VKA-resistant patients (n = 34, 97%) possessed at least one VKORC1*3 (n = 29, 83%) or VKORC1*4 (n = 15, 43%) haplotypes. In a 70-year-old man atrial fibrillation patient on the daily acenocoumarol dose of 16 mg, a novel p.Ile123Met (c.369C>G) VKORC1 mutation was found. In-silico analysis showed that the p.Ile123Met can functionally underlie the acenocoumarol resistance, presumably by altering VKA binding. To our knowledge this is the first cohort of Polish patients resistant to VKA evaluated for the causal genetic background. We found one new detrimental mutation underlying VKA resistance. Our study highlights a key role of unidentified environmental factors in VKA resistance in daily clinical practice.

Bleeding predictors in patients following venous thromboembolism treated with vitamin K antagonists: Association with increased number of single nucleotide polymorphisms.

INTRODUCTION: Single nucleotide polymorphisms (SNP) in genes encoding proteins involved in metabolism and action of vitamin K antagonists (VKA) affect anticoagulation stability. We investigated how those polymorphisms influence bleeding rates in patients following venous thromboembolism (VTE). MATERIALS AND METHODS: In 324 patients following unprovoked VTE, 143 (44%) on warfarin and 181 (56%) on acenocoumarol, we recorded bleeds within the preceding 24 months. We assessed eight SNP, including those in cytochrome P450 isoform 2C9 (CYP2C9) and isoform 4F2 (CYP4F2), vitamin K epoxide reductase complex subunit 1 (VKORC1), gamma-glutamyl carboxylase (GGCX), apolipoprotein E (APOE) and multidrug resistance gene 1 (MDR1). RESULTS: Within 48 months before enrolment, bleeding events occurred in 80 (25%) patients, including 14 (4%) major bleeds. Patients with bleeds had 16.2% lower median time in therapeutic range (TTR) and were more often carriers of CYP2C9*3 variant (26 [33%] vs. 19 [8%], p < 0.001) compared with the remainder. Bleeding occurred more frequently in patients with ≥4 SNP compared with the remainder (27 [34%] vs. 47 [19%], p = 0.009) with no intergroup differences of TTR. Number of SNP was one of the predictors of any bleeding. The regression model for major bleeding including factors such as CYP2C9*3 c. 1075 C, VKORC1 c. -1639 A and APOE c. 388 C showed good predictive ability (area under the curve - 0.79). CONCLUSIONS: In VTE patients on the maintenance treatment with VKA, bleeding episodes are associated with CYP2C9 gene variations and increased number of SNP of genes involved in the action and metabolism of VKA.

Warfarin Metabolites in Patients Following Cardiac Valve Implantation: A Contribution of Clinical and Genetic Factors.

INTRODUCTION: Warfarin, a racemic mixture of S- and R-enantiomers, is the cornerstone of therapy in patients following cardiac valve replacement. S-warfarin is metabolized to 7-S-hydroxywarfarin by the cytochrome P450 isoform 2C9 encoded by CYP2C9 gene. R-warfarin is metabolized by multiple cytochromes P450. We sought to assess the impact of clinical and genetic factors on circulating warfarin metabolites following valve implantation. MATERIAL AND METHODS: Venous blood was collected from 120 patients after 3 months since elective mitral and/or aortic valve replacement. Plasma S-warfarin, R-warfarin, S-7-hydroxywarfarin, and R-7-hydroxywarfarin were determined using high-performance liquid chromatography. The S-7-hydroxywarfarin/S-warfarin and S-warfarin/R-warfarin (S/R) ratios, along with warfarin sensitivity index (WSI), defined as INR/S-warfarin ratio, were calculated. Vitamin K epoxide reductase complex subunit 1 (VKORC1) c.-1639A, CYP2C9*3 and CYP2C9*2 alleles were determined using real-time polymerase chain reaction. RESULTS: The S-warfarin was higher in former smokers (p = 0.047) and the VKORC1 c.-1639A allele carriers (p < 0.0001). The S-7-hydroxywarfarin was lower in carriers of the VKORC1 c.-1639A allele (p = 0.0005) and CYP2C9*3 (p = 0.047). The S-7-hydroxywarfarin/S-warfarin ratio was lower in the carriers of CYP2C9*3 (p = 0.008), but not in those with VKORC1 -c.1639A allele. The S/R ratio was higher in patients with hypertension (p = 0.01). The independent predictors of elevated S/R ratio defined as the upper quartile were diabetes (p = 0.045), CYP2C9*3 (p < 0.0001) and CYP2C9*2 (p = 0.0002). The independent predictors of elevated WSI were current smoking (p = 0.049), implantation of mechanical valve (p = 0.006) and VKORC1c.-1639A allele (p = 0.007). CONCLUSION: We conclude that not only genetic, but also several clinical factors affect warfarin metabolites in patients following cardiac valve implantation.

An automated method for fibrin clot permeability assessment.

The fibrin clot permeability coefficient (Ks) is a useful measure of porosity of the fibrin network, which is determined by a number of genetic and environmental factors. Currently available methods to evaluate Ks are time-consuming, require constant supervision and provide only one parameter. We present an automated method in which drops are weighed individually, buffer is dosed by the pump and well defined clot washing is controlled by the software. The presence of a straight association between drop mass and their dripping time allows to shorten the measurement time twice. In 40 healthy individuals, Ks, the number of drops required to reach the plateau (DTP), the time to achieve the plateau (TTP) and the DTP/TTP ratio (DTR) were calculated. There was a positive association between Ks (r = 0.69, P < 0.0001) evaluated by using the manual [median of 4.17 (3.60-5.18) ·10⁻9 cm²) and the automated method [median of 4.35 (3.74-5.38) ·10⁻9 cm²]. The correlation was stronger (r = 0.85, P < 0.001) in clots with DTP of 7 or less (n = 12). DTP was associated with total homocysteine (tHcy) (r = 0.35, P < 0.05) and activated partial thromboplastin time (APTT) (r = -0.34, P < 0.05), TTP with Ks (r = -0.55, P < 0.01 for the manual method and r = -0.44, P < 0.01 for the automated method) and DTP (r = 0.75, P < 0.0001), and DTR with Ks (r = 0.70, P < 0.0001 for the manual method and r = 0.76, P < 0.0001 for the automated method), fibrinogen (r = -0.58, P < 0.0001) and C-reactive protein (CRP) (r = -0.47, P < 0.01). The automated method might be a suitable tool for research and clinical use and may offer more additional parameters describing fibrin clot structure.

Genetic determinants of acenocoumarol and warfarin maintenance dose requirements in Slavic population: a potential role of CYP4F2 and GGCX polymorphisms.

INTRODUCTION: VKORC1 and cytochrome CYP2C9 genetic variants contribute largely to inter-individual variations in vitamin K antagonists (VKAs) dose requirements. Cytochrome P450 4F2 isoform (CYP4F2), gamma-glutamyl carboxylase (GGCX) and apolipoprotein E (APOE) polymorphisms have been suggested to be of minor significance. MATERIALS AND METHODS: We sought to assess the impact of those polymorphisms on dose requirements in Central-Eastern European cohort of 479 patients receiving acenocoumarol (n=260) or warfarin (n=219). RESULTS: There were no differences between the acenocoumarol and warfarin groups with regard to the gender, age, body mass index and international normalized ratio. The VKORC1 c.-1639A allele carriers required a lower dose of acenocoumarol and warfarin than the non-carriers (28.0 [21.0-35.0] vs. 42.0 [28.0-56.0] mg/week, p<0.0001; 35.0 [28.0-52.0] vs. 52.0 [35.0-70.0] mg/week, p=0.0001, respectively). Carriers of 2 and/or 3 variant alleles for CYP2C9 also required a lower dose of warfarin as compared with 1 1 carriers (35.0 [31.5-52.5] vs. 43.8 [35.0-60.2] mg/week, p=0.02; 35.0 [23.5-35.0] vs. 43.8 [35.0-60.2] mg/week, p<0.0001, respectively). Similarly, possession of G allele of GGCX c.2084+45 polymorphism was associated with lower warfarin dose (35.0 [26.3-39.2] vs. 45.5 [35.0-65.1] mg/week, p=0.03). No effect of CYP2C9*2,-*3 and GGCX c.2084+45G>C polymorphisms on acenocoumarol dosage was observed. Interestingly, carriers of CYP4F2 c.1297A variant required a higher dose of acenocoumarol and warfarin than non-carriers (43.8 [35.0-60.2] vs. 35.0 [35.0-52.5] mg/week, p=0.01; 35.0 [28.0-52.5] vs. 28.0 [28.0-42.0] mg/week, p=0.05). CONCLUSIONS: We have shown for the first time, that besides VKORC1 and CYP2C9 genetic variants, the CYP4F2 c.1297A and GGCX c.2084+45G have a moderate effect on VKAs dose requirements in Slavic population from Central-Eastern Europe.

Influence of fatty acids on mitochondrial metabolism of adipocyte progenitors and endothelial cells.

CONTEXT: In obesity, the cells are exposed to excessive amounts of nutrients, especially free fatty acids (FFAs) that induce a variety of metabolic changes. OBJECTIVE: We investigated the effect of FFAs on the mitochondrial function in different cell populations under stress conditions. METHODS: Human adipose tissue progenitor cells (SVF) or endothelial cells (HUVECs) were incubated with 30µM of selected saturated or unsaturated FFA for 24 h, at times supplemented with 5ng/mL tumour necrosis factor alpha (TNFα) for the last 4 h. Changes in oxygen respiration rate, mitochondrial membrane potential (mitoMP) and total ATP content were monitored. RESULTS: Saturated palmitic acid demonstrated no effect, while a selection of unsaturated FFAs ameliorated metabolism of the progenitor SVF cells. TNFα either did not affect or nullified some of the favourable FFA-induced effects. CONCLUSIONS: The mitoMP was the most sensitive parameter reflecting positive impact of the unsaturated FFA on the adipose SVF cells' metabolism.