Vitamin K Epoxide Reductase Complex Subunit 1 Research Articles

Genetic polymorphisms and major bleeding risk during vitamin K antagonists treatment: The BLEEDS case-cohort.

Major bleeding occurs annually in 1%-3% of patients on vitamin K antagonists (VKAs), despite close monitoring. Genetic variants in proteins involved in VKA response may affect this risk. To determine the association of genetic variants (cytochrome P450 enzymes 2C9 [CYP2C9] and 4F2 [CYP4F2], gamma-glutamyl carboxylase [GGCX]) with major bleeding in VKA users, separately and combined, including vitamin K epoxide reductase complex subunit-1 (VKORC1). A case-cohort study was established within the BLEEDS cohort, which includes 16,570 patients who initiated VKAs between 2012 and 2014. We selected all 326 major bleeding cases that occurred during 17,613 years of follow-up and a random subcohort of 978 patients. We determined variants in CYP2C9, CYP4F2, GGCX, VKORC1 and evaluated the interaction between variant genotypes. Hazard ratios for major bleeding with 95% confidence intervals (95% CI) were estimated by weighted Cox regression. Genotype was determined in 256 cases and 783 subcohort members. Phenprocoumon was the most prescribed VKA for both cases and the subcohort (78% and 75%, respectively). Patients with major bleeding were slightly older than subcohort patients. CYP4F2-TT carriership was associated with a 1.6-fold (95% CI 0.9-2.8) increased risk of major bleeding compared with CC-alleles, albeit not statistically significant. For the CYP2C9 and GGCX variants instead, the major bleeding risk was around unity. Carrying at least two variant genotypes in CYP2C9 (poor metabolizer), CYP4F2-TT, and VKORC1-AA was associated with a 4.0-fold (95%CI 1.4-11.4) increased risk, while carriers of both CYP4F2-TT and VKORC1-AA had a particularly increased major bleeding risk (hazard ratio 6.7, 95% CI 1.5-29.8) compared with carriers of CC alleles in CYP4F2 and GG in VKORC1. However, the number of major bleeding cases in carriers of multiple variants was few (8 and 5 patients, respectively). CYP4F2 polymorphism was associated with major bleeding, especially in combination with VKORC1 genetic variants. These variants could be considered to further personalize anticoagulant treatment.

Is a Vitamin K Epoxide Reductase Complex Subunit 1 (VKORC1) Polymorphism a Risk Factor for Nephrolithiasis in Sarcoidosis?

Sarcoidosis is a systemic inflammatory disorder characterized by granuloma formation in various organs. It has been associated with nephrolithiasis. The vitamin K epoxide reductase complex subunit 1 (VKORC1) gene, which plays a crucial role in vitamin K metabolism, has been implicated in the activation of proteins associated with calcification, including in the forming of nephrolithiasis. This study aimed to investigate the VKORC1 C1173T polymorphism (rs9934438) in a Dutch sarcoidosis cohort, comparing individuals with and without a history of nephrolithiasis. Retrospectively, 424 patients with sarcoidosis were divided into three groups: those with a history of nephrolithiasis (Group I: n = 23), those with hypercalcemia without nephrolithiasis (Group II: n = 38), and those without nephrolithiasis or hypercalcemia (Group III: n = 363). Of the 424 sarcoidosis patients studied, 5.4% had a history of nephrolithiasis (Group I), only two of whom possessed no VKORC1 polymorphisms (OR = 7.73; 95% CI 1.79-33.4; p = 0.001). The presence of a VKORC1 C1173T variant allele was found to be a substantial risk factor for the development of nephrolithiasis in sarcoidosis patients. This study provides novel insights into the genetic basis of nephrolithiasis in sarcoidosis patients, identifying VKORC1 C1173T as a potential contributor. Further research is warranted to elucidate the precise mechanisms and explore potential therapeutic interventions based on these genetic findings.

A physiologically-based pharmacokinetic/pharmacodynamic modeling approach for drug-drug-gene interaction evaluation of S-warfarin with fluconazole.

Warfarin is a widely used anticoagulant, and its S-enantiomer has higher potency compared to the R-enantiomer. S-warfarin is mainly metabolized by cytochrome P450 (CYP) 2C9, and its pharmacological target is vitamin K epoxide reductase complex subunit 1 (VKORC1). Both CYP2C9 and VKORC1 have genetic polymorphisms, leading to large variations in the pharmacokinetics (PKs) and pharmacodynamics (PDs) of warfarin in the population. This makes dosage management of warfarin difficult, especially in the case of drug-drug interactions (DDIs). This study provides a whole-body physiologically-based pharmacokinetic/PD (PBPK/PD) model of S-warfarin for predicting the effects of drug-drug-gene interactions on S-warfarin PKs and PDs. The PBPK/PD model of S-warfarin was developed in PK-Sim and MoBi. Drug-dependent parameters were obtained from the literature or optimized. Of the 34 S-warfarin plasma concentration-time profiles used, 96% predicted plasma concentrations within twofold range compared to observed data. For S-warfarin plasma concentration-time profiles with CYP2C9 genotype, 364 of 386 predicted plasma concentration values (~94%) fell within the twofold of the observed values. This model was tested in DDI predictions with fluconazole as CYP2C9 perpetrators, with all predicted DDI area under the plasma concentration-time curve to the last measurable timepoint (AUClast ) ratio within twofold of the observed values. The anticoagulant effect of S-warfarin was described using an indirect response model, with all predicted international normalized ratio (INR) within twofold of the observed values. This model also incorporates a dose-adjustment method that can be used for dose adjustment and predict INR when warfarin is used in combination with CYP2C9 perpetrators.

Frequencies of VKORC1-1639G>A and rs397509427 in Patients on Warfarin and Healthy Syrian Subjects.

Vitamin K epoxide reductase complex subunit 1 (VKORC1) gene encodes a key enzyme with multiple cellular activities, namely, the reduction of vitamin K to its active form. VKORC1-1639G>A (rs9923231) is a common single nucleotide polymorphism with a crucial impact on warfarin dosing and possibly other physiological functions. This study aimed at investigating the frequencies of VKORC1-1639G>A alleles and genotypes in Syrian healthy subjects and patients on warfarin for different indications. A total of 138 individuals were enrolled in this cross-sectional study. Genomic DNA was extracted from both patients on warfarin and healthy subjects, and polymerase chain reaction (PCR) specific amplicons were genotyped via standard sequencing which also allowed the detection of rs397509427. Comparisons of -1639G>A frequency with other populations were drawn. Of 94 patients on warfarin, 53 (56.38%) were with idiopathic venous thromboembolism (VTE). Despite comparable frequencies of the -1639A allele (47% and 50%), the AA and GA genotypes were at disparate frequencies of 93.2% versus 79.8% in the healthy subjects (n = 44) versus patients on warfarin, respectively. Carriers of the GG genotype were at a four-fold increased risk of VTE in comparison with those of the AA and GA genotypes (odds ratio (OR) = 4, 95% CI = 1.105 - 13.6, P = 0.0469). All study subjects were wild-type for the rs397509427 variant. Our results prove a high -1639A prevalence in Syrian healthy subjects and patients on warfarin at frequencies comparable to other Mediterranean and Middle Eastern populations. The A allele carriers are at a lower VTE risk, whereas a global prevalence gradient of the G allele is suggested to be associated with VTE risk.

The vitamin-k-cycle-enzyme VKORC1L1, but not its isoenzyme VKORC1, reduces inflammation in coronary endothelial cells

Abstract Introduction Vascular inflammation is a crucial contributor to atherosclerosis, in which oxidative stress, endoplasmic reticulum (ER) stress and transition of endothelial to mesenchymal cells (EndMT) are of critical importance. Vitamin-K-antagonists (VKA) promote vascular dysfunction, while vitamin K intake was proposed to reduce incidence of coronary artery disease. Key enzyme for regeneration of vitamin K is the Vitamin K epoxide reductase complex subunit 1 (VKORC1), which also represents the pharmacological target of VKA. VKOR-like1 (VKORC1L1) is an isoenzyme of VKORC1, which resides at the ER-membrane, exerts antioxidative properties and is involved in vitamin K maintenance. Aim of this study was to investigate the role of VKORC1 and VKORC1L1 in inflammation of human coronary artery endothelial cells (HCAEC). Methods and Results In silico analyses of the proteome of human coronary atherosclerosis revealed increased expression of VKORC1L1, but not of VKORC1. In vitro induction of oxidative stress (H2O2) and ER-Stress (tunicamycin) in HCAEC promoted time- and dose-dependent enhanced expression of VKORC1L1, without altering VKORC1 expression (H2O2: 2.3-fold vs. 0.8-fold after 40 minutes, p=0.04; tunicamycin: 1 μg/ml: 1.43-fold vs. 0.8-fold, p<0.01). Moreover, inducing EndMT by treatment with a differentiation medium (containing TGF-ß2, IL-1ß) resulted in a reduction of VKORC1L1, but not of VKORC1 (Fig.1). To analyze the relevance of the VKOR-enzymes on endothelial cell function, VKORC1 and VKORC1L1 were downregulated by siRNA. VKORC1L1 downregulation increased formation of reactive oxygen species (ROS) (DCFDA signal: 0.98-fold vs. 1.31-fold, p = 0.03) and reduced proliferation (EdU signal: 0.56 ± 0.02 vs. control, p < 0.01), whereas viability of HCAEC remained unchanged. ELISA and qPCR experiments revealed enhanced expression of a variety of markers of vascular inflammation after VKORC1L1 (e.g., IL-6: 3.42-fold ± 0.53, NF-κB: 1.95-fold ± 0.13, VCAM-1: 1.59-fold ± 0.12, vs. control). Additionally, expression of ER-stress markers was increased (e.g., GRP78; 1.32-fold ± 0.04 vs. control, p<0.01) (Fig.1). In contrast, VKORC1 downregulation significantly promoted proliferation (EdU signal: 3.9 ± 1.7 vs. control, p<0.01) as well as viability (alamarBlue® absorbance: 1.07 ± 0.08 vs. control, p=0.04), and decreased ROS (0.88 ± 0.17 vs. control, p=0.03). Pro-inflammatory and ER-stress proteins were either unaltered or decreased after VKORC1 knockdown (e.g., GRP78: 0.89-fold, VCAM-1: 0.68-fold) (Fig.2). To further elucidate these findings, we will perform overexpression experiments and stable knockdown of the VKOR-enzymes. Conclusion VKORC1 and its isoenzyme VKORC1L1 exhibit divergent effects on endothelial cell inflammation. Further studies are warranted to shed light on the regulation of the enzymes to improve our understanding regarding vascular side effects of VKA treatment and beneficial effects of vitamin K supplementation.

Effect of asciminib and vitamin K2 on Abelson tyrosine-kinase-inhibitor-resistant chronic myelogenous leukemia cells

BackgroundAbelson (ABL) tyrosine kinase inhibitors (TKIs) are effective against chronic myeloid leukemia (CML); however, many patients develop resistance during ABL TKI therapy. Vitamin K2 (VK2) is a crucial fat-soluble vitamin used to activate hepatic coagulation factors and treat osteoporosis. Although VK2 has demonstrated impressive anticancer activity in various cancer cell lines, it is not known whether VK2 enhances the effects of asciminib, which specifically targets the ABL myristoyl pocket (STAMP) inhibitor.MethodIn this work, we investigated whether VK2 contributed to the development of CML cell lines. We also investigated the efficacy of asciminib and VK2 by using K562, ponatinib-resistant K562 (K562 PR), Ba/F3 BCR–ABL, and T315I point mutant Ba/F3 (Ba/F3 T315I) cells.ResultsBased on data from the Gene Expression Omnibus (GEO) database, gamma-glutamyl carboxylase (GGCX) and vitamin K epoxide reductase complex subunit 1 (VKORC1) were elevated in imatinib-resistant patients (GSE130404). UBIA Prenyltransferase Domain Containing 1 (UBIAD1) was decreased, and K562 PR cells were resistant to ponatinib. In contrast, asciminib inhibited CML cells and ponatinib resistance in a dose-dependent manner. CML cells were suppressed by VK2. Caspase 3/7 activity was also elevated, as was cellular cytotoxicity. Asciminib plus VK2 therapy induced a significantly higher level of cytotoxicity than use of each drug alone. Asciminib and VK2 therapy altered the mitochondrial membrane potential.ConclusionsAsciminib and VK2 are suggested as a novel treatment for ABL-TKI-resistant cells since they increase treatment efficacy. Additionally, this treatment option has intriguing clinical relevance for patients who are resistant to ABL TKIs.

Polymorphism Detection of VKORC1 (Vitamin K Epoxide Reductase Complex, Subunit 1) Gene for Warfarin Dose Adjustment in Egyptian Patients with Budd Chiari and/or Portal Vein Thrombosis: A Single Center Experience

Background Budd–Chiari syndrome is a heterogeneous group of disorders characterized by hepatic venous outflow obstruction at the level of the hepatic venules, the large hepatic veins, the inferior vena cava till its junction with the right atrium. The classic triad of abdominal pain, ascites, and hepatomegaly is observed in the vast majority of patients with Budd-Chiari syndrome, but it is nonspecific. Warfarin was introduced into clinical practice in the 1950s, and has been the most widely prescribed oral anti-coagulant for prevention and treatment of thrombotic diseases despite its significant side effects as (bleeding tendency, drug drug interaction). Vitamin K epoxide reductase complex subunit1 (VKORC1), a membrane protein located in hepatocytes, has an important role in the anabolism of vitamin K, which is essential for blood coagulation. Objectives The aim beyond this study is to evaluate effect of the allelic frequency of the VKORC1 (G1639A) polymorphic genes by real time PCR on response of Egyptian patients with Budd–Chiari syndrome or PVT to Warfarin. Patients and Methods The studied patients were selected from patients presented to Budd chiari study group clinic.tropical medicine department in Ain Shams University hospitals. Within 6 months from july 2020 to December 2020, total number of cases during the study period was 50 cases fulfilling the inclusion criteria Results The mean age of the patients was (32.40 ± 9.69) slightly more common in males. The chronic presentation represented 60% of the patients and the acute represented 32%.The most common cause of budd chiarri syndrome was MTHFRD (52%) followed by FVLM (36%) then Primary ant phospholipid antibody syndrome (26%). Patients with the −1639 promoter polymorphism GG genotype had lower dose requirements, whereas the AG/AA genotypes had higher dose. Conclusion Identifying these polymorphisms in patients prior to starting the therapy may help clinicians choose the appropriate warfarin dose & decrease its adverse effects.

The Association Between Bone Remodeling Markers, genes polymorphisms with Incidence of Osteopenia Among Young Saudi Female.

Osteopenia and osteoporosis, are prevalent skeletal systemic conditions, cause weaker bones and an increased risk of fragility fractures. This work is aimed to evaluate the relation between bone-remolding markers and genotypes of four single nucleotide polymorphisms in young Saudi females (rs2297480 of farnesyl diphosphate synthase (FDPS), rs3736228 of Low-density lipoprotein receptor-related protein 5(LRP5), rs1234612 of sclerostin (SOST), and rs9934438 of Vitamin K epoxide reductase complex subunit 1 (VKORC1) ). For this purpose, 750 premenopausal females aged 18 to 40 years old, either university students, postgraduates, or university employees were recruited and divided into three groups according to bone mineral density BMD (g/cm2) divided by T score into osteoporosis (n = 12), osteopenia (n = 147), and normal (n = 591). Serum SOST, BALP, calcium, phosphate, ALP, albumin, beta-CTXs and human VDR levels were determined. TaqMan SNP Genotyping assays were used to genotype four polymorphisms using real-time PCR (applied biosystem). Results showed that BALP, CTX-1 and SOST were significantly higher in the osteoporosis and osteopenia groups than in the normal group. Bone mineral density readings were considerably lower in females with the GG genotype in FDPS rs2297480 and TT genotype in LRP5 rs3736228, which increase the risk for osteopenia by 3. 6-fold and 3. 06-fold than control respectively. Also, females with the TT genotype in LRP5 rs3736228 have decreased average values for Bone Mineral Density. In conclusion, the GG genotype of FDPS rs2297480 and the TT genotype of LRP5 rs3736228 was shown to be strongly associated with osteopenia in young Saudi females with low bone mineral density and SOST levels.

Prediction of a Stable Complex of Compounds in the Ethanol Extract of Celery Leaves (Apium graveolens L.) Function as a VKORC1 Antagonist

The vitamin K cycle, specifically the protein VKORC1 (Vitamin K epoxide reductase complex subunit 1), is closely linked to coagulation mechanisms in the body. Disruptions in this cycle will be associated with vascular diseases such as myocardial infarction and stroke. Warfarin is commonly used as an anticoagulant, but it still has side effects in interactions with numerous drug compounds; thus, it is necessary to search for safer candidate compounds. The ethanol extract of celery leaves (Apium graveolens L.) has potential anticoagulant activity, but the compound responsible for this activity has not been identified yet. The CADD method has been developed to predict in silico a compound's potential to interact with binding sites. This study aimed to predict interactions and obtain a stable complex of the compounds in the ethanol extract of celery leaves, which function as a VKORC1 antagonist. A total of 23 compounds were simulated using Autodock 4.2, and AMBER 18 was then used to simulate the stability of the five compounds with the best interactions. The docking simulation results of 17 test compounds (ligands) yielded five selected compounds, namely 6-isopentenyloxy-isobergapten (S1), Heratomin (S2), Apigenin (S3), Lanatin (S4), and Isoimperatorin (S5), with G values of −9.27, −9.26, −9.22, −9.13, and −8.94 kcal/mol, respectively. The MD simulation continued to produce 6-isopentenyloxy-isobergapten as the most effective ligand for stabilizing the complex for 100 ns from these five ligands. In conclusion, the 6-isopentenyloxy-isobergapten from celery leaf has the potential as a candidate anticoagulant, VKORC1.

Mechanism of VKORC1 and VKORC1L1 signaling in the effects of sodium dehydroacetate on coagulation factors in rat hepatocytes

Sodium dehydroacetate (Na-DHA) is widely used as an antibacterial and preservative additive in food and cosmetics. Previously, we reported that repeated oral administration of Na-DHA induces coagulation disorders, and inhibited liver vitamin K epoxide reductase complex subunit 1 (VKORC1) and VKORC1-like protein 1 (VKORC1L1) in rats. However, the effects of Na-DHA on coagulation factors in rat hepatocytes and the mechanism of VKORC1 and VKORC1L1 signaling in that process are unclear. Here, we constructed stable Vkorc1 and Vkorc1l1 overexpressing cell lines using lentiviruses and transfected small interfering RNAs into buffalo rat liver BRL3A cells for Vkorc1 and Vkorc1l1 overexpression and silencing, respectively. After treatment with 5 mmol/L Na-DHA for 24 h, VKORC1 and VKORC1L1 expression levels were detected by real-time PCR and western blotting. Vitamin K (VK) and factor IX (FIX) contents were detected using enzyme linked immunosorbent assays. We observed that Na-DHA inhibited VKORC1 and VKORC1L1 expression levels and reduced VK and FIX levels in rat hepatocytes. Overexpression or silencing of Vkorc1 and Vkorc1l1 increased or decreased, respectively, the production and secretion of VK and FIX in rat hepatocytes, and alleviated or aggravated the inhibitory effects of Na-DHA on VKORC1 and VKORC1L1 expression levels. Taken together, the results indicated that both VKORC1 and VKORC1L1 signaling play regulatory roles in the effects of Na-DHA on coagulation factors in rat hepatocytes.

Novel Gamma-Glutamyl Carboxylase Mutation Causing Hemorrhagic and Non-Hemorrhagic VKCFD1 Phenotypes

Background: Vitamin K clotting factor deficiency (VKCFD) types 1 and 2 are very rare autosomal recessive disorders associated with reduced clotting factors activities for all vitamin K dependent clotting factors. Less than 30 cases are reported with defects in GGCX (gamma-glutamyl carboxylase) leading to VKCFD Type 1 (VKCFD1) or VKORC1 (vitamin K epoxide reductase complex subunit 1) leading to VKCFD Type 2 (VKCFD2). In addition to clotting factor deficiency, GGCX mutations can be associated with skeletal abnormalities such as bone hypoplasia, conductive hearing loss, mental retardation, and skin hyperlaxity. Here, we report an Arabic family with VKCFD1 harboring an unreported, new pathogenic variant in the GGCX gene. The proband presented at 17 months of age with severe bruising following trauma and work up demonstrated deficiencies of all Vitamin K dependent (VKD) coagulation proteins. Another sibling born 8 years later was also diagnosed with the same condition with abnormal coagulation parameters at birth. Additionally, the non-hemorrhagic manifestations diagnosed in both siblings were mid-facial hypoplasia with flat nasal bridge, dental abnormalities, and shortened distal phalanges representing a keutel syndrome-like phenotype. Both patients are currently managed with oral vitamin K supplementation with resolution of bleeding symptoms. Here we present the in-vitro assessment of the mutations effects to vitamin K supplementation and potential clinical implications. Methods: The proband, affected brother and mother, all underwent genetic evaluation at the University Clinic Bonn in Germany. DNA was isolated from the patient's blood and Sanger Sequencing was performed for the genes VKORC1 and GGCX. The identified new pathogenic variant in GGCX was further characterized by the in-vitro assay published by Ghosh et al. in 2021. Results: The brothers were found to be homozygous for c.[416G>A];[416G>A] p.(Cys139Tyr), a genetic variant in exon 4 of the GGCX gene. Mutations in VKORC1 gene leading to reduced activity for the VKD clotting factors were excluded confirming the diagnosis of VKCFD Type 1 (VKCFD1). The mother was found to be heterozygous for the same GGCX variant. The affected siblings and the mother were further genotyped for the promoter single nucleotide polymorphism in VKORC1 gene NG_011564.1:g.3588G>A (VKORC1:c.-1639 G>A) since this might correlate with the severity of the phenotypes in patients with skeletal manifestations. The brothers and mother were found to harbor VKORC1:c.-1639 GA, which has been associated with a 33% reduced VKORC1 expression. Treatment with 5mg of vitamin K resulted in increased activities of the VKD clotting factors (Table 1). However, clotting factor activities did not normalized after vitamin K supplementation. The in vitro characterization of the new identified pathogenic variant GGCX:p.C139Y (Figure 1) showed reduced activities for all VKD clotting factors, which did not reach wild type levels despite increasing concentrations of vitamin K. The levels of Matrix Gla protein (MGP), Bone Gla protein (BGLAP), and Gla-rich protein (GRP) showed almost no response to increasing amount of vitamin K. Discussion: Here, we report a new pathogenic variant in exon 4 of GGCX c.[416G>A];[416G>A] p.(Cys139Tyr) leading to VKCFD1 with reduced clotting factor activities and non-hemorrhagic manifestations. The pre and post vitamin K supplementation factor levels in the patients correlate with the in-vitro data and demonstrate failure of the clotting factor levels to normalize despite vitamin K supplementation. The in vitro data for the non-hematological VKD proteins indicate that the patients might develop a pseudoxanthoma elasticum (PXE)-like phenotype resulting into hyperlaxity of skin due to the almost zero levels of carboxylated GRP. The skeletal/bone phenotypes can be explained by the almost zero values for carboxylated MGP and GRP. To assess this in-vivo, levels of human MGP and BGLAP are currently being analyzed in the patients and mothers blood samples. These patients highlight the nuances associated with the diagnosis and management of patients with VKCFD. The antenatal management and timing of Vitamin K supplementation to prevent intrauterine bleeding is also unknown, and there may be benefit to starting early to prevent the bone and skin manifestations. Further research is warranted in rare bleeding disorders such as VKCFD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Gene variants of coagulation related proteins that interact with SARS-CoV-2.

Thrombosis is a recognized complication of Coronavirus disease of 2019 (COVID-19) and is often associated with poor prognosis. There is a well-recognized link between coagulation and inflammation, however, the extent of thrombotic events associated with COVID-19 warrants further investigation. Poly(A) Binding Protein Cytoplasmic 4 (PABPC4), Serine/Cysteine Proteinase Inhibitor Clade G Member 1 (SERPING1) and Vitamin K epOxide Reductase Complex subunit 1 (VKORC1), which are all proteins linked to coagulation, have been shown to interact with SARS proteins. We computationally examined the interaction of these with SARS-CoV-2 proteins and, in the case of VKORC1, we describe its binding to ORF7a in detail. We examined the occurrence of variants of each of these proteins across populations and interrogated their potential contribution to COVID-19 severity. Potential mechanisms, by which some of these variants may contribute to disease, are proposed. Some of these variants are prevalent in minority groups that are disproportionally affected by severe COVID-19. Therefore, we are proposing that further investigation around these variants may lead to better understanding of disease pathogenesis in minority groups and more informed therapeutic approaches.

Identification and Enzymatic Activity Evaluation of a Novel CYP2C9 Allelic Variant Discovered in a Patient.

Warfarin is a widely prescribed anticoagulant but the doses required to attain the optimum therapeutic effect exhibit dramatic inter-individual variability. Pharmacogenomics-guided warfarin dosing has been recommended to improve safety and effectiveness. We analyzed the cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase complex subunit 1 (VKORC1) genes among 120 patients taking warfarin. A new coding variant was identified by sequencing CYP2C9. The novel A > G mutation at nucleotide position 14,277 led to an amino acid substitution of isoleucine with valine at position 213 (I213V). The functional consequence of the variant was subsequently evaluated in vitro. cDNA of the novel variant was constructed by site-directed mutagenesis and the recombinant protein was expressed in vitro using a baculovirus–insect cell expression system. The recombinant protein expression was quantified at apoprotein and holoprotein levels. Its enzymatic activities toward tolbutamide, warfarin and losartan were then assessed. It exhibited changed apparent Km values and increases of 148%, 84% and 67% in the intrinsic clearance of tolbutamide, warfarin and losartan, respectively, compared to wild-type CYP2C9*1, indicating dramatically enhanced in vitro enzymatic activity. Our study suggests that the amino acid at position 213 in wild-type CYP2C9*1 may be important for the enzymatic activity of CYP2C9 toward tolbutamide, warfarin and losartan. In summary, a patient taking high-dose warfarin (6.0 mg/day) in order to achieve the target international normalized ratio was found to have a mutation in the CYP2C9 gene.

AtreMorine Treatment Regulates DNA Methylation in Neurodegenerative Disorders: Epigenetic and Pharmacogenetic Studies

Background: Neurodegenerative disorders are one of the major health problems in Western countries. Genetic and epigenetic mechanisms play crucial roles in the origin and progression of these disorders. DNA methylation is the most widely studied epigenetic mark and is an important regulator of gene expression. Objective: Little is known about the influence of bioactive dietary components on epigenetic mechanisms in neurodegenerative diseases. In this study, we investigated the effects of E-PodoFavalin-15999 (AtreMorine®), a bioproduct with potent neuroprotective and dopamine enhancing capabilities, on DNA methylation patterns in Alzheimer’s (AD) and Parkinson’s Disease (PD). We also aimed to assess, in patients with PD, the effects that genetic variation across candidate pharmacogenes may have on dopamine synthesis and release in response to treatment with AtreMorine. Methods: We analyzed global DNA methylation and de novo DNA methyltransferase (DNMT) expression in a transgenic (3xTg) mouse model of AD, and further examined global DNA methylation in blood samples from patients with PD. Results: AtreMorine treatment increased global DNA methylation in 3xTg mice and in patients with Parkinson´s disease, and produced high DNMT3a expression in AD mice. We observed varied responses to AtreMorine across the following pharmacogenetic genophenotypes analyzed, cytochrome P450 oxidases (CYP2D6, CYP2C19, CYP2C9, CYP3A4, CYP3A5, CYP1A2), human arylamine N-acetyltransferase 2 (NAT2), the vitamin K epoxide reductase complex subunit 1 (VKORC1), ATP-binding cassette subfamily B member 1 (ABCB1), and solute carrier organic anion transporter family member 1B1 (SLCOB1). Conclusion: Our results suggest that AtreMorine regulates DNA methylation in neurodegenerative disorders and may constitute a new therapeutic option for the treatment of these pathologies.

Genetic Variation in VKORC1 and Risk for Osteoporosis

The vitamin K epoxide reductase complex subunit 1 (VKORC1) plays an important role in bone development and bone metabolism by influencing the vitamin K cycle. The aim of this study was to investigate the association between VKORC1 gene polymorphisms and bone mineral density and the risk of osteoporosis. We determined VKORC1 gene polymorphisms (rs9923231 and rs9934488) by using polymerase chain reaction-restriction and Sanger sequencing method in 606 postmenopausal women including 288 osteoporosis patients and 318 healthy controls. No significant differences were observed in the rs9934488 polymorphisms between the osteoporosis group and controls. Subgroup analyses also indicated no positive result. VKORC1 rs9923231 polymorphism increased the risk of osteoporosis in the homozygous and allelic models. A significant correlation was observed in a subgroup of the elderly (age ≥55years). Additionally, the genotypes of the rs9923231 polymorphism were significantly associated with low body mass index, and T-score, but not with age, serum calcium, or phosphorus. In conclusion, VKORC1 rs9923231 polymorphism is a genetic contributor to osteoporosis risk and it should be confirmed in large well-designed studies.

Vitamin K epoxide reductase complex subunit 1 (VKORC1) gene polymorphism as determinant of differences in Covid-19-related disease severity

Covid-19, caused by SARS-CoV-2, has major world-wide health-related and socio-economic consequences. There are large disparities in the burden of Covid-19 with an apparent lower risk of poor outcomes in East Asians compared to populations in the West. A recent study suggested that Covid-19 leads to a severe extrahepatic vitamin K insufficiency, which could lead to impaired activation of extrahepatic proteins like endothelial anticoagulant protein S in the presence of normal hepatic procoagulant activity. This would be compatible with the enhanced thrombogenicity in severe Covid-19. The same study showed that vitamin K antagonists (VKA) that inhibit vitamin K recycling, had a greater impact on procoagulant activity than on the activation of extrahepatic vitamin K-dependent proteins during SARS-CoV-2 infections. A genetic polymorphism in the vitamin K epoxide reductase complex 1, VKORC1 −1639A, is particularly prevalent in East Asia and associates with low vitamin K recycling rates. Carriage of the allele may be regarded as bioequivalent to low-dose VKA use. We speculate that VKORC1 −1639A confers protection against thrombotic complications of Covid-19 and that differences in its allele frequency are partially responsible for the differences in Covid-19 severity between East and West.

The point mutation analysis of Cyp2C9*2 (Arg144Cys C>T), Cyp2C9*3 (Ile359Leu A>C) and VKORC1 (1639G>A) in women with cervical cancer related to HPV: A case-control study.

Human papillomavirus (HPV) is the most common sexually transmitted viral infection worldwide. HPV tumorigenesis genotypes are the causative agents of cervical cancer and genital malignancies. The scientific literaturehas demonstrated that life style, environmental, epigenetic accompanied with HR-HPV genotypes arepotential risk factors forcervical cancer progression. The frequencies of the Cyp2C9*2 , Cyp2C9*3 , and vitamin K epoxide reductase complex subunit 1 (VKORC1) genotypes as potential molecular biomarkers have been investigated on Iranian women with cervical malignancy related to HPV genotypes. As a case-control study, the mutations wereappraised using a polymerase chain reaction-restriction fragment length polymorphism procedure on women sufferingfrom HPV infection (60 cases), CC (46 cases), and 40 subjects of as healthy control. The outcomes demonstrated that Cyp2C9*3 showed a meaningful relationship between women diagnosed with cervical cancer and the healthy population (AA vs. AC; OR, 7.15; 95% CI, 1.94-26.3; p = .003). It was also observed that the Cyp2C9*3 mutation in women with cervical cancer and VKORC1 in healthy population with HPV (+), did not follow the Hardy-Weinberg equilibrium. Our findings aidunderstanding the genetic polymorphism distribution of Cyp2C9*2 , Cyp2C9*3 , and VKORC1 in women with genital malignancies. Thiscan also be useful in predicting the susceptibility risk factors for developing cervical cancer. However, allelic discrimination as a molecular biomarker requires further research.

The Contribution of VKORC1 and CYP2C9 Genetic Polymorphisms and Patients' Demographic Characteristics with Warfarin Maintenance Doses: A Suggested Warfarin Dosing Algorithm.

The requirement of varying doses of warfarin for different individuals can be explained by environmental and genetic factors. We evaluated the frequency of vitamin K epoxide reductase complex subunit 1 (VKORC1) and cytochrome P450 2C9 (CYP2C9) variants together with patientdemographic characteristics and investigated their association with warfarin dose requirement with the objective to suggest a warfarin dosing algorithm. In this study, 185 patients with heart valve replacement from West Azerbaijan, Iran were genotyped for VKORC1 (-1639 G>A) and CYP2C9 (*2 and *3 alleles) by PCR-RFLP. Multiple linear regression was performed to create a new warfarin dosing algorithm. The frequency of variants in studied subjects was 12% for CYP2C9 *2, 25.8% for CYP2C9 *3, and 60% for -1639A. The patients who carried the A allele at position -1639 VKORC1 and the variants CYP2C9 *2 and *3 required a significantly lower daily mean warfarin dosage (P = 0.001). Statistical analysis also indicated a significant relationship between the daily maintenance dose of warfarin with age and blood pressure among the studied patients’ cohort (P < 0.001). This study showed that in the heart valve replacement patients considering VKORC1 and CYP2C9 polymorphisms beside demographic characteristics such as age will be helpful in pre-treatment dosing of warfarin which in turn reduces the complications associated with inappropriate warfarin dosing.

Genetic risk assessment towards warfarin application: Saudi Arabia study with a potential to predict and prevent side effects.

Warfarin doses are greatly affected by polymorphism altering cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase complex subunit 1 (VKORC1) gene. This study evaluated the prevalence of alleles (either single or double) and carriers of single nucleotide polymorphisms (SNPs) in both genotypes CYP2C9 and VKORC1 in alkharj area, Saudi Arabia and its association with warfarin use risk. Total 112 samples were collected and genotyped using FlexiGene DNA Kit for isolation and StepOnePlus Real-Time PCR System by TaqMan allelic discrimination methods. The results indicated the frequency of 11%, 8% and 45% for CYP2C9 *2 *3 and VKORC1-1639 G > A polymorphism. And as a combination genotype it was 15.18% For both CYP2C9 and VKORC1 polymorphism, 27.67% for CYP2C9 and 42.86% for VKORC1. Non-carriers rate came to be at 30.3%. According to previously published dosing changes in warfarin for polymorphism carriers (single-double-triple). The predicted warfarin doses reduction in order of 1–1.6, 2–2.9, 2.9–3.7 mg/day. It was found that 72.3% of the study population was carrier of a type of polymorphism, 15.18% for two types of polymorphisms. These findings predict changes in warfarin metabolism and eventually dosing alteration among patients on warfarin. Both genotypes (CYP2C9 and VKORC1) require different dosing of warfarin than non-carriers in order to minimize the risk of warfarin overdosing and avoidance of the drug-related problems (DRPs).

CYP2C9, VKORC1, and CYP4F2 polymorphisms and pediatric warfarin maintenance dose: a systematic review and meta-analysis.

Studies on the effect of cytochrome P450 2C9 (CYP2C9), vitamin K epoxide reductase complex subunit 1 (VKORC1), and cytochrome P450 4F2 (CYP4F2) polymorphisms on warfarin maintenance dose in children are conflicting. We conducted a systematic review and meta-analysis to evaluate the effect of these polymorphisms on warfarin maintenance dose in children. We searched relevant literature using the MEDLINE, EMBASE, and Cochrane Central Register of Controlled Trial libraries without any language restrictions from their inception to 23 July 2017. Dose differences are expressed as standardized mean difference (SMD) or mean difference (MD) with 95% confidence intervals (CI). This review was registered in the PROSPERO prospective register of systematic reviews (CRD42015016172). We included a total of nine studies (745 participants) in the meta-analysis. Patients with CYP2C9 *1/*2, *1/*3, *2/*2, *2/*3, or *3/*3 required a lower warfarin maintenance dose compared with patients with CYP2C9 *1/*1 (SMD = -0.610, 95% CI: -0.802 to -0.419, I2 = 0%). Patients with VKORC1-1639GA or AA required a lower warfarin maintenance dose compared with patients with VKORC1-1639GG (SMD = -0.666, 95% CI: -0.887 to -0.445, I2 = 33%). However, no associations were observed between CYP4F2 polymorphisms and warfarin maintenance dose (MD = 0.005 mg/kg/day, 95% CI: -0.006 to 0.015, I2 = 0%). These results were not affected by a sensitivity analysis. Our meta-analysis provides evidence that CYP2C9 and VKORC1 variant statuses affect warfarin maintenance dose in children, but not CYP4F2.