Multiplexed measurement of variant abundance and activity reveals VKOR topology, active site and human variant impact
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ABSTRACT:
ORGANISM(S): synthetic construct Homo sapiens
PROVIDER: GSE149922 | GEO | 2020/08/10
REPOSITORIES: GEO
Similar Datasets
Project description:Warfarin targets human vitamin K epoxide reductase (hVKOR), a redox enzyme in the membrane of endoplasmic reticulum (ER), to prevent the formation of blood clots. Although warfarin has been a popular medication since 1954, its mechanism of action is still unclear. A fundamental issue is the controversial orientation of transmembrane helices (TM) in hVKOR. Stable isotope-coded reagents were usedto label VKOR in free, mutated, and warfarin-binding states directly in the cellular environment, followed by LC-MS/MS bottom-up approach to investigate the warfarin binding mechanism.
2017-03-30 | PXD003774 | Pride
Project description:<p>Warfarin is an oral anticoagulant and one of the most used drugs in the world accounting for up to 1.5% of prescriptions globally. Warfarin inhibits vitamin K epoxide reductase complex 1 (VKORC1), an enzyme responsible for the vitamin K recycling that is required for the activation of clotting factors. Warfarin is prescribed at an average dose of 5 mg/day for most of the indications but significant inter-patient variability has been observed. Single nucleotide variations at the VKORC1 (<a href="https://www.ncbi.nlm.nih.gov/gene/?term=VKORC1">GeneID: 79001</a>) and CYP2C9 (<a href="https://www.ncbi.nlm.nih.gov/gene/?term=CYP2C9">GeneID: 1559</a>) genes alone explain from 28 to 35% of variability in warfarin dose requirements and up to 56% when clinical factors (age, gender, body surface area, diabetes and heart valve) are incorporated into regression analysis. However, genetic-guided dosing algorithms are mostly derived from studies in Europeans. The objective of the present study was to identify genetic polymorphisms that can explain variability in warfarin dose requirements among Caribbean Hispanics of Puerto Rico. VKORC1 (<a href="https://www.ncbi.nlm.nih.gov/gene/?term=VKORC1">GeneID: 79001</a>) and CYP2C9 (<a href="https://www.ncbi.nlm.nih.gov/gene/?term=CYP2C9">GeneID: 1559</a>), the most important genetic predictors of warfarin response, were sequenced using Next Generation sequencing. Other candidate genes in DMET Plus Assay were assessed to identify genetic variants with relevance in warfarin dose requirements among Puerto Ricans. Our study used the Extreme Discordant Phenotype approach to perform a case-control association analysis that were confirmed with univariate and conditioned regressions. Accordingly, patients were stratified based on stable warfarin doses as sensitive (<4 mg/day), control (4-6 mg/day) and resistant (>6 mg/day). Genetic variants associated with warfarin response among the study cohort were used to perform a multivariate regression analyses to develop a genetic-guided dosing algorithm tailored for Puerto Ricans. The CYP2C9rs2860905 SNP was found to be strongly associated with warfarin dose requirements in Puerto Ricans. The CYP2C9 rs2860905 SNP tags four haplotypes that represent the trihybrid admixture of Puerto Ricans. Therefore, haplotypes harboring the rs2860905 variant are more informative in predicting warfarin dose among Puerto Ricans than the common single segregating SNPs (i.e., CYP2C9*2 and CYP2C9*3) relevant to Europeans. DMET Plus array confirmed the strong association of VKORC1 (<a href="https://www.ncbi.nlm.nih.gov/gene/?term=VKORC1">GeneID: 79001</a>) and CYP2C9 (<a href="https://www.ncbi.nlm.nih.gov/gene/?term=CYP2C9">GeneID: 1559</a>) with warfarin dose requirements, but also identified other polymorphisms in CES2 (<a href="https://www.ncbi.nlm.nih.gov/gene/?term=CES2">GeneID: 8824</a>) and ABCB1 (<a href="https://www.ncbi.nlm.nih.gov/gene/?term=ABCB1">GeneID: 5243</a>) associated with warfarin resistance (>6 mg/day). Ancestry analysis showed that Puerto Ricans from Veteran Affairs Caribbean Healthcare System (VACHS) using warfarin had higher Native American proportions than Puerto Ricans from the 1000 Genomes project; however, this difference was not significant. Incorporation of rs2860905 in a regression model (R2=0.60 MSE=0.38) that also included additional genetic predictors (i.e., VKORC1-1639G>A; CYP2C9 rs1856908; ABCB1 rs10276036; CES2 rs4783745) and non-genetic factors (i.e., hypertension, diabetes and age) showed better prediction of warfarin dose requirements than CYP2C9*2 and CYP2C9*3 combined (partial R2=0.132 versus 0.023 and 0.007, respectively, p<0.001). Interestingly, deep vein thrombosis and diabetes were found associated with high warfarin dose requirements among Puerto Ricans. Our results support the use of CYP2C9 rs2860905 along with other genetic markers [e.g., VKORC1(<a href="https://www.ncbi.nlm.nih.gov/gene/?term=VKORC1">GeneID: 79001 </a>)] and clinical covariates to predict warfarin dose in Puerto Ricans. Although our findings need further replication, this study contributes to the field of Pharmacogenetics and to improve anticoagulation therapy among Puerto Ricans.</p>
| phs001496 | dbGaP
Project description:CYP2C9 encodes a cytochrome P450 enzyme responsible for metabolizing up to 15% of small molecule drugs, and CYP2C9 variants can alter the safety and efficacy of these therapeutics. In particular, the anti-coagulant warfarin is prescribed to over 15 million people annually and polymorphisms in CYP2C9 can affect patient response leading to an increased risk of hemorrhage. We developed Click-seq, a pooled yeast-based activity assay to test thousands of variants. Using Click-seq, we measured the activity of 6,142 missense variants expressed in yeast. We also measured the steady-state cellular abundance of 6,370 missense variants expressed in a human cell line using Variant Abundance by Massively Parallel sequencing (VAMP-seq). These data revealed that almost two-thirds of CYP2C9 variants showed decreased activity, and that protein abundance accounted for half of the variation in CYP2C9 function. We also measured activity scores for 319 previously unannotated human variants, many of which may have clinical relevance.
2021-03-13 | GSE165412 | GEO
Project description:Vitamin K epoxide reductase (VKOR) drives the vitamin K cycle, activating vitamin K-dependent blood clotting factors. VKOR is also the target of the widely used anticoagulant drug, warfarin. Despite VKOR's pivotal role in coagulation, its structure and active site remain poorly understood. In addition, VKOR variants can cause vitamin K-dependent clotting factor deficiency or alter warfarin response. Here, we used multiplexed, sequencing-based assays to measure the effects of 2,695 VKOR missense variants on abundance and 697 variants on activity in cultured human cells. The large-scale functional data, along with an evolutionary coupling analysis, supports a four transmembrane domain topology, with variants in transmembrane domains exhibiting strongly deleterious effects on abundance and activity. Functionally constrained regions of the protein define the active site, and we find that, of four conserved cysteines putatively critical for function, only three are absolutely required. Finally, 25% of human VKOR missense variants show reduced abundance or activity, possibly conferring warfarin sensitivity or causing disease.
| S-EPMC7462613 | biostudies-literature
Project description:Pharmacogenetic Optimization of Anticoagulant Response (POAT) and Genetic and Environmental Determinants of Warfarin Response (GEDWR)
| PRJNA236403 | ENA
Project description:Pharmacogenetic Optimization of Anticoagulant Response (POAT) and Genetic and Environmental Determinants of Warfarin Response (GEDWR)
| PRJNA236404 | ENA
Project description:Genetic aberrations of the maternal UBE3A allele, which encodes the E3 ubiquitin ligase E6AP, are the cause of Angelman syndrome (AS), an imprinting disorder. In most cases, the maternal UBE3A allele is not expressed. Yet, approximately 10 percent of AS individuals harbor distinct point mutations in the maternal allele resulting in the expression of full-length E6AP variants that frequently display compromised ligase activity. In a high-throughput screen, we identified cyanocobalamin, a vitamin B12-derivative, and several alloxazine derivatives as activators of the AS-linked E6AP-F583S variant. Furthermore, we show by cross-linking coupled to mass spectrometry that cobalamins affect the structural dynamics of E6AP-F583S and apply limited proteolysis coupled to mass spectrometry to obtain information about the regions of E6AP that are involved in, or are affected by, binding cobalamins and alloxazine derivatives. Our data suggest that dietary supplementation with vitamin B12 could be beneficial for AS individuals.
2024-05-09 | PXD048963 | Pride
Project description:Vitamin K (VK), a fat-soluble vitamin, functions as a co-factor during the gamma-carboxylation reaction that converts glutamic acid residues to gamma-carboxyglutamic acid residues in proteins transiting through the endoplasmic reticulum. Two enzymes are involved which together form the VK cycle: gamma-glutamyl carboxylase (GGCX), and vitamin K oxidoreductase (VKORC1). GGCX requires reduced VK (VKH2) as an essential cofactor, which upon carboxylation, is oxidized to VK epoxide and then reconverted to VKH2 by VKORC1. Physiologically, gamma-carboxylation is essential in the liver for the activity of several coagulation factors (e.g., prothrombin, factor IX, etc.), and in arteries and cartilage to modulate the activity of Matrix Gla Protein (MGP) which prevents extra-osseous tissue mineralization. Gamma-carboxylation also negatively regulates the function of osteocalcin, a bone-derived hormone that promotes glucose homeostasis. Our objective is to identify unrecognized gamma-carboxylated proteins.
2023-05-10 | PXD032920 | Pride
Project description:Trypanosoma brucei Lister 427 bloodstream forms were cultured in HMI-11 medium. Total RNA was prepared using Qiagen RNAeasy kits for single sample RNAseq to estimate VSG mRNA abundance (and not to reconstruct the transcriptome). The cDNA libraries were prepared and sequenced at the Beijing Genomics Institute (Shenzhen, China). Polyadenylated RNA was purified from total RNA, converted to cDNA using random hexamer primers sheared and size selected for fragments ~200 bp in length using the Illumina TruSeq RNA Sample Preparation Kit v2. RNAseq of the resulting libraries was used for the determination of transcript abundances. Sequencing was performed on an Illumina Hiseq 2000 (Illumina, CA) platform and 90 base paired end reads obtained. Four samples were analysed: 1. Trypanosoma brucei Lister 427 expressing VSG2 2. Trypanosoma brucei Lister 427 expressing VSG6 3. Trypanosoma brucei Lister 427 expressing VSG6 and a VSG2 transgender located in the active bloodstream expression site 28 days after electroporation 4. Trypanosoma brucei Lister 427 expressing VSG6 and a VSG2 transgender located in the active bloodstream expression site 44 days after electroporation.
2020-06-03 | E-MTAB-9122 | biostudies-arrayexpress