Coumarin derivatives such as warfarin represent the therapy of choice for the long-term treatment and prevention of thromboembolic events. Coumarins target blood coagulation by inhibiting the vitamin K epoxide reductase multiprotein complex (VKOR). This complex recycles vitamin K 2,3-epoxide to vitamin K hydroquinone, a cofactor that is essential for the post-translational gamma-carboxylation of several blood coagulation factors. Despite extensive efforts, the components of the VKOR complex have not been identified. The complex has been proposed to be involved in two heritable human diseases: combined deficiency of vitamin-K-dependent clotting factors type 2 (VKCFD2; Online Mendelian Inheritance in Man (OMIM) 607473), and resistance to coumarin-type anticoagulant drugs (warfarin resistance, WR; OMIM 122700). Here we identify, by using linkage information from three species, the gene vitamin K epoxide reductase complex subunit 1 (VKORC1), which encodes a small transmembrane protein of the endoplasmic reticulum. VKORC1 contains missense mutations in both human disorders and in a warfarin-resistant rat strain. Overexpression of wild-type VKORC1, but not VKORC1 carrying the VKCFD2 mutation, leads to a marked increase in VKOR activity, which is sensitive to warfarin inhibition.
Anticoagulant compounds, i.e., derivatives of either 4-hydroxycoumarin (e.g., warfarin, bromadiolone) or indane-1,3-dione (e.g., diphacinone, chlorophacinone), have been in worldwide use as rodenticides for Ͼ50 years. These compounds inhibit blood coagulation by repression of the vitamin K reductase reaction (VKOR). Anticoagulant-resistant rodent populations have been reported from many countries and pose a considerable problem for pest control. Resistance is transmitted as an autosomal dominant trait although, until recently, the basic genetic mutation was unknown. Here, we report on the identification of eight different mutations in the VKORC1 gene in resistant laboratory strains of brown rats and house mice and in wild-caught brown rats from various locations in Europe with five of these mutations affecting only two amino acids (Tyr139Cys, Tyr139Ser, Tyr139Phe and Leu128Gln, Leu128Ser). By recombinant expression of VKORC1 constructs in HEK293 cells we demonstrate that mutations at Tyr139 confer resistance to warfarin at variable degrees while the other mutations, in addition, dramatically reduce VKOR activity. Our data strongly argue for at least seven independent mutation events in brown rats and two in mice. They suggest that mutations in VKORC1 are the genetic basis of anticoagulant resistance in wild populations of rodents, although the mutations alone do not explain all aspects of resistance that have been reported. We hypothesize that these mutations, apart from generating structural changes in the VKORC1 protein, may induce compensatory mechanisms to maintain blood clotting. Our findings provide the basis for a DNA-based field monitoring of anticoagulant resistance in rodents.
Background: Coumarin derivatives have been in world-wide use for rodent pest control for more than 50 years. Due to their retarded action as inhibitors of blood coagulation by repression of the vitamin K reductase (VKOR) activity, they are the rodenticides of choice against several species. Resistance to these compounds has been reported for rodent populations from many countries around the world and poses a considerable problem for efficacy of pest control.
House mouse tissue samples from 30 populations in Germany, Switzerland and the Azores were analyzed for sequence changes in the gene VKORC1, which potentially confer resistance to anticoagulant rodenticides. Except for one population originating from south Germany, sequence variants were found in individuals from all locations analyzed (29 out of 30 sites surveyed), with less than 10 % of the individuals matching the wild-type genotype. The most frequent and widespread amino acid substitutions were Leu128Ser, Tyr139Cys and a group of linked sequence changes (Arg12Trp/Ala26Ser/Ala48Thr/Arg61Leu). These three genotypes occurred either alone or in combination with each other or with other less frequent sequence changes. Where they occurred as the sole variant, the proportion of homozygous animals was 72-83 %, suggesting a high selection pressure due to permanent pest control in these populations.An evaluation of published data revealed that the three frequent sequence changes found are associated with a substantial loss of rodenticide efficacy of first generation anticoagulants (e.g. warfarin, coumatetralyl) as well as the second generation compound bromadiolone and most probably also difenacoum. Further studies are required to investigate the effect on compounds of higher potency, in particular, where combinations of sequence changes occur in one individual.
In theory, genes under natural selection can be revealed by unique patterns of linkage disequilibrium (LD) and polymorphism at physically linked loci. However, given the effects of recombination and mutation, the physical extent and persistence of LD patterns in natural populations is uncertain. To assess the LD signature of selection, we survey variation in 26 microsatellite loci spanning an Ϸ32-cM region that includes the warfarin-resistance gene (Rw) in five wild rat populations having resistance levels between 0 and 95%. We find a high frequency of heterozygote deficiency at microsatellite loci in resistant populations, and a negative association between gene diversity (H) and resistance. Contrary to previous studies, these data suggest that directional rather than overdominant selection may predominate during periods of intense anticoagulant treatment. In highly resistant populations, extensive LD was observed over a chromosome segment spanning Ϸ14% of rat chromosome 1. In contrast, LD in a moderately resistant population was more localized and, in conjunction with likelihood ratios, allowed assignment of Rw to a 2.2-cM interval. Within this genomic window, a diagnostic marker, D1Rat219, assigned 91% of rats to the correct resistance category. These results further demonstrate that ''natural selection mapping'' in field populations can detect and map major fitness-related genes, and question overdominance as the predominant mode of selection in anticoagulant-resistant rat populations.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.