The genetic basis of resistance to anticoagulants in rodents

Abstract

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 >50 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.

Figure 1.—

Geographic origin of resistant rodent populations. Warfarin resistance areas in Europe are shown and locations where resistant rats were trapped in the wild are indicated. Different mutations are represented by different hatching.

Figure 2.—

VKORC1 activity after recombinant expression in HEK293 cells. HEK293 cells were transfected by VKORC1 cDNA constructs and enzyme activity was measured as described in materials and methods. Activity of the wild-type construct in the absence of warfarin was set to 100%. All experiments were run in triplicate.

Figure 3.—

Sequence alignment of selected VKORC1 genes and related sequences. Sequence alignments were done by MultAlin (http://prodes.toulouse.inra.fr/multalin/multalin.html) and Boxshade (http://bioweb.pasteur.fr/seqanal/interfaces/boxshade.html). Predicted hydrophobic α-helical domains are represented by solid bars below the amino acid sequence. Fully conserved positions (as described by Goodstadt and Ponting 2004) are indicated by a + sign; the Thr-Tyr-Ala motif is marked by ***. Mutations observed in resistant animals are given by their single-letter amino acid code below the rat wild-type sequence.