Dear Dr. Ahmed,

The following publications cover the answer to your question.

1-http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1449767/

Genetics. 2005 Aug; 170(4): 1839–1847.

doi:  10.1534/genetics.104.040360

PMCID: PMC1449767

The Genetic Basis of Resistance to Anticoagulants in Rodents

Hans-Joachim Pelz,*,1 Simone Rost,†,1 Mirja Hünerberg,† Andreas Fregin,† Ann-Charlotte Heiberg,‡ Kristof Baert,§ Alan D. MacNicoll,** Colin V. Prescott,†† Anne-Sophie Walker,‡‡ Johannes Oldenburg,§§ and Clemens R. Müller†,2

Author information ► Article notes ► Copyright and License information ►

This article has been cited by other articles in PMC.

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 ofVKORC1 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.

MATERIALS AND METHODS

Animals:

 R. norvegicus:

Rats of both sexes were analyzed. Animals of the laboratory outbred strains HW and HH were obtained from two independent laboratory stocks [Central Science Laboratory (CSL), York and Reading University], strains HS and HB1 were from the CSL, and strain HB2 from Reading. Four of these strains were derived from wild stock and were crossed with warfarin-susceptible Wistar-derived Tolworth albino susceptible to incorporate homozygous genes for Scottish (HS; Greaves and Ayres 1973), Welsh (HW; Greaves and Ayres 1969), Hampshire (HH; Greaves and Cullen-Ayres 1988), and Berkshire (HB1) resistance. The fifth strain was derived from wild Berkshire stock and crossed for four generations onto a warfarin-susceptible line of CD rats (obtained from Charles River UK) to incorporate homozygous genes for Berkshire resistance (HB2; Hussain 1998). A German strain was derived from wild stock and crossed with Wistar susceptibles to incorporate genes for at least warfarin resistance (Kohn and Pelz 1999). Wild rats were caught over the past years in the course of regional resistance monitoring programs and immediately analyzed for resistance by blood clotting response (BCR). They originated from the following geographic areas (Figure 1): Belgium—Flanders; Denmark—Bornholm, Fünen, Jutland, and Zealand; France—Yonne, Eure and Loire; Germany—Münsterland and Emsland; United Kingdom—Yorkshire.

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.

M. musculus/domesticus:

The Reading susceptible mouse strain (MHS) was the Swiss mouse, an outbred strain obtained from Charles River UK. The Reading resistant mouse strain (MHR) was homozygous for a resistance gene that had been transferred through six generations onto the Reading susceptible mouse MHS background (C. V. Prescott, unpublished results). The mouse resistance gene was obtained from a wild population of house mice (M. musculus domesticus) originally trapped near Reading, United Kingdom (Prescott 1996).

At the CSL (York, UK) a homozygous warfarin-resistant mouse strain was derived by crossing mice of a warfarin-resistant PBI strain, which were direct descendants of the wild mice reported in 1976 (Wallace and MacSwiney 1976), with warfarin-susceptible mice of a LAC-gray strain. Offspring were selected for warfarin resistance and backcrossed to the LAC-gray strain for six generations. Warfarin-resistant mice of the sixth generation were mated with each other and homozygous warfarin-resistant individuals selected to produce a homozygous Cambridge resistant strain.

Blood clotting response test:

BCR resistance testing with warfarin was done as described (Martin et al. 1979) with modifications (MacNicoll and Gill 1993). BCR testing with bromadiolone was performed according to Gill et al.(1994)(1993). In Germany and Denmark, the test solution was administered by intraperitoneal injection instead of oral application, which had proved equally effective (H. J. Pelz, unpublished data) and is not confounded by any mechanism of reduced intestinal absorption. With each series of tests, the activity of the test solution was checked by including a Wistar albino rat as a known susceptible individual.

DNA sequence determination and mutation screening assays:

DNA was prepared from fresh or frozen tissue samples by standard procedures. The three exons and flanking intronic sequences of the VKORC1 gene were amplified by PCR and sequenced as described (Rostet al. 2004). For the mutation Tyr139Cys, an amplification refractory mutation system (ARMS)-PCR test was developed (Ye et al. 2001) using the outer primers F: ATC CTG AGT TCC CTG GTG TCT GTC GCT G and R: TCA GGG CTT TTT GAC CTT GTG TTC TGG C (2 pmol each) and the inner primers F: TGA TTT CTG CAT TGT TTG CAT CAC CAC ATG and R: CAA CAT CAG GCC CGC ATT GAT GGA AT (10 pmol each). PCR reactions were performed with Taq polymerase and 1× buffer (Invitrogen, San Diego) in the presence of 1.5 mm MgCl2 and 200 mm betaine for 3 min at 95° followed by 32 cycles of 20 sec at 95°, 20 sec at 62°, 10 sec at 70°, and a final 3 min at 70°. The outer primer pair generates a control band of 168 bp while the inner primers result in a band of 123 bp for the wild type and 101 bp for the mutated sequence, respectively.

The mutation Leu120Gln creates a novel StuI restriction site in exon 3. Digestion of the PCR product specific for exon 3 results in fragments of 195 and 135 bp in the presence of the mutation and a single band of 330 bp in the wild type (Rost et al. 2004). The mutation Leu128Gln generates a novel site for BsrI with fragments of 170 and 160 bp in mutant PCR products. The mutation Tyr139Ser can be detected by MnlI digestion as a fragment of 110 bp (plus several bands