Genomic assignment of the warfarin resistance locus, Rw, in the rat
Michael H. Kohn,
Department of Organismic Biology, Ecology, and Evolution (OBEE), University of California, Los Angeles, 621 Charles E. Young Drive South,
Los Angeles, California 90095-1606, USA
Federal Biological Research Center for Agriculture and Forestry, Institute for Nematology and Vertebrate Research, Toppheideweg 88,
D-48161 Mu¨nster, Germany
Received: 30 December 1998 / Accepted: 17 March 1999
Abstract. The locus responsible for resistance to the anticoagu-
lants warfarin and bromadiolone (locus symbol Rw) was integrated
into the rat (Rattus norvegicus) microsatellite genome map. Sev-
enth-generation offspring of a segregating strain of rats heterozy-
gous resistant to both compounds were tested with a blood-
clotting-response (BCR) test. No recombination between resis-
tance to warfarin and bromadiolone was observed, indicating a
common genetic basis. No recombinants were found between Rw
and D1Arb18 (Myl2) located at the MIT-microsatellite map posi-
tion 95.90 (SHRSP × BN F
-cross) or 82.24 (FHH × ACI F
cross). Resistance segregated in a ratio expected for single, dom-
inant gene responses. An equal number of females and males were
resistant, but females retained higher percentage blood coagulation
activities (PCA) after anticoagulant administration. Partial synteny
between rat, mouse, and human suggests that Myl2 may serve as
anchor to map the Rw homologs in mouse and human.
Warfarin [3-(␣-acetonylbenzyl)-4-hydroxycoumarin] was intro-
duced in the late 1940s to control rodents (Jackson et al. 1988).
Resistant rat (Rattus norvegicus) populations were reported in En-
gland beginning 1958 and subsequently in many different parts of
the world (e.g., Jackson et al. 1988; Pelz 1990). To overcome
control problems, more potent anticoagulants such as bromadio-
propyl]-4-hydroxy-2H-1-benzopyan-2-one) were developed.
However, rat populations resistant to these “second-generation”
anticoagulants have now also been reported (e.g. Pelz et al. 1995).
Resistance has also developed in roof rats (Rattus rattus) and
house mice (Mus musculus and Mus domesticus; e.g., MacNicoll
1986; Endepols and Schuster 1991).
It is generally thought that resistance is mediated by a single
dominant gene, Rw (Greaves and Ayres 1969, 1982). Male rats
seem to be more sensitive to anticoagulants than females, conceiv-
ably owing to the action of sex-linked modifiers (Wallace and
MacSwiney 1976). It is not clear whether resistance to different
anticoagulants is due to allelic variants at the Rw locus or whether
different loci are involved. Cross-resistance to various anticoagu-
lants has now been documented (e.g., Pelz et al. 1995). Biochemi-
cal experiments suggest that in resistant rats the liver enzyme
complex vitamin K 2,3-epoxide reductase (VKOR), which is com-
posed of a microsomal epoxide hydrolase (mEH) and a glutathi-
one-S-transferase (GST; Cain et al. 1998; Guenther et al. 1998),
exhibits a decreased affinity to the anticoagulant poison (Hilde-
brandt and Suttie 1982). Different types of resistance (i.e., strains)
are distinguished based on their biochemical profiles (MacNicoll
To understand the molecular basis for resistance, positional
cloning of the Rw gene is needed. This is important because ge-
netic resistance poses immense problems for rodent control, re-
sulting in severe loss of crop worldwide and public health concern.
Knowledge of the structure of the gene product would allow the
molecular modeling of more effective formulations. A PCR-based
test would facilitate the detection of resistance. Furthermore, in
human medicine, warfarin is the most used substance in antico-
agulation prophylaxis, but patients with hereditary resistance do
not respond to administration (O’Reily et al. 1983). A genetic
assay for allelic variants at the human homolog to Rw would allow
a prediction of treatment outcomes. Finally, warfarin resistance
has become a paradigm for overdominant selection (Greaves et al.
1977; Partridge 1979), but further details are needed to dissect the
evolutionary forces that govern frequencies of the gene.
Resistance was measured and microsatellites were typed in a
segregating backcross population of resistant rats to identify a
microsatellite that co-segregates with resistance. We examined
whether resistance to warfarin and bromadiolone was due to a
single locus by measuring the recombination frequency between
both resistant phenotypes. Finally, we tested whether resistance
segregates in a 1:1 ratio, as is expected for a single dominant gene,
and we discuss sex differences observed in our strain.
Materials and methods
Origin of rats and resistance testing.
A male rat caught in the wild
from the Mu¨nsterland area, Germany, homozygous resistant to warfarin
(RwRw) and bromadiolone (RbRb), was crossed with a Wistar albino sus-
ceptible (WAS; Rw+Rw+/Rb+Rb+) female and all seven F
zygous resistant to both anticoagulants (RwRw+/RbRb+; Pelz et al. 1995).
male was used to establish a segregating backcross population, of
which all rats were tested for warfarin and bromadiolone resistance. Most
of the colony was abandoned at the time when the program for genetic
analysis was initiated. However, for 67 of 74 offspring of seven matings,
each between a 7th generation heterozygous resistant (RwRw+ RbRb+)
male with a WAS female, tissue samples were available for genetic typing.
Rats were tested for warfarin and bromadiolone resistance with the
blood-clotting-response (BCR) method as described in Pelz et al. (1995)
and following the references given therein. Individuals that retained a
percentage clotting activity (PCA) > 17.0% after warfarin administration
and >12.5% after bromadiolone administration respectively were classified
About 50 ng of phenol/chloroform purified
DNA (Sambrook et al. 1989) was used as template for PCR amplification
of the microsatellite markers D1Mit2, D1Arb18, D1Mit13, D1Rat67,
D1Rat130, D1Rat193, D1Rat289, D1Uwm6, D1Uwm7, and D1Mgh21
(purchased from Research Genetics, Huntville, Ala.) following a standard
protocol provided by the supplier.
Correspondence to: M.H. Kohn
Mammalian Genome 10, 696–698 (1999).
© Springer-Verlag New York Inc. 1999