Brief report A gene-anchored map position of the rat warfarin-resistance locus, Rw, and its orthologs in mice and humans Michael H. Kohn and Hans-Joachim Pelz The locus underlying hereditary resis- the markers Myl2 (zero recombinants) chromosome 7 at about 60 to 63 cM tance to the anticoagulant warfarin (sym- and Itgam, Il4r, and Fgf2r (one recombi- and onto one of the human chromo- bol in the rat, Rw) was placed in relation nant each) during linkage analysis in a somes 10q25.3-26, 12q23-q24.3, and to 8 positionally mapped gene-anchored congenic warfarin- and bromadiolone- 16p13.1-p11. (Blood. 2000;96:1996-1998) microsatellite loci whose positions were resistant laboratory strain of rats. Com- known in the genome maps of the rat, parative ortholog mapping between rat, mouse, and human. Rw segregated with mouse, and human placed Rw onto mouse © 2000 by The American Society of Hematology Introduction Warfarin, or 3-(␣-acetonylbenzyl)-4-hydroxycoumarin, and its 8 genes whose locations were known in the genomic maps of the structural relatives are tied to human health and economics. rat, mouse, and human.8 We also use the recently published Hereditary resistance to warfarin (locus symbol, Rw), and to radiation hybrid map of the rat to delineate regions of homology several of its related compounds, has been observed in humans and with the mouse and human.9 rodents, foremost Rattus norvegicus, R rattus, and Mus spp.1,2 The mechanism of warfarin resistance in humans, rats, and mice is thought to have orthologous genetic underpinnings; that is, in all Study design species mutations within the same enzyme complex (presumably a vitamin K epoxide reductase) mediate resistance.3 Origin of rats and resistance tests Resistance is a concern both in human medicine and rodent A previously described laboratory strain was used that was derived from a control. In human medicine, warfarin is a frequently used orally 1 cross between a wild-caught male rat from the Mu¨nsterland area in administered anticoagulant substance. Resistance may cause fail- Germany, homozygous resistant to the anticoagulants warfarin and broma- ure of oral anticoagulant therapy, which may lead to thrombosis diolone, with a Wistar albino susceptible (WAS) female.7,10 All rats were and stroke. Rodent pest control worldwide relies largely on the use tested for warfarin and bromadiolone resistance with the blood clotting of warfarin-based rodenticides. The evolution of resistance has led response (BCR) method.7,10,11,12 The observed segregation pattern in the to locally severe management problems resulting in public health back-cross population was compatible with the hypothesis that resistance to concerns and economic loss.2,4 The gene is also of specific interest both compounds was due to the same dominant gene or, alternatively, 2 because it is a central yet-to-be isolated enzyme of the vitamin K tightly linked genes.7 Genetic typing was done on 67 offspring of the cycle.3,5,6 seventh back-crossing generation. Rw has been recently mapped with anonymous microsatellite markers and one gene-anchored locus (Myl2) in the rat.7 An Mapping with microsatellite loci approximately 6-cM spanning interval on rat chromosome 1 (Chr About 50 ng of phenol/chloroform purified DNA13 was used as template for 1), located between the microsatellites D1Rat193 and D1Rat130, polymerase chain reaction (PCR) amplification of the gene-anchored defined the approximate map position of the Rw gene. However, microsatellite markers D1Smu2 (adrenergic receptor ␣-2a, Adra2a), D1Smu4 Myl2 is not well suited for comparative mapping either in the (cytochrome P-450 2e1 ethanol indicuble; Cyp2e1), D1Smu5 (fibroblast mouse or human. It has no mapped ortholog in the mouse other growth factor receptor 2; Fgfr2), D1Smu6 (interleukin-4 receptor; Il4r), than the synthetic probe, Mylpf, which hybridized to mouse D1Smu7 (sodium channel, nonvoltage gated ␤, Scnn1b), D1Smu9 (protein chromosome 7 (Chr 7). In humans, the use of Myl2 is misleading as kinase C ␤; Pkcb), and D1Smu10 (integrin ␣-M; Itgam)—all reference 8—following standard protocols.7,8 Previously generated genotype data for anchor because any of the 5 chromosomes 9, 10, 11, 12, and 16 may the markers D1Arb18 (myosin light chain, skeletal muscle; Myl2), D1Rat193, be partially homologous to the 6-cM interval. Thus, it is currently D1Mit13, D1Mgh21 were included into the analysis for reference.7 impossible to even tentatively place Rw in the human genome map. The Rw locus was mapped by haplotype analysis14 as implemented in Here, a warfarin- and bromadiolone-resistant congenic strain of Map Manager Classic, version 2.6.5.15 Briefly, markers were ordered and laboratory rats (Rattus norvegicus) is used to place Rw in relation to Rw integrated such that the total map length and the number of double From the Department of Organismic Biology, Ecology, and Evolution (OBEE), Reprints: Michael H. Kohn, Department of Organismic Biology, Ecology, and University of California at Los Angeles, Los Angeles, CA; Federal Biological Evolution (OBEE), University of California at Los Angeles, 621 Charles E. Research Center for Agriculture and Forestry, Institute for Nematology and Young Dr South, Los Angeles, CA 90095-1606; e-mail: michaelk@ Vertebrate Research, Mu¨nster, Germany. biology.ucla.edu. Submitted February 4, 2000; accepted May 2, 2000. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby M.H.K and the project were partially funded by a nonresident tuition fellowship, marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734. an OBEE departmental fellowship, an ISOP fellowship (all UCLA), and an NSF dissertation improvement grant. © 2000 by The American Society of Hematology 1996 BLOOD, 1 SEPTEMBER 2000 ⅐ VOLUME 96, NUMBER 5 BLOOD, 1 SEPTEMBER 2000 ⅐ VOLUME 96, NUMBER 5 ORTHOLOG MAPPING OF WARFARIN RESISTANCE 1997 crossovers were minimized while retaining the positions of published markers.14 Linkage was assessed at the 99% confidence level. Map positions were tested with the “find best position” and “rearrange” (at 95% confidence level using ␹2 statistics) commands, and LOD scores were deduced with the aid of the likelihood calculator. Recombination fractions less than or equal to 0.25 were directly converted into map distances given as cM Ϯ SE and a 95% confidence interval (95% CI).16 Mapping resources Most of this analysis used (1) the Mouse Genome Database (MGD), World Wide Web (URL:http://www.informatics.jax.org/), January 2000, at the Jackson Laboratory, Bar Harbor, ME; (2) the Rat Genome Database (RATMAP), World Wide Web (URL:http://www.ratmap.gen.gu.edu), 1998, at the University of Gothenborg, Gothenborg, Sweden; and (3) the UniGene genome resource on human, mouse and rat, World Wide Web (URL:http: www.ncbi.nlm.nih.gov/UniGene), January 2000, at the National Center for Biotechnology Information, National Institutes of Health, Bethesda, MD. As additional resources, see references 8 and 9, and those listed previously.7 Results and discussion Rw and Myl2 cosegregated without recombination,7 and their linkage position was delineated by one recombinant to the markers Figure 1. Genotyping results and comparative mapping of Rw in rat, mouse, Itgam, Il4r, and Fgfr2, and 2 recombinants to D1Rat193, respec- and human. (A) Haplotypes and their respective frequencies. Black boxes represent WAS-derived alleles, white boxes a combination of the wild-derived allele with a WAS tively (Figure 1A). To accommodate for a 2% error associated with allele. Haplotype frequencies are given underneath each column (n ϭ 67). LOD the BCR method,7,12 and to adjust the mapping resolution accord- scores are presented on the right panel. (B) The distal q-arm of rat Chr 1 containing ing to sample size,14,15 the genomic location of Rw is better Rw, and its orthologs in mouse and human. The map interval containing Rw is shaded in gray. In the rat, map distances and their corresponding 95% CI are given in described as a 6.0 Ϯ 4.0-cM interval, with an associated 95% CI of cM. The centromere is depicted as a black circle, and broken double lines separate 0.8 to 20.8 cM (Figure 1B), which includes Myl2, Itgam, Il4r, and linkage groups. In the mouse, chromosomal assignments are noted in boldface, and Fgfr2. Data from linkage to coat color,17 microsatellites,7 and these locus positions are given in cM (in brackets). In humans, chromosomal assignments gene-anchored loci now independently affirm the map location for are denoted as above, but locus positions are as in the cytogenetic human genome map.1 indicates allelic heterogeneity between laboratory and wild strain;2, conflicting Rw in the rat. Fgfr2, Itgam, and Il4r cannot be further resolved with information on chromosomal location of Cyp2e1, see references 8 and 9. our strain. However, previous work suggests that Fgfr2 is located ϳ1.6 cM distal of the latter 2.8 Scnn1b was placed as published8 10q25.3-q26, 12q23-24.3, and 16p11-13 (Figure 1B). With the (Figure 1B) because allelic heterogeneity precluded genotype available resolution of our strain we cannot discriminate among scoring (not shown). Cyp2e1 was weakly associated with Rw (13 these 3 possibilities. However, to further narrow the location of Rw recombinants; Figure 1A), and Adra2a was essentially unlinked to in the human genome, we suggest that MYL2, ITGAM, IL4R, and Rw (24 recombinants; data not shown). FGFR2 may be useful as anchor markers during genetic typing Gene order is highly conserved between rat and mouse within experiments that use cohorts of patients resistant to warfarin. this chromosomal region, and the close spacing of Itgam, Il4ra, and In conclusion, the gene-anchored map position of Rw in the rat, Fgfr2 in both species reflects this.8,9 When these loci are used as mouse, and human will allow for targeted searches for candidate anchor, the genetic map interval 60 to 63 cM on mouse Chr 7 genes and expressed sequence tagged sites on the specified emerges as likely location of war (Figure 1B).