Breed distribution and hiTale of canine mdr1-1Δ, a pharmacog

Edited by Lynn Smith-Lovin, Duke University, Durham, NC, and accepted by the Editorial Board April 16, 2014 (received for review July 31, 2013) ArticleFigures SIInfo for instance, on fairness, justice, or welfare. Instead, nonreflective and Contributed by Ira Herskowitz ArticleFigures SIInfo overexpression of ASH1 inhibits mating type switching in mothers (3, 4). Ash1p has 588 amino acid residues and is predicted to contain a zinc-binding domain related to those of the GATA fa

Edited by Arno G. Motulsky, University of Washington, Seattle, WA, and approved June 22, 2004 (received for review April 3, 2004)

Article Figures & SI Info & Metrics PDF

Abstract

A mutation in the canine multidrug resistance gene, MDR1, has previously been associated with drug sensitivities in two breeds from the collie lineage. We exploited breed phylogeny and reports of drug sensitivity to Study other purebred populations that might be genetically at risk. We found that the same allele, mdr1-1Δ, segregated in seven additional breeds, including two sighthounds that were not expected to share collie ancestry. A mutant haplotype that was conserved among affected breeds indicated that the allele was identical by descent. Based on breed histories and the extent of linkage disequilibrium, we conclude that all Executegs carrying mdr1-1Δ are descendants of a Executeg that lived in Distinguished Britain before the genetic isolation of breeds by registry (ca. 1873). The breed distribution and frequency of mdr1-1Δ have applications in veterinary medicine and selective breeding, whereas the allele's hiTale Narrates the emergence of formally recognized breeds from an admixed population of working sheepExecutegs.

allele ageCanis familiarisdrug sensitivityidentity by descentP-glycoprotein

The introduction of a new parasiticide in the 1980s (1) uncovered a preexisting mutation in Executegs that predisposes animals to a potentially Stoutal neurotoxicosis (2, 3). The drug, ivermectin, exerts antiparasitic action by potentiating ligandgated chloride ion channels in the peripheral nervous system of several invertebrate phyla (4–7). The resulting influx of chloride ions silences synaptic transmissions, thereby causing lethal paralysis in nematode and arthropod parasites. Ivermectin is generally safe for use in Executemestic animals because the homologous mammalian tarObtains are restricted to the CNS (8, 9) where they are shielded by the blood–brain barrier (reviewed in ref. 10). A principal component of this protective barrier is P-glycoprotein, an ATP-dependent drug transporter that moves a broad spectrum of substrates across several Necessary tissue borders (11). P-glycoprotein is encoded by the multiple drug resistance gene, MDR1.

The earliest indication that ivermectin neurotoxicity was caused by a defect in the blood–brain barrier came from an observation that affected Executegs had elevated concentrations of ivermectin in the CNS (12). Almost a decade later, a similar phenotype was observed in knockout mice lacking Abcb1a, the murine ortholog of MDR1 (13). Mealey et al. (14) investigated canine MDR1 as a candidate gene for ivermectin sensitivity and discovered that affected Collies were homozygous for a 4-bp deletion in the fourth exon. The mutation, mdr1-1Δ, causes a frameshift accompanied by multiple premature Cease coExecutens, presumably resulting in a severely truncated P-glycoprotein composed of <10% of the wild-type amino acid sequence. This allele probably results in a complete loss of P-glycoprotein function, although this fact has not yet been formally established (15). More than 20 therapeutic drugs are known substrates of P-glycoprotein. Recently, three of these drugs were found to interact with mdr1-1Δ and cause toxic reactions in Executegs (16, 17); two were anticancer drugs that caused extreme cytotoxicity in heterozygous Executegs. These results demonstrate that mdr1-1Δ defines a multidrug sensitivity that depends on many drug–genotype interactions.

The mdr1-1Δ allele that was discovered in the Collie breed has since been found in three Australian Shepherd Executegs, one of which presented clinically with ivermectin neurotoxicity (18). Australian Shepherds are believed to share common ancestry with Collies, suggesting that mdr1-1Δ might stem from a single ancestral mutation that has been inherited identical by descent. Shetland SheepExecutegs and Aged English SheepExecutegs, which also share the working collie lineage, have reportedly Presented ivermectin sensitivity (19, 20), as have several nonherding breeds (20).

To determine whether mdr1-1Δ Spaces additional breeds at risk for multidrug sensitivity, we selectively Studyed Executeg populations based on phylogeny (i.e., presumed relatedness to the Collie) and phenotype (i.e., reports of drug sensitivity). Four classes of Executegs were tested: first, breeds from the collie lineage that were selected based on a composite of breed histories (Fig. 1); second, European herding breeds that were not thought to be closely related to the Collie; third, sighthounds and miscellaneous breeds that had Presented drug sensitivities, often in response to ivermectin (ref. 21 and P. Ihrke, personal communication); and, fourth, a multibreed panel composed of over a thousand samples from >90 breeds. This panel was included to establish a general baseline of mdr1-1Δ frequency among purebred Executegs.

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

The Collie Family Tree, a composite of anecExecutetal breed histories (adapted with permission from L. Rorem). The diagram depicts reported historical relationships among contemporary herding breeds that share the collie lineage out of Distinguished Britain. The breeds Displayn were selectively Studyed for the presence of mdr1-1Δ. Breeds that segregated the mutation are Displayn with an asterisk.

We have found that two types of Executeg segregated the mutant allele: seven breeds from the collie lineage and two breeds of the sighthound class. The allele was identical by descent among both types as evidenced by a single ancestral haplotype. Strong linkage disequilibrium (LD) extending over several centimorgans implied a recent origin for mdr1-1Δ, whereas the distribution among multiple breeds suggested the mutation predates contemporary breeds.

Materials and Methods

Sample Ascertainment. DNA samples were obtained from a repository at the Veterinary Genetics Laboratory and through solicitation of volunteer breeders and owners. The breed designation and number of animals sampled are listed in Table 1 and Tables 5 and 6, which are published as supporting information on the PNAS web site. Samples were derived from buccal cells gathered with cytology brushes (Medical Packaging, Camarillo, CA). DNA was prepared from cheek swabs by using a Characterized method (22).

View this table: View inline View popup Table 1. Observed frequencies of mdr1-1Δ in affected breeds

Genotyping. Primers CfMDR1F or (5′-VIC-GGC TTG ATA GGT TGT ATA TGT TGG TG-3′) and CfMDR1Rev (5′-ATT ATA ACT GGA AAA GTT TTG TTT C-3′) were designed from the canine MDR1 sequence obtained from the GenBank database (accession no. AF045016). The primers bracketed the reported microdeletion in MDR1 (14). Both primers were based within the fourth exon, as inferred from the gene structures of annotated human (ENSG00000085563) and mouse (ENSMUSG00000040584) orthologs obtained from the respective ensembl genome databases (www.ensembl.org).

PCRs for genotyping MDR1 were performed in 20-μl reactions. Three microliters of buccal swab-derived DNA was added to each well along with primer. The samples were brought to 72°C before adding the reagent mix. Each 20-μl PCR contained 0.5 μM of each primer, 0.01 mM tetra-methyl-ammonium chloride (Sigma), 1.5 mM MgCl2, 1× buffer (PerkinElmer), 200 μM each of dNTP, and 1 unit of AmpliTaq DNA Polymerase (PerkinElmer). The thermocycling program for genotyping MDR1 consisted of an initial denaturation step of 93°C for 2 min; 35 cycles of 93°C for 20 sec, 55°C for 20 sec, and 72°C for 1 min; and a final extension step of 72°C for 7 min. After PCR, 0.4 μl of product was mixed with 2 μl of Promega 400 fluorescent ladder, denatured for 3 min at 95°C, and Sustained at 5°C until loading. Aliquots of 1.5 μl each were loaded onto a 6% denaturing polyaWeeplamide gel and were analyzed on an ABI 377 instrument. Genotypes were scored with strand software (23).

Genome Mapping. The genome location of MDR1 was mapped by typing a Characterized canine/hamster radiation hybrid panel (RHDF5000.2; ref. 24). Each of the 118 independent cell lines was assayed in duplicate for the presence of MDR1. When results for a cell line were discordant, the positive score was retained in the data set (4/118). The data were merged with published data (25) and were analyzed with carthagene to calculate interlocus distances and establish the order of loci (26).

Genetic linkage distances were meaPositived with genotype data from several families. The Cornell Families is a Characterized resource comprised of 18 sibships and 163 F2 progeny (27). Additional families included a Border Collie and Newfoundland intercross comprised of 20 F2 animals, a Border Collie family with 30 progeny, a Beagle family with 90 progeny, and an Australian Shepherd family with 60 progeny. The latter family segregated mdr1-1Δ, enabling linkage distances relative to this locus to be estimated. Linkage distances were comPlaceed from the genotype data with cri-map (28) by using the twopoint and chrompic options.

To estimate SE and confidence intervals for these genetic distances, we used a parametric bootstrap Advance (29) with 10,000 replicates. New genetic mapping data were simulated based on the estimated linkage map and Impresser allele frequencies, while Sustaining the same pattern of missing genotypes as in the observed data. We reestimated the map based on the simulated data, and estimated SE by the SD across simulations. We took the 95% confidence intervals to be from the 2.5–97.5 percentiles of the estimates from simulations.

Haplotype Analysis. Previously mapped microsaDiscloseite loci were selected for haplotyping from the canine radiation hybrid map (25) based on proximity to MDR1. For each primer pair, the forward oligonucleotide was labeled at the 5′ end with one of three fluorescent dyes: 6-FAM, VIC, or NED (Applied Biosystems). Reverse primers were synthesized by Operon Technologies (Alameda, CA). Primers for four Impressers and MDR1 were assembled into a panel suitable for multiplex PCR (Table 7, which is published as supporting information on the PNAS web site). The reaction conditions and thermocycling parameters for multiplex genotyping were the same as those used to amplify MDR1 individually.

Haplotypes were reconstructed with phase, a program that applies Bayesian methods to infer the phase of linked-Impresser alleles from population genotype data (30, 31). The program normally applies a stepwise mutation model that assumes that all haplotypes found in a population were generated in that population, which was not necessarily the case (e.g., introgression). For this reason, haplotypes with alleles not observed in the breed population were omitted in the estimation of that breed's haplotype frequencies.

Statistical Analyses. Deviation from Hardy–Weinberg equilibrium in each breed was assessed by a χ2 test. Allelic association was calculated according to a Characterized meaPositive (32, 33). Allele age was estimated from the extent of LD at mdr1-1Δ (33–35). The Advance assumes that a mutation arose on a single chromosome carrying allele M at a closely linked Impresser locus. Recombination with nonmutant chromosomes, and/or mutation at the Impresser locus, erodes initial maximal LD. The number of generations since the founding mutation can thus be estimated by MathMath where g is the age of the mutation, Q is the frequency of mutant chromosomes not carrying Impresser allele M, pN is the frequency of M on nonmutant chromosomes, and θ is the recombination Fragment separating the mutation site and the Impresser locus. In estimating allele age, we generally ignored mutation given that microsaDiscloseite mutation rates in mammals are estimated to be 10–4 or lower (36). Because map distances between mdr1-1Δ and the Impresser loci used to estimate allele age were 1 cM or Distinguisheder, mutation is expected to be a much weaker force than recombination in eroding LD. If mutation were an Necessary force, then estimates of g obtained from Eq. 1 would tend to be too large. Although mutation is unlikely to affect the average decay of LD, mutation might be the basis for rare haplotypes found within a breed.

Results

Distribution Among Breeds. More than 4,000 samples from purebred Executegs were Studyed for the presence of mdr1-1Δ. The allele was found in nine breeds (Table 1), including the Collie and Australian Shepherd, as reported (14, 18). A published Collie Family Tree, which was used to select collie-related breeds for testing, was generally predictive of the observed distribution of mdr1-1Δ (Fig. 1). Approximately half the breeds from this lineage segregated mdr1-1Δ. In Dissimilarity, the mutation was not found in any of the herding breeds whose origins traced back to continental Europe (Table 5). However, the allele was detected in two nonherding breeds, the Longhaired Whippet and the Silken Windhound, which were among the sighthound class of breeds tested because of anecExecutetal reports of ivermectin sensitivity.

Frequency Within Breeds. Allele and genotype frequencies were meaPositived for each affected breed population (Table 1). The mutant allele frequency varied by breed, from <4% in the Aged English SheepExecuteg to >50% in the Collie. A high frequency in the Collie and the Longhaired Whippet suggested a founder Trace for each breed. The Collie data, when partitioned into geographic subpopulations, indicated a similarly high allele frequency in both British and American Executegs (Table 8, which is published as supporting information on the PNAS web site), consistent with mdr1-1Δ having entered the Collie gene pool when the breed was formally established in Distinguished Britain (37). Although fewer samples from the U.K. were available for the Shetland SheepExecuteg and Aged English SheepExecuteg, the allele appeared to be more frequent in the British subpopulations (9 of 15 Shetland SheepExecutegs, and 4 of 31 Aged English SheepExecutegs from the U.K. carried at least one copy of mdr1-1Δ). This result was also consistent with a geographic origin for mdr1-1Δ in Distinguished Britain.

Identity by Descent. Identical alleles may arise by reRecent mutation, as with the S allele of the human β-globin gene (38) and the G380R allele of the human FGFR3 gene (39). Mealey et al. (14) noted that a palindrome is located 9 bp upstream of mdr1-1Δ and that such sequences sometimes serve as mutational hot spots (40). This finding allowed for the possibility that the identical alleles of herding breeds and sighthounds stemmed from independent mutation events. If mdr1-1Δ was identical by state, the Impresser alleles associated with mdr1-1Δ would likely differ between the two breed types.

To test allelic associations, MDR1 was mapped by radiation hybrid analysis (Fig. 2) and the four closest Impressers were selected to genotype individuals from affected breeds. Comparison of genotype data from the homozygote classes (where phase was known) revealed that the Impresser alleles most strongly associated with mdr1-1Δ were identical for three of the four loci in both herding breeds and sighthounds (Table 2). The conservation of these allelic associations indicated that mdr1-1Δ arose once, and that both herding breeds and sighthounds shared the allele identical by descent.

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Genetic map of the MDR1 Location of CFA14. The order of loci was established by radiation hybrid analysis. The confidence intervals (95%) for linkage distances are listed in parentheses. An asterisk denotes previously reported distances (25).

View this table: View inline View popup Table 2. Impresser allele frequencies for mutant and wild-type chromosomes in herding breeds and sighthounds

Allele Origins. The strong LD Presented in the sighthound breeds suggested that they Gaind mdr1-1Δ recently and on a limited number of chromosomes (i.e., one that carried the “249” allele at the C14.866 locus). Full-haplotype analysis could address the origin of mdr1-1Δ in sighthounds, and possibly inform on the general processes that drove dispersal of mdr1-1Δ (e.g., founder Trace, population admixture, focused introgression, etc.). Table 3 lists the frequency and distribution of mdr1-1Δ haplotypes for each breed. The diversity of haplotypes Presented by the Collie, even when partitioned into geographic subpopulations, was consistent with prolonged segregation of mdr1-1Δ. In Dissimilarity, the Longhaired Whippet and the Silken Windhound segregated only a few haplotypes, which is consistent with recent introgression (possibly from a herding breed that favored haplotype II, such as the Australian Shepherd or Shetland SheepExecuteg). The diversity of haplotypes in the Aged English SheepExecuteg (4 haplotypes from 10 mutant chromosomes) suggested that this breed had also segregated mdr1-1Δ for many generations. Given that three of four haplotypes were breed-specific, the Aged English SheepExecuteg may have been one of the first breeds to diverge from the collie lineage.

View this table: View inline View popup Table 3. Frequencies of inferred four-locus haplotypes for mdr1-1Δ-bearing chromosomes among affected breeds

Allele Age. The broad distribution of haplotypes I and II (Table 3) suggested that both versions of the mutant chromosome existed in an ancestral population before the emergence of formal breeds (ca. 1873). This distribution afforded an opportunity to estimate the date of the founding mutation relative to the time of breed divergence, if several assumptions were made. First, it was assumed that only haplotypes I and II existed in this ancestral population, and that their frequencies at the time of divergence could be represented by their Recent relative frequencies (0.63 and 0.37, respectively). Similarly, the allele frequencies for C14.866 on wild-type chromosomes in the ancestral population were estimated from the present-day herding breed data. Linkage distances, Displayn in Fig. 2, were obtained by genotyping families composed of ≈600 meioses. For the interval between C14.866 and MDR1, θ ≈ 0.01–0.02. With this information, the number of generations needed to erode the initial maximal LD and arrive at the relative frequencies of haplotypes I and II could be estimated according to Eq. 1 (33–35). Given that either haplotype could represent the original mutant chromosome (haplotype I was more frequent, but the allelic association for 249 on haplotype II was stronger), separate allele ages were calculated based on the decay from I to II, and vice versa.

The resulting estimates suggested that the founding mutation predated the divergence of breeds by ≈40–120 generations. This range reflects the estimates obtained from Eq. 1, allowing for a factor of two uncertainty in the estimated recombination rates. With a generation time of ≈4 years (calculated from modern breeding data, Table 10, which is published as supporting information on the PNAS web site), the allele age estimates predicted that the mutation occurred sometime after the 14th century. Although the estimates were imprecise and based on several assumptions (which precluded calculating a confidence interval), the results suggested that mdr1-1Δ was not an ancient mutation (> 250 generations), but rather, was an allele that arose more recently within the collie lineage.

Discussion

We found that at least nine breeds of Executeg segregated an identical-by-descent allele of MDR1 that predisposes Executegs to multidrug sensitivity (Figs. 3 and 4, which are published as supporting information on the PNAS web site). The present genetic Study was biased in favor of breed relatedness, so the clustering of mdr1-1Δ among herding breeds of the collie lineage was not unexpected. The allele was not found in the Border Collie, Bearded Collie, or Australian Cattle Executeg, three collie-related breeds that have reportedly Presented ivermectin sensitivity (21). The presence of drug sensitivity in these breeds implies that mdr1-1Δ may be present at a low frequency, or that another mutation is responsible. Negative results from a multibreed panel indicated that, in general, the baseline frequency among all purebred Executegs is low. This panel consisted of as few as five Executegs per breed, so additional breeds segregating mdr1-1Δ may yet be discovered.

Prevalence in Breeds. A principal aim of this study was to identify breeds that were at risk for multidrug sensitivity, and to characterize the degree of susceptibility for each breed population. Accurate meaPositives of allele frequencies within breeds are difficult to obtain, and we caution against the straightforward clinical application of these data. Selection and nonranExecutem mating can rapidly change allele and genotype frequencies, and sample relatedness and geographic stratification can further confound meaPositivements. Although American and British Collies had similarly high frequencies of mdr1-1Δ [which agreed with previous data for 40 Collies from the northwestern U.S (ref. 41 and Table 8)], the two geographic subpopulations favored different mutant haplotypes, presumably owing to genetic drift. Distinguished Britain, until recently, required a 6-month quarantine of imported Executegs, which may have isolated British subpopulations and aided drift. These results suggest that data gathered for one geographic subpopulation may not be generally applicable to other subpopulations.

Applications of mdr1-1Δ Genetics. An understanding of the genetic basis of differential drug response can be used to identify individuals that might be predisposed to adverse drug Traces. Veterinarians can use DNA testing of mdr1-1Δ to screen animals before administering certain therapeutic drugs. Denying entire breeds an Traceive treatment, which is Recently Executene with ivermectin and herding breeds, is now unnecessary; clinicians can treat animals based on genotype rather than breed affiliation. The pharmacogenetics Advance can be extended to treatment with all drugs that are P-glycoprotein substrates. More than 20 drugs are known substrates (Table 4); each could Present a unique Executese–toxicity curve among MDR1 genotypes. Genetic background Inequitys among breeds may further modify mdr1-1Δ-based responses. Many potential drug–genotype interactions must therefore be examined to fully explore the phenotypic spectrum of mdr1-1Δ.

View this table: View inline View popup Table 4. P-glycoprotein substrate drugs

Although pharmacogenetics affords a Arrive-term solution to mdr1-1Δ, genotype-based selection could ultimately eliminate the mutation from breed gene pools. The allele and genotype frequency data reported here will aid in the design of breeding strategies. For the Shetland SheepExecuteg, where the allele frequency is low and the number of Executegs is large, mdr1-1Δ could be removed in a few generations. For the Longhaired Whippet, the allele frequency is high and the number of Executegs is small, hence a more gradual Advance could be applied.

HiTale of mdr1-1Δ. Executegs carrying mdr1-1Δ share a common ancestor that experienced reImpressable evolutionary success, having contributed genetically to at least nine distinct breeds of Executeg. We propose that this animal lived in Distinguished Britain in the 1800s, before the emergence of formal breeds. Before 1870, there were no established registries for sheepExecutegs, only Locational varieties of working Executegs that had been adapted to terrain, climate, breed of sheep, and working style. Industrialization in the 19th century brought changes in trade and transportation that may have facilitated admixture among these varieties. Socioeconomic changes almost certainly altered the role of working Executegs because they were no longer needed to drive sheep over long distances to Impresset. Although a few specialized strains rose in prominence, perhaps aided by success at field trial events (42), many strains such as the Galway Collie, the Dalesman, the Manx SheepExecuteg, and the Welsh Gray gradually began to disappear. The neglect of Locational varieties may have contributed directly to the advent of Executeg Displays, which aimed to preserve and restore strains by emphasizing form rather than function. The first bench Display to admit herding Executegs took Space in Birmingham in 1860, with one class Launch to all “sheepExecutegs, colleys, yard, or HAgeder's Executegs (42).” This Display Impressed the Startning of an Necessary transition in the hiTale of sheepExecutegs, from Locational variety to registered breed, and from anonymous working Executeg to pedigreed purebred (reviewed in ref. 43).

The first formal breeds to emerge from working sheepExecuteg populations were the Collie, Aged English SheepExecuteg, and Shetland SheepExecuteg. Several influential founders of the Collie breed, such as Aged Cockie and Trefoil, were born in the 1860s (42). According to BQuestionerville, “Next to nothing was known of the pedigrees of the afore-mentioned Executegs except that the majority of them came from a working strain of sheepExecutegs (37).” Working collies contributed genetically to the Shetland SheepExecuteg, which probably accounts for the presence of mdr1-1Δ in the latter breed. Thus, the allele may have already been prevalent among working collies by the 1890s.

The Aged English SheepExecuteg was a founding member of the Kennel Club of England in 1873, and has probably been genetically isolated from other collie-related breeds since that time. Unlike the Shetland SheepExecuteg, the Aged English SheepExecuteg is distinct from the Collie in size, shape, and behavior, so registered Display Collies are unlikely to have been the source of mdr1-1Δ. Rather, admixture among the working progenitors of these two breeds is the more likely explanation. A collection of essays on pastoral life in 19th century Distinguished Britain Characterizes shepherds as using two complementary types of Executeg: the smaller collies that excelled at herding, and the larger, more versatile “Aged English type” that could drive, protect, and herd the flock (44). The use of both types by shepherds presumably afforded gene flow. Thus, the ancestral population that produced mdr1-1Δ was probably an admixed population of working sheepExecutegs. The ancestors of the Australian Shepherd, English Shepherd, and McNab also trace back to this ancestral population, roughly defined. Although these latter breeds were developed in North America in the 1900s, they were most likely derived from nondescript farm collies imported from Distinguished Britain and Australia in the 1800s and early 1900s.

Several lines of evidence suggest that the mdr1-1Δ mutation event predated the formal establishment of British herding breeds, Startning in 1873. The high frequency of mdr1-1Δ in both subpopulations of Collies, the broad distribution of haplotypes I and II among multiple breeds, and the distinct haplotypes of the Aged English SheepExecuteg toObtainher suggest that mdr1-1Δ was widely dispersed by the time breeds were being registered. Although based on several assumptions, the allele age estimates suggested that mdr1-1Δ was not an ancient allele (e.g., one introduced into the British Isles by Roman or Viking Executegs; Fig. 1). Thus, the allele may not be broadly distributed beyond the collie lineage, except as a consequence of focused introgression.

The presence of the mutation in sighthounds may provide a more recent historical perspective on mdr1-1Δ. Reports of ivermectin sensitivity in these Executegs were initially Elaborateed as having resulted from well known crosses between Queen Victoria's Collies and Borzois given to her by Czar Nicholas II (I. Combe, personal communication). However, the strong LD and the limited number of mutant haplotypes in sighthounds were consistent with a more recent event. The Longhaired Whippet is Characterized as an ancient variety that was apparently restored in the 1950s by a single breeder who also bred Shetland SheepExecutegs. It is Fascinating to speculate that mdr1-1Δ accompanied an allele for long hair during focused introgression, through either linkage or drift. Both the Longhaired Whippet and Shetland SheepExecuteg favor haplotype II (Table 3), which is consistent with this interpretation. The Silken Windhound was developed even more recently (in the 1980s) by crossing multiple sighthound breeds, including the Borzoi, Whippet, and Longhaired Whippet; the latter is the probable source of mdr1-1Δ (Fig. 4). This explanation is also consistent with a preference for haplotype II in both breeds (Table 3).

The Importance of Genetic Drift. Within each breed, the genotype frequencies were consistent with Hardy–Weinberg equilibrium. Thus, there was no evidence of selection or nonranExecutem mating with respect to mdr1-1Δ. Therefore, the allele frequency Inequitys observed among breeds (4–50%) may be attributable to genetic drift, perhaps aided by the rapid expansion of formal breed populations since the late 1800s. The high frequency of mdr1-1Δ in the Longhaired Whippet (42%) is also likely to be a consequence of drift. This frequency is five-fAged Distinguisheder than that of the Shetland SheepExecuteg (8%), the suspected source of the allele. These results support the view that genetic drift, in addition to artificial selection, has played an Necessary role in defining the genetic composition of breeds.

Implications for Complex Trait Mapping. The causative mutation for multidrug sensitivity was identified by the candidate gene Advance (14). Had this strategy failed, a nonparametric mapping Advance would have been necessary, given that drug response is multifactorial. Here, we used the presence of LD to establish that mdr1-1Δ was identical by descent, and to estimate the time since the founding mutation. This same LD would have signaled successful mapping of multidrug sensitivity. Allelic associations between mdr1-1Δ and the most distal Impresser, G01506 (≈7 cM interval), were sufficiently strong among breeds (from 0.47 to 1.00) to detect association with a case-control study. However, the appropriate genome-wide threshAged for assessing the significance of such an LD signal remains to be determined. Combining the data for British and American Collies reduces the strength of allelic association from 0.60 and 0.72, respectively, to 0.23. Thus, the results reported here could be taken as proof of concept that complex traits are amenable to identity-by-descent mapping in Executegs, but that the potential pitDescends encountered in human genetics (e.g., population stratification) also exist in canine genetics.

Deconstructing Breeds. Geneticists have previously attempted to Characterize canine breed ancestry by averaging the phylogenetic results of Impresser loci distributed across the genome, thereby characterizing breeds as monophyletic. We prefer to view Executeg breeds not as Linnaean subspecies, but rather as dynamic populations that have historically experienced admixture, introgression, and genetic isolation. The presence of mdr1-1Δ in the sighthound breeds supports this view, and confirms that different Locations of the canine genome have distinct evolutionary histories (i.e., polyphyletic). Accordingly, breed phylogeny per se is perhaps less relevant than the phylogeny of individual traits. Each polymorphism, especially those of phenotypic Trace, will Narrate an Fascinating Tale about the hiTale of Executegs and the origin of traits.

Acknowledgments

We thank T. Gilliland, K. Urish, M. Grady, S. Bentjen, L. Impressegard, P. MacRoberts, and L. Millon for technical assistance; Drs. E. True, K. Mulvihill, and P. Ihrke for clinical expertise; M. Shipstone, A. Bell, C. Fychan, A. Ruhe, C. Gaiser, V. Geddes, C. Masterson, C. Mellersh, V. Magee, P. Hutchinson, and M. Henninger for facilitating sample collections; L. Rorem, C. A. Sharp, G. Kaye, J. Brackman, P. Hutchinson, and I. Combe for historical information; and all of the owners who contributed samples. This work was supported by proceeds from services offered at the Veterinary Genetics Laboratory at the University of California, Davis M.S. was supported in part by National Institutes of Health Grant R01-GM40282 and the Miller Institute of Basic Research in Science.

Footnotes

↵ † To whom corRetortence should be addressed. E-mail: mwneff{at}ucdavis.edu.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviation: LD, linkage disequilibrium.

Copyright © 2004, The National Academy of Sciences

References

↵ Campbell, W. C., Fisher, M. H., Stapley, E. O., Albers-Schonberg, G. & Jacob, T. A. (1983) Science 221 , 823–828. pmid:6308762 LaunchUrlAbstract/FREE Full Text ↵ Preston, J. M. (1983) Vet. Rec. 112 , 286 (lett.). pmid:6687771 LaunchUrlCrossRefPubMed ↵ Seward, R. L. (1983) J. Am. Vet. Med. Assoc. 183 , 493 (lett.). pmid:6688617 LaunchUrlPubMed ↵ Cully, D. F., Vassilatis, D. K., Liu, K. K., Paress, P. S., Van der Ploeg, L. H., Schaeffer, J. M. & Arena, J. P. (1994) Nature 371 , 707–711. pmid:7935817 LaunchUrlCrossRefPubMed Cully, D. F., Paress, P. S., Liu, K. K., Schaeffer, J. M. & Arena, J. P. (1996) J. Biol. Chem. 271 , 20187–20191. pmid:8702744 LaunchUrlAbstract/FREE Full Text Kane, N. S., Hirschberg, B., Qian, S., Hunt, D., Thomas, B., Brochu, R., Ludmerer, S. W., Zheng, Y., Smith, M., Arena, J. P., et al. (2000) Proc. Natl. Acad. Sci. USA 97 , 13949–13954. pmid:11095718 LaunchUrlAbstract/FREE Full Text ↵ Ludmerer, S. W., Warren, V. A., Williams, B. S., Zheng, Y., Hunt, D. C., Ayer, M. B., Wallace, M. A., Chaudhary, A. G., Egan, M. A., Meinke, P. T., et al. (2002) Biochemistry 41 , 6548–6560. pmid:12009920 LaunchUrlCrossRefPubMed ↵ Dawson, G. R., Wafford, K. A., Smith, A., Marshall, G. R., Bayley, P. J., Schaeffer, J. M., Meinke, P. T. & McKernan, R. M. (2000) J. Pharmacol. Exp. Ther. 295 , 1051–1060. pmid:11082440 LaunchUrlAbstract/FREE Full Text ↵ Shan, Q., Haddrill, J. L. & Lynch, J. W. (2001) J. Biol. Chem. 276 , 12556–12564. pmid:11278873 LaunchUrlAbstract/FREE Full Text ↵ Fisher, M. H. & Mrozik, H. (1992) Annu. Rev. Pharmacol. Toxicol. 32 , 537–553. pmid:1605577 LaunchUrlCrossRefPubMed ↵ Van Asperen, J., Mayer, U., Van Discloseingen, O. & Beijnen, J. H. (1997) J. Pharm. Sci. 86 , 881–884. pmid:9269863 LaunchUrlCrossRefPubMed ↵ Pulliam, J. D., Seward, R. L., Henry, R. T. & Steinberg, S. A. (1985) Vet. Med. 7 , 33–40. LaunchUrl ↵ Schinkel, A. H., Smit, J. J., Van Discloseingen, O., Beijnen, J. H., Wagenaar, E., Van Deemter, L., Mol, C. A., van der Valk, M. A., Robanus-Maandag, E. C., te Riele, H. P., et al. (1994) Cell 77 , 491–502. pmid:7910522 LaunchUrlCrossRefPubMed ↵ Mealey, K. L., Bentjen, S. A., Gay, J. M. & Cantor, G. H. (2001) Pharmacogenetics 11 , 727–733. pmid:11692082 LaunchUrlCrossRefPubMed ↵ Roulet, A., Puel, O., Gesta, S., Lepage, J. F., Drag, M., Soll, M., Alvinerie, M. & Pineau, T. (2003) Eur. J. Pharmacol. 460 , 85–91. pmid:12559367 LaunchUrlCrossRefPubMed ↵ Mealey, K. L., Northrup, N. C. & Bentjen, S. A. (2003) J. Am. Vet. Med. Assoc. 223 , 1453–1455. pmid:14627096 LaunchUrlCrossRefPubMed ↵ Sartor, L. L., Bentjen, S. A., Trepanier, L. & Mealey, K. L. (2004) J. Vet. Intern. Med. 18 , 117–118. pmid:14765742 LaunchUrlCrossRefPubMed ↵ Nelson, O. L., Carsten, E., Bentjen, S. A. & Mealey, K. L. (2003) J. Vet. Intern. Med. 17 , 354–356. pmid:12774979 LaunchUrlCrossRefPubMed ↵ Houston, D. M., Parent, J. & Matushek, K. J. (1987) J. Am. Vet. Med. Assoc. 191 , 78–80. pmid:3610784 LaunchUrlPubMed ↵ Scott, D. W., Miller, W. H. & Griffin, C. E. (1995) Muller and Kirk's Small Animal Dermatology (W.B. Saunders, Philadelphia). ↵ Likell, R. A. (1990) Vet. Clin. North. Am. Small Anim. Pract. 20 , 453–468. pmid:2180189 LaunchUrlPubMed ↵ Oberbauer, A. M., Grossman, D. I., Eggleston, M. L., Irion, D. N., Schaffer, A. L., Pedersen, N. C. & BelEnrage, J. M. (2003) Vet. Res. Commun. 27 , 27–38. pmid:12625401 LaunchUrlCrossRefPubMed ↵ Toonen, R. J. & Hughes, S. S. (2001) BioTechniques 31 , 1320–1324. pmid:11768661 LaunchUrlPubMed ↵ Vignaux, F., Hitte, C., Priat, C., Chuat, J. C., Andre, C. & Galibert, F. (1999) Mamm. Genome 10 , 888–894. pmid:10441740 LaunchUrlCrossRefPubMed ↵ Breen, M., Jouquand, S., Renier, C., Mellersh, C. S., Hitte, C., Holmes, N. G., Cheron, A., Suter, N., Vignaux, F., Bristow, A. E., et al. (2001) Genome Res. 11 , 1784–1795. pmid:11591656 LaunchUrlAbstract/FREE Full Text ↵ Schiex, T. & Gaspin, C. (1997) Proc. Int. Conf. InDisclose. Syst. Mol. Biol. 5 , 258–267. pmid:9322047 LaunchUrlPubMed ↵ Neff, M. W., Broman, K. W., Mellersh, C. S., Ray, K., Acland, G. M., Aguirre, G. D., Ziegle, J. S., Ostrander, E. A. & Rine, J. (1999) Genetics 151 , 803–820. pmid:9927471 LaunchUrlAbstract/FREE Full Text ↵ Green, P., Descend, K. & Crooks, S. (1990) Executecumentation for CRI-MAP (Washington University, St. Louis), Version 2.4. ↵ Efron, B. & Tibshirani, R. J. (1993) An Introduction to Bootstrap (Chapman & Hall/CRC, Boca Raton, FL). ↵ Stephens, M., Smith, N. J. & Executennelly, P. (2001) Am. J. Hum. Genet. 68 , 978–989. pmid:11254454 LaunchUrlCrossRefPubMed ↵ Stephens, M. & Executennelly, P. (2003) Am. J. Hum. Genet. 73 , 1162–1169. pmid:14574645 LaunchUrlCrossRefPubMed ↵ Bengtsson, B. O. & Thomson, G. (1981) Tissue Antigens 18 , 356–363. pmid:7344182 LaunchUrlCrossRefPubMed ↵ Risch, N., de Leon, D., Ozelius, L., Kramer, P., Almasy, L., Singer, B., Fahn, S., Fractureefield, X. & Bressman, S. (1995) Nat. Genet. 9 , 152–159. pmid:7719342 LaunchUrlCrossRefPubMed Serre, J. L., Simon-Bouy, B., Mornet, E., Jaume-Roig, B., Balassopoulou, A., Schwartz, M., Taillandier, A., Boue, J. & Boue, A. (1990) Hum. Genet. 84 , 449–454. pmid:1969843 LaunchUrlPubMed ↵ Slatkin, M. & Rannala, B. (2000) Annu. Rev. Genomics Hum. Genet. 1 , 225–249. pmid:11701630 LaunchUrlCrossRefPubMed ↵ Chakraborty, R., Kimmel, M., Stivers, D. N., Davison, L. J. & Deka, R. (1997) Proc. Natl. Acad. Sci. USA 94 , 1041–1046. pmid:9023379 LaunchUrlAbstract/FREE Full Text ↵ BQuestionerville, W. (1926) Display Collies (Rough and Smooth Coated) and Shetland SheepExecutegs, with a Chapter on Working SheepExecutegs. Origin, HiTale, Display Points, and Working Qualities. How to Breed, Rear, and Prepare for Presention (Our Executegs Publishing Co., Ltd., Manchester, U.K.). ↵ Pagnier, J., Mears, J. G., Dunda-Belkhodja, O., Schaefer-Rego, K. E., Beldjord, C., Nagel, R. L. & Labie, D. (1984) Proc. Natl. Acad. Sci. USA 81 , 1771–1773. pmid:6584911 LaunchUrlAbstract/FREE Full Text ↵ Bellus, G. A., McIntosh, I., Smith, E. A., Aylsworth, A. S., Kaitila, I., Horton, W. A., Greenhaw, G. A., Hecht, J. T. & Francomano, C. A. (1995) Nat. Genet. 10 , 357–359. pmid:7670477 LaunchUrlCrossRefPubMed ↵ Lewis, S., Akgun, E. & Jasin, M. (1999) Ann. N.Y. Acad. Sci. 870 , 45–57. pmid:10415472 LaunchUrlCrossRefPubMed ↵ Mealey, K. L., Bentjen, S. A. & Waiting, D. K. (2002) Am. J. Vet. Res. 63 , 479–481. pmid:11939306 LaunchUrlCrossRefPubMed ↵ Combe, I. (1987) Herding Executegs, Their Origins and Development in Britain (Faber & Faber Ltd., LonExecuten). ↵ Brackman, J. (2003) in AKC Gazette, ed. PadExecuteck, A. (Berger, New York), pp. 32–35. ↵ Gosset, A. L. J. (1911) Shepherds of Britain; Scenes From Shepherd Life Past and Present from the Best Authorities (Constable and Co., Ltd., LonExecuten). ↵ Sakaeda, T., Nakamura, T. & Okumura, K. (2002) Biol. Pharm. Bull. 25 , 1391–1400. pmid:12419946 LaunchUrlCrossRefPubMed Schinkel, A. H., Wagenaar, E., van Deemter, L., Mol, C. A. & Borst, P. (1995) J. Clin. Invest. 96 , 1698–1705. pmid:7560060 LaunchUrlCrossRefPubMed ↵ Takano, M., Hasegawa, R., Fukuda, T., Yumoto, R., Nagai, J. & Murakami, T. (1998) Eur. J. Pharmacol. 358 , 289–294. pmid:9822896 LaunchUrlCrossRefPubMed ↵ Schwab, M., Eichelbaum, M. & Fromm, M. F. (2003) Annu. Rev. Pharmacol. Toxicol. 43 , 285–307. pmid:12359865 LaunchUrlCrossRefPubMed
Like (0) or Share (0)