Slov Vet Res 2007; 44 (1/2): 19-24 UDC 575.117:575.224:631.527.3:636.74
Original Research Paper
MUTATION OF MDR1 GENE ASSOCIATED WITH MULTIDRUG SENSITIVITY IN AUSTRALIAN SHEPHERDS IN SLOVENIA
Marko Cotman, Jelka Zabavnik *
Institute of Anatomy, Histology and Embryology; Veterinary Faculty, Gerbiceva 60, 1000 Ljubljana, Slovenia * Corresponding author, E-mail: jelka.zabavnik@vf.uni-lj.si
Summary: The multidrug-resistance 1 (MDR1) transport protein plays an important protective role at blood-tissue barriers by limiting the entry of MDR1 protein substrates to brain, testis, fetus and other tissues. For subpopulation of Collies and related dog breeds increased susceptibility to neurotoxic side effects of several drugs including ivermectin, moxidectin and loperamide was detected. It was demonstrated that in ivermectin susceptible dogs the deletion mutation in MDR1 gene produces a frame shift resulting in the production of severly truncated non-functional MDR1 protein. In order to evaluate the occurrence and frequency of the mutated mdr1-1A allele in the population of Australian Shepherds in Slovenia we have screened 10 dogs representing approximately one third of the Slovenian population. The results of our study indicate very high frequency of mdr1-1A allele (70%) in Australian Shepherds in Slovenia. In 40% of studied population homozygous mutated genotype was determined. Considering the important role of MDR1 protein in drug disposition and blood-brain barrier protection, testing of the MDR1 genotypes and MDR1 genotype-based breeding programs are recommended for improving the safety of drug therapy with MDR1 protein substrates in Australian Shepherds.
Key words: molecular biology-genetics; P-glycoprotein-genetics; ivermectin-adverse effetcs; genes, MDR-genetics; neurotoxicity syndromes-etiology-genetics; pedigree; dogs-genetics
Introduction
The multidrug-resistance 1 (MDR1) transport protein is a large transmembrane P-glycoprotein (Pgp) encoded by the MDR1 gene. MDR1 is a member of the ATP-binding cassette (ABC) superfamily of transporters that use the energy derived from ATP hydrolysis to export various molecules including a variety of drugs across cell membranes from the cytosol to the extracellular medium and therefore performing the protective role in cells. MDR1 is expressed in a variety of tissues with excretory function, e.g. small intestine, liver, kidney, and at blood-tissue barriers, such as blood-brain barrier, blood-testis barrier and placenta. This protein is thought to have an important role in removing toxic metabolites from the cells, it limits the absorption of orally administered drugs, promotes drug elimina-
Received: 13 March 2007 Accepted for publication: 22 June 2007
tion into bile and urine and protects various tissues, e.g. brain, testis and fetus (1, 2, 3, 4).
Unbalanced level of MDR1 protein expression in the cells can cause disorders in the availability of MDR1 substrates in the cells. The expression of the MDR1 gene is frequently amplified in multidrug-resistant cells, resulting in a large overproduction of the MDR1 protein. If these cells are exposed to toxic compounds they can develop resistance to several drugs. This is well documented in humans, where the over-expression of human MDR1 protein in tumor cells is causing resistance of these cells to various chemotherapeutic drugs (5). The opposite, lack of MDR1 protein is observed in dogs - because of the mutation in the MDR1 gene the functional MDR1 protein is not expressed and the protective role is impaired.
More than 20 therapeutic drugs are known substrates of MDR1 protein, one of these is ivermectin that is used extensively in veterinary medicine as parasiticide toxic for nematodes and arthropod
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parasites, in dogs it is often used for prevention and therapy of heartworm (Dirofilaria immitis). In some Collies and Collie related dog breeds ivermectin treatment causes neurotoxicity (6, 7). In one study 1/200 of the lethal dose of ivermectin for beagles was lethal for Collies (6). Mealey et al. (8) discovered that the affected dogs were homozygous for a 4-bp deletion of the fourth exon of MDR1 gene. This mutation causes a frameshift causing premature stop codon, presumably resulting in truncated MDR1 protein with loss of function and therefore defective brain-blood barrier.
MDR1 deletion mutation associated with the ivermectin sensitivity has been reported in Collies and related breed dogs in the northwestern United States (9) and in France (10), in herding breeds in Australia (11), Japan (12) and in Germany (13). MDR1 deletion mutation was observed in many pure-breed dogs: in Australian Shepherds, Collies, English Shepherds, Longhaired Whippets, McNabs, Old English Sheepdogs, Shetland Sheepdogs and Silken Windhounds (14).
Population of Australian Shepherds in Slovenia is relatively small, it comprises only about thirty dogs. In Slovenia it is a relatively new breed, the dogs are imported from different countries. The aim of our study was to determine if mdr1-1A allele is present in our population of Australian Shepherds.
Material and methods
Animals
13 dogs were included in the study (10 Australian Shepherds, 3 mixed breed dogs). Samples were taken from dogs whose owners were interested in determining the MDR1 genotype of their dogs or with the consent of the owners to use the samples for research. Owners were informed about the study mainly through announcements made at the meetings of the owners of Australian Shepherds. Samples from mixed breed dogs were taken from a repository at the Laboratory for Molecular Biology and Molecular Genetic at the Veterinary Faculty in Ljubljana.
DNA isolation
Genomic DNA was isolated from blood samples collected in tubes containing anticoagulant (EDTA or acid citrate dextrose solution B) by a standard phenol-chloroform protocol as described by Sam-
brook et al. (15) or by commercially available Wizard Genomic DNA Purification Kit (Promega).
MDR1 gene amplification
MDR1 gene was amplified by use of primers (5' - GGC TTG ATA GGT TGT ATA TGT TGG TG - 3' and 5' - ATT ATA ACT GGA AAA GTT TTG TTT C - 3') described by Neff et al. (14) in polymerase chain reaction (PCR). The primers bracketed the reported 4 bp deletion in MDR1. The PCR consisted of 35 cycles with denaturing (20 seconds at 93oC), primers annealing (20 seconds at 55oC) and primers extension (1 minute at 72oC) in thermocycler (Biometra).
PCR product analyses
PRC products were separated by capillary elec-trophoresis on the ABI PRISM 310 apparatus to detect the size of the PCR products and analyzed by the programme GeneMapper 3.7.
Results
The allele mdr1-1A was found in 7 dogs of Australian Shepherd breed. The samples originated from five dogs with one known parent that was shown to be homozygous for the mdr1-1A allele. All five offspring had mdr1-1A allele, three were homozygous for this allele, two were heterozygous. In another family the parent was shown to be homozygous for mdr1-1A allele as well as its single offspring that was tested. The results of genotyping in Australian Shepherds are shown in Table 1 and in Figure 2.
In addition to Australian Shepherds some non-pure breed dogs were tested, one sample was obtained from the dog related to Collie breed. This dog was heterozygous for MDR1/mdr1-1A. Two other samples obtained from mixed breed dogs were homozygous for the wild type MDR1 allele.
Discussion
Ivermectin sensitivity connected to mdr1-1A homozygous genotype in dogs was first described in dogs of Collie breed (16, 17). Initial studies performed to determine the frequencies of mdr1-1A allele responsible for ivermectin toxicity were done on Collies (9, 10), however, recent studies include also other dog breeds of Collie lineage. The MDR1 gene frequencies in dogs of Australian Shepherd breed have already been determined in some countries. In
Mutation of MDR1 gene associated with multidrug sensitivity in Australian shepherds in Slovenia
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Table 1: Observed allele and genotype frequencies of gene encoding MDR1 protein in studied dogs of the Australian Shepherds breed
		Allele %		Genotype %		
Breed	No. of dogs	MDR1	mdr1-1A	mdr1-1A/ mdr1-1A	mdr1-1A/ MDR1	MDR1/ MDR1
Australian Shepherd	10	45	55	40	30	30
Figure 1: Electropherogram of PCR products after PCR amplification using the primers neighbouring the 4 bp deletion in MDR1 gene. Top - homozygous wild type genotype MDR1/MDR1; middle - homozygous mutated genotype mdr1-1A/mdr1-1A; bottom - heterozygous genotype mdr1-1A/ MDR1
7 CI	
w	m
• 1 « en tt o— 0 Ci	léÉè et t) « □ - n m o C3 Clfl
Figure 2: The results of genotyping for the MDR1 and mdrl-lA alleles in Australian Shepherds dogs. The samples originated from two families and two non-related dogs. The examined dogs represented approximately one third of all Slovenian dog population of Australian Shepherds breed (MDR1/MDR1 - white; mdrl-lA/mdrl-1A- black; mdrl-lA/MDRl - shadowed). Symbols labeled with question-marks represent the dogs that were not tested, the genotypes were only predicted
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the United States 178 dogs were examined, obtained mdr1-1A allele frequency was 16.6 % (14). In Japan 9 Australian Shepherds were tested and the obtained mdr1-1A allele frequency was 33.3 % (12), in Australia, 17 Australian Shepherds were tested for the same mutation in the MDR1, the mdr1-1A allele frequency was 42.86 % (11). The most extensive study that included 1500 dogs from 7 different dog breeds was performed in Germany. Frequency of the mutated genotype was the highest in Collies (54.6%), followed by Shetland Sheepdog (30.0%) and Australian Shepherd (19.5%). In this study 333 Australian Shepherds were included (13). We have included in our study 10 Australian Shepherds, this is small sample, however, it represents about 30% of Slovenian population of this breed. A very high frequency of the mdr1-1A allele was detected (55%), 40% of examined animals were homozygous for the mdr1-1A allele and 30% were carriers of mdr1-1A allele. Only a minority of studied dogs, 30%, was free of the mutated mdr1-1A allele, responsible for the multidrug sensitivity (Table 1, Figure 2).
MDR1 gene is displaying the pattern of Mende-lian inheritance, the mutation was detected also in some pure breeds related to Collies as well as in non-pure breeds that previously did not have mdr1-1A allele (11, 12, 13, 14). In our study we have observed one mdr1-1A allele in one sample obtained from non-pure breed dog that was descendent of Collie. In this dog, most probably the mutated allele was obtained from the Collie parent.
Sensitivity of Collies to ivermectin was initially studied in the United States in the regions where heartworm Dirofilaria immitis is endemic and therefore ivermectin was widely used in low preventive doses and in high doses for therapy of heartworm in dogs. Neurotoxicoses were often observed in some Collies after the application of ivermectin in therapeutic doses (7). In Slovenia, heartworm Dirofilaria immitis is present in Primorska region and in the neighbouring Italy and Croatia, the preventive application of ivermectin is indicated for the dogs living or visiting these regions.
Ivermectin is the most studied substrate of the transmembrane transporter P-gp encoded by the MDR1 gene, however, other drugs used in therapy of dogs are also MDR1 protein substrates. MDR1 protein substrates in previously documented interactions with canine mdr1-1 A are antimicrobial agents (erythromycin, grepafloxacin), anticancer agents (doxorubicin, vincristine), immunosuppres-sants (cyclosporin A, tacrolimus), steroids (dexam-
ethasone, hydrocortisone), gastrointestinal drugs (loperamide, domperidon), cardiac drugs (quinidine, digoxin) (14). Probably there are many more MDR1 protein substrates that can cause neurotoxicoses if the blood-brain barrier is not efficient because of non-functional product of mdr1-1A allele.
Based on the determined frequency of mdrl-1A allele in Australian Shepherds in Slovenia we would suggest the verification of the presence of the mdr1-1A allele before using therapeutic doses of ivermectin or other MDR1 protein substrates. The method used in our study to detect the mutated allele is reliable, fast and affordable. According to the known presence of the mdr1-1A allele in some dog breeds in countries where the allele frequencies in different dog populations were already determined (9, 10, 11, 12, 13, 14) the same practice would be suggested for dog breeds related to Collie.
In conclusion, the results of this study indicate that very high percentage of Australian Shepherds in Slovenia (70%) contains at least one mdr1-1A allele responsible for sensitivity to ivermectin and other substrates of P-gp transporter. Considering the important role of MDR1 protein in drug distribution to the cells and in particular for blood-brain barrier protection special care should be taken when treating Australian Shepherds with drugs that are P-gp substrates. Detection of dogs with mutated mdr1-1A allele in Slovenia based on MDR1 genotyping are recommended to increase the safety of drug therapy with P-gp substrates and to prepare specific breeding programmes to lower the frequencies of the ho-mozygous mdr1-1A allele in a dog population.
Acknowledgements
We thank Mrs. Jasna Sporar and dog owners for the blood samples and Mrs. Magdalena Dobravec for her technical assistance. This research was supported by the Slovenian Research Agency in the frame of Research Programme P4 0053.
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MUTACIJA GENA MDR1, POVEZANA S PREOBČUTLJIVOSTJO NA RAZLIČNA ZDRAVILA PRI AVSTRALSKIH OVČARJIH V SLOVENIJI
M. Cotman, J. Zabavnik
Povzetek: Transportni protein MDR1 (multidrug-resistance 1 protein - protein za odpornost proti različnim drogam) igra pomembno zaščitno vlogo v različnih krvno-tkivnih pregradah in s tem omejuje dostop MDRI-substratov do možganov, testisov, zarodka in drugih tkiv. Pri deležu populacije škotskih ovčarjev in njim sorodnih pasmah je bila opažena povečana pojavnost nevrotoksikoz ob obdelovanju z različnimi zdravili - z ivermektinom, moksidektinom in loperamidom. Pri psih s povečano občutljivostjo na ivermektin je bila ugotovljena delecijska mutacija gena (alel mdr1-1A). Zaradi mutacije pride do zamika bralnega okvira gena, kar povzroči proizvodnjo zelo skrajšanega nedejavnega proteina MDR1. Z raziskavo smo želeli ugotoviti pojavnost in frekvenco mutiranega alela mdr1-1A v populaciji avstralskih ovčarjev v Sloveniji. Preiskali smo 10 psov pasme avstralski ovčar, kar predstavlja približno eno tretjino populacije te pasme v Sloveniji. Rezultati naše študije kažejo zelo visoko frekvenco alela mdr1-1A (70%) v slovenski populaciji avstralskih ovčarjev. V 40 % proučevanih avstralskih ovčarjev se je mutirani alel pojavil v homozigotni obliki. Glede na pomembno vlogo proteina MDR1 pri razporejanju drog v organizmu in pri zaščiti v krvno-možganski pregradi priporočamo ugotavljanje genotipov MDR1 in uvedbo rejskega programa za usmerjeno izbiranje genotipov MDR1, kar bi povečalo varnost ob obdelovanju avstralskih ovčarjev z zdravili, ki so substrat proteina MDR1.
Ključne besede: molekularna biologija-genetika; P-glikoprotein-genetika; ivermektin-škodljivi učinki; gen MDR-genetika, nevrotoksični sindromi-etiologija-genetika; rodovnik; psi-genetika