COMUNICACIÓN BREVE

The search for the mdr1-1Δ mutation of the MDR1 gene in four canine breeds in Uruguay (preliminary study)

Búsqueda de la mutación mdr1-1Δ del gen MDR1 en cuatro razas caninas en Uruguay (estudio preliminar)

Rosa Gagliardi B,¹* M.Sc, Diana Martínez A,² MV, Beatriz Tellechea H,² MV, Pedro Sitjar Q,² MV, Silvia Llambí D,¹ Ph.D, María Arruga L,³ Ph.D.

¹Universidad de la República, Facultad de Veterinaria, Área Genética. Montevideo, C.P. 11600, Uruguay.
²Veterinarios con ejercicio liberal de la profesión.
³Universidad de Zaragoza, Facultad de Veterinaria, Laboratorio de Citogenética y Genética Molecular. Zaragoza, C. P. 50013, España.

*Correspondence: rgagliar@gmail.com

Received: Received: May 2014; Accepted: October 2014.

ABSTRACT

Objective. The objective of this study is to analyze the frequency of mdr1-1Δ mutation in German Shepherd, Doberman, Border Collie and Greyhound dog breeds in Uruguay. Materials and methods. A total of 95 animals from the four breeds mentioned above were studied. DNA was isolated from blood using potassium acetate with a subsequent degradation from RNA with RNAsaH. The concentration and quality of the DNA obtained was evaluated with a Nanodrop, ND-1000 spectrophotometer. To determine the presence or absence of the mdr1-1Δ mutation, DNA samples were sent to Gene Seek, Neogen Corporation of Chicago, United States, for genotyping. Results. In all 95 animals studied, the mdr1-1Δ mutation was not present. Conclusions. Based on the preliminary results obtained, other elements that may cause adverse drug reactions must be considered: unidentified mutations in other regions of the MDR1 gene; mutations in other genes involved in the transport of drugs from the same subfamily or another; mutations in enzymes involved in drug metabolism (e.g. Cytochrome P450). Moreover, especially with Border Collies and Greyhounds, it is advisable to increase the number of animals in the study.

Key words: Dogs, ivermectine, P-glycoprotein (Source: CAB).

RESUMEN

Objetivo. En este trabajo se realizó la búsqueda de la mutación mdr1-1Δ en caninos de las razas Ovejero Alemán, Doberman, Galgo y Border Collie de Uruguay. Materiales y métodos. Se estudió un total de 95 animales, de las cuatro razas caninas mencionadas. El ADN se aisló a partir de muestras sanguíneas empleando acetato de potasio 8M con posterior degradación de ARN con RNAsaH. Se evaluó la concentración y calidad del ADN en un espectrofotómetro Nanodrop, ND-1000. Para conocer la presencia o ausencia de la mutación mdr1-1Δ, los ADN de las muestras extraídas se enviaron a Gene Seek, (Neogen Corporation) de Chicago, Estados Unidos. Resultados. En ninguno de los 95 animales estudiados se encontró la mutación mdr1-1Δ. Conclusiones. Dados los resultados preliminares obtenidos, se deberían considerar otros elementos que puedan causar reacciones adversas a fármacos: mutaciones aun no identificadas en otras regiones del gen MDR1; mutaciones en otros genes que intervengan en el transporte de medicamentos de la misma subfamilia u otra; mutaciones en enzimas que participan en el metabolismo de los medicamentos (ej. Citocromo P450). Por otra parte, y en particular en las razas Border Collie y Galgo es aconsejable aumentar el número de animales en estudio.

Palabras clave: Glucoproteína-P, ivermectina, perros (Fuente: CAB).

INTRODUCTION

Antiparasitic treatments in canines, both external and internal, are one of the routine tasks in a Veterinary Clinic. Different types of drugs are used for this treatment. Macrocyclic lactone is part of a family of endectocide antiparasitics that has frequently been used, particularly ivermectine. It increases the release and union of gamma-amino butyric acid (GABA) in nerve synapses, provoking the blockage of transmission impulses and paralysis of the parasite. GABA is an inhibiting neurotransmitter in the nervous system of nematodes and arthropods, while in mammals it acts on the central nervous system (CNS). In these latter ones the blood-brain barrier plays a protective role in preventing xenobiotics from entering, and for this reason drugs such as ivermectine present a wide safety margin.

P-glycoprotein, part of said barrier, is responsible for transporting different compounds outside the CNS and plays, in this case, a protective role (1). Starting in the 1980s, when ivermectine began to be used as an antiparasitic, cases began to be reported of canines that had neurologic symptoms (lethargy, ataxia, coma, blindness, convulsions, etc.) which resulted in respiratory paralysis and death of the animal. Generally Collies, Shetland Sheepdogs, Australian Shepherds, White Shepherds, Longhaired Whippets or dogs interbred with these breeds were affected. It was later seen that these animals lacked functional P-glycoprotein, caused by the presence of a 4pb deletion (mdr1-1Δ mutation) in the gene that codifies it (MDR1: multiple drug resistant gene) (2-5).

Merola et al (1) performed drug neurotoxicity communications on different breeds and mixed breeds of dogs, including some where the abovementioned mutation has not been detected. Among the breeds that have been studied are German Shepherds, Dobermans, Border Collies and Greyhounds. Of these breeds, the one that showed mdr1-1Δ mutation was Border Collies and some breeds related to Greyhounds. In spite of these findings, it is important to consider that in some of these studies very few animals were analyzed (Doberman: 2 cases) (1).

In this study, the preliminary search for mdr1-1Δ mutation in canines of the German Shepherd, Doberman, Greyhound and Border Collie breeds in Uruguay is carried out.

MATERIALS AND METHODS

Samples analyzed. A total of 95 animals from four breeds were studied: German Shepherd (34 animals), Doberman (31 animals), Greyhound (21 animals) and Border Collie (9 animals). The samples were random, from different regions of the country, particularly to the south, east and northeast (departments of Montevideo, Canelones, San José and Maldonado), due to animal density in these departments. Blood was drawn from the anterobrachial or saphenous vein, in conditions of asepsia, using K3 EDTA as an anticoagulant, preserved frozen until processed in the laboratory. Extractions were performed in the presence of the animal's owners, at their home or during a routine veterinary visit, with minimal stress, following CHEA regulations (Honorary Commission on Animal Experimentation).

DNA Isolating. DNA was isolated using an extraction technique with potassium acetate 8M and later degradation of RNA with RNAsaH (6). A spectrophotometer (Nanodrop, ND-1000) was used to evaluate the concentration and quality of DNA obtained.

Search for the mutation. To find the genotype of the MDR1 gene, DNA from extracted samples was sent to the Gene Seek laboratory, Neogen Corporation from Chicago, United States. In table 1 a sample from a region of the MDR1 gene that includes the studied mutation is shown.

RESULTS

As can be seen in table 2, where as an example a sample is shown of the result of the genotypes of 10 of samples studied, none of the cases that were analyzed in this work presented the mdr1-1Δ mutation.

DISCUSSION

The absence of the mdr1-1Δ mutation in the 95 genotyped samples belonging to the four canine breeds could be due in first place to the low number of genotyped samples for each breed, considering that the frequency of the mutation is low. Merola (1) studied dogs of different races that presented sensibility to ivermectine. Among these dogs, 20% of cases with German Shepherds presented sensibility to the drug, and in Greyhounds, 7.6%. In these cases, a direct relation between the existence of sensibility and the administered dose of ivermectine is shown. Animals that have the studied mutation lack normal P glycoprotein, being susceptible to ivermectine even in the low doses (0.15 to 0.34 mg/kg) (1) used in routine treatments in veterinary clinics.

In reference to this study, the mdr1-1Δ mutation was sought in four breeds that have shown adverse reactions to ivermectine or that are related to the Collie Border breed.

In the case of German Shepherds, in the United States the mdr1-1Δ mutation was described specifically in white animals (7,8). In that country, white is a variety within the breed. On the other hand, in Europe the mutation has not been detected in German shepherds. However, white animals make up the White Swiss Shepherd breed, which does present the mdr1-1Δ mutation in an elevated frequency (2,3). In Uruguay, German Shepherds are found widely throughout the country. Additionally, White Swiss Shepherd is not clearly defined as a separate breed. The majority of the samples taken for this study correspond to the southeast area of the country, where there are no white animals. At any rate, it is unknown if the animals studied are related to animals of this color. These preliminary results lead to the prediction that the mutation problem would not be present in German Shepherds without the introgression of White Swiss Shepherds.

Regarding Dobermans, there is little previous information regarding the presence or absence of the mdr1-1Δ mutation (1). In spite of the fact that cases reporting sensitivity to the drug exist, the presence of the aforementioned mutation have not been communicated. In this breed, around the world, two cases of drug poisoning with ivermectine were discovered. In the first case, the animal accidentally ingested an excessive amount of the drug (1). This case was described in the 1990s, and since the mutation had not been described, its presence could not be determined. The second case involved animals with demodicosis that were treated with ivermectine and presented neurotoxicity. These cases were genotyped for the MDR1 gene, normal homozygotes for this gene (9).

In reference to Greyhounds, 23 samples were studied and all showed a negative result for the mdr1-1Δ mutation. According to previous studies (8), in certain breeds related to these animals (Longhair Whippet), the mutation is found in elevated frequencies at more than 40%. The fact that it was not found in the analyzed samples could be attributed to the fact that in our country there are no pure animals of this breed, since the majority are a cross between creole greyhound and English Greyhound, as well as others. At any rate, it would be important to increase the number of animals analyzed, considering the possibility that the mutation is rarely found in the population of greyhounds in our country. On the other hand, given the mix in the breed, it would be interesting to continue studying these animals in order to have a better idea of the situation respecting this mutation.

In the case of Border Collies, where 9 animals were studied, the mdr1-1Δ mutation wasn't found either. In this breed, Neff et al (8) described the absence of the mutation. Other authors (10) detected it in frequencies of only 0.6%. In Uruguay previous studies were done to analyze wild dogs and collies, finding the mdr1-1Δ mutation only in collies (11,12). Given the existing relationship between Border Collies and collies, where the studied mutation was first described, and the fact that there are authors that found it in very low frequency, it would be interesting to continue studying this breed and increase the number of sampled animals to know their situation in the country.

As previously mentioned, former studies (1) associated toxicity to ivermectine with the dose that was administered, and keeping in mind that in the four breeds that were studied there were cases of sensitivity to ivermectine, it was considered that drug sensitivity could be due to yet unidentified mutations in other regions of the MDR1 gene, mutations in other genes that intervene in the transport of drugs from the same subfamily or another one, or mutations in enzymes that participate in the metabolism of these drugs (e.g. cytochrome P450) (9,13).

Based on the results obtained in this study and the elements previously mentioned, the possibility that animals from the studied breeds could present adverse reactions to drugs such as ivermectine cannot be discarded, and it is advisable for more studies to be done in order to be able to analyze different causes responsible for the mentioned drug sensitivity, among others. This acquires greater interest if the amount of drugs that underlie P glycoprotein are considered as well as the relationship this has to other types of treatments such as those with antitumor drugs.

Acknowledgments

The authors thank the Kennel Club Uruguayo (FVet, UdelaR agreement), the breeders of the animals that were studied, and individual veterinarians, all of whom worked to obtain samples of the animals studied.

REFERENCES

1. Merola V, Khan S, Gwaltney-Brant S. Ivermectin toxicosis in dogs: A restrospective study. J Am Anim Hosp Assoc 2009; 45 (3):106-111.         [ Links ]

2. Gramer I, Leidolf R, Döring B, Klintzsch S, Krämer E-M, Yalcin E, Petzinger E, Geyer J. Breed distribution of the nt230 (del4) MDR1 mutation in dogs. Vet J 2011; 189 (1):67-71.         [ Links ]

3. Geyer J, Klintzsch S, Meerkamp K, Wöhlke A, Distl O, Moritz A, Petzinger E . Detection of the nt230 (del4) MDR1 mutation in White Swiss Shepherd dogs: case reports of doramectin toxicosis, breed predisposition, and microsatellite analysis. J Vet Pharmacol Therap 2007; 30 (5):482-485.         [ Links ]

4. Roulet A, Puel O, Gesta S, Lepage J-F, Drag M, Soll M, Alvinerie M, Pineau T. MDR1-deficient genotype in Collie dog hypersensitive to the Pglycoprotein substrate ivermectin. European J of Pharmacol 2003; 460 (2-3):85-91.         [ Links ]

5. Mealey KL, Bentjen SA, Gay JM, Canthor GH. Ivermectin sensitivity in collies is associated with a deletion mutation of the MDR1 gene. Pharmacogenetics 2001; 11 (8):727-733.         [ Links ]

6. Fraga J, Rodríguez J, Fuentes O, Castex M, Fernández-Calienes A. Comparación entre 5 métodos para la extracción de ADN de triatomíneos: su utilización en la técnica de ADN polimórfico amplificado al azar Rev Cubana Med Trop 2004; 56 (3):208-13.         [ Links ]

7. Mealey KL. Farmacogenetica. Clin Vet NA Med Peq Anim 2006; 36 (5):961-973.         [ Links ]

8. Neff MW, Robertson KR, Wong AK, Safra N, Broman KW, Slatkin M, Mealey KL, Pedersen NC. Breed distribution and history of canine mdr1-1Δ a pharmacogenetic mutation that marks the emergence of breed from the collie lineage. Proc Nat Acad Sci 2004; 101 (32):11725-11730.

9. Bissonnette S, Paradis M, Daneau I, Silverside DW. The ABCB1-1Δ mutation is not responsible for subchronic neurotoxicity seen in dogs of noncollie breeds following macrocyclic lactone treatment for generalized demodicosis. Vet Dermatol 2008; 20(1):60-66.

10. Geyer J, Döring B, Godoy JR, Leidolf R, Moritz A, Petzinger E. Frecuency of the nt230 (del4) MDR1 mutation in Collies and related dog breeds in Germany. J Vet Pharmacol Therap 2005; 28 (6):545-551.         [ Links ]

11. Gagliardi R, García C, Llambí S, Arruga MV. Analysis of mdr1-1Δ mutation of MDR1 gene in the “Cimarron Uruguayo” dog. Rev MVZ Córdoba 2013; 18(2):3480-3483.

12. Gagliardi R. Estudios geneticos en caninos de raza Cimarron Uruguayo (Canis familiaris). [Tesis de Maestría]. Montevideo: Universidad de la República, Programa de Desarrollo de las Ciencias Básicas (PEDEClBA); 2009.         [ Links ]

13. Tomiyasu H, Goto-Koshino Y, Fujino Y, Ohno K, Tsujimoto H. Epigenetic regulation of the ABCB1 gene in drug-sensitive and drug-resistant lymphoid tumour cell lines obtained from canine patients. Vet J 2014; 199 (1): 103-109.         [ Links ]