Search tips
Search criteria 


Logo of jvetsciJournal of Veterinary ScienceAbout J Vet SciInstructions to AuthorsSubmission of ManuscriptThis Article
J Vet Sci. 2010 December; 11(4): 341–344.
Published online 2010 December 3. doi:  10.4142/jvs.2010.11.4.341
PMCID: PMC2998746

Novel insertion mutation of ABCB1 gene in an ivermectin-sensitive Border Collie


P-glycoprotein (P-gp) is encoded by the ABCB1 gene and acts as an efflux pump for xenobiotics. In the Border Collie, a nonsense mutation caused by a 4-base pair deletion in the ABCB1 gene is associated with a premature stop to P-gp synthesis. In this study, we examined the full-length coding sequence of the ABCB1 gene in an ivermectin-sensitive Border Collie that lacked the aforementioned deletion mutation. The sequence was compared to the corresponding sequences of a wild-type Beagle and seven ivermectin-tolerant family members of the Border Collie. When compared to the wild-type Beagle sequence, that of the ivermectin-sensitive Border Collie was found to have one insertion mutation and eight single nucleotide polymorphisms (SNPs) in the coding sequence of the ABCB1 gene. While the eight SNPs were also found in the family members' sequences, the insertion mutation was found only in the ivermectin-sensitive dog. These results suggest the possibility that the SNPs are species-specific features of the ABCB1 gene in Border Collies, and that the insertion mutation may be related to ivermectin intolerance.

Keywords: ABCB1, Border Collie, insertion mutation, polymorphism


P-glycoprotein (P-gp), encoded by the ABCB1 gene (formerly known as MDR1), is a membrane transport protein in the ATP-binding cassette superfamily [16]. P-gp is normally expressed in various mammalian tissues including the apical border of intestinal epithelial cells, brain capillary endothelial cells, biliary canalicular cells, renal proximal tubular epithelial cells, placenta, and testes [4,9,14,17,21]. P-gp functions as an efflux pump on the cell membrane, and thus protects the cell from potentially toxic xenobiotics [16]. Several polymorphisms of the ABCB1 gene are known to cause deformations and dysfunctions of P-gp in humans [3,6,12,18], while in ivermectin-sensitive Collies, a frame shift mutation has been found which causes a premature stop to P-gp synthesis [7,19].

We examined a Border Collie with depression, hypersalivation, and several other adverse reactions which developed following ivermectin intake for heartworm treatment. None of its family members showed any adverse reactions to ivermectin administration. DNA analysis, however, did not reveal the previously described frame shift mutation in the ivermectin-sensitive Border Collie. For this reason, we examined the full-coding sequence of the ABCB1 gene in the ivermectin-sensitive Border Collie, and compared the sequence to those of its family members and to wild-type Beagles in order to investigate the possibility of different mutations.

Materials and Methods

Peripheral blood samples were collected from the cephalic veins of the ivermectin-sensitive Border Collie and its seven family members. Total RNA was extracted from the peripheral blood using TRIzol reagent (Invitrogen, USA). The total RNA was reverse transcribed into first-strand cDNA using a random hexamer primer and the Superscript first-strand synthesis system of the RT-PCR kit (Invitrogen, USA).

To verify the family relationship between the Border Collies, a 261-bp mitochondrial D-loop region was amplified using the primers L15910 and H16498 as previously described [5]. All PCR products were sequenced using an ABI Prism BigDye terminator cycle sequencing ready reaction kit v.5.1 (PE Applied Biosystems, USA) and the sequences were compared.

We designed primers to amplify the coding sequence of the ABCB1 gene; these were based on the wild-type Beagle ABCB1 gene sequence (GenBank accession number NM_001003215). Each primer was designed to contain overlapping sequences of 100~200 bp at both ends of the PCR products. The conditions of the designed primers were verified using DNAMAN software v.4.16 (Lynnon, Canada). Primer information is listed in Table 1. PCR amplification was performed as follows: one cycle at 94[degree celsius] for 5 min; 35 cycles at 94[degree celsius] for 30 sec at the primer annealing temperature (55~57[degree celsius]) for 30 sec, followed by at 72[degree celsius] for 1 min. The sizes of the resultant PCR products were analyzed using 2% agarose gel electrophoresis.

Table 1
Oligo-sequences of the primers used in this study

All PCR products were sequenced using the ABI Prism BigDye terminator cycle sequencing ready reaction kit v.5.1 (PE Applied Biosystems, USA). Finally, all identified sequences were merged (except the overlapping sequences) and compared using CLC sequence viewer v.4.6.2 (CLC Bio, Denmark).


Fig. 1 shows the family pedigree of the Border Collies. All Border Collies except for the father showed the same nucleotide sequence in the 261-bp mitochondrial D-loop region. There was a 99% similarity between the father and the rest of the family.

Fig. 1
Pedigree analysis showing the relationships of the ivermectin-sensitive Border Collie and its seven family members. The black square indicates the ivermectin-sensitive Border Collie.

When compared to the coding sequence of the ABCB1 gene in wild-type Beagle dogs, one insertion mutation and eight single nucleotide polymorphisms (SNPs) were found at the nucleotide level in the ivermectin-sensitive Border Collie (Fig. 2). Three nucleotides ('AAT') were inserted between the 72nd and 73rd nucleotide of the ABCB1 coding sequence. The eight SNPs were characterized as G574A, C635G, A985T, G996A, G1595A, T2082A, T2086C, and A3817G. At the protein level, the insertion mutation led to the addition of an asparagine between the 24th and 25th amino acids. Six of the SNPs lead to amino acid exchanges (Val192Ile, Pro212Arg, Thr329Ser, Arg532Gln, Ser696Pro, and Ile273Val), while G996A and T2082A resulted in silent mutations. All SNPs were also examined in all of the Border Collie family members; however, the insertion mutation was observed only in the ivermectin-sensitive Border Collie.

Fig. 2
Diagram showing the location of mutations in the coding sequence of the ABCB1 gene and P-glycoprotein (P-gp). The arrow indicates the position of the insertion mutation and arrowheads show the positions of single nucleotide polymorphisms. Underbars indicate ...


P-gp was first discovered in 1976 in a Chinese hamster ovary (CHO) cell line that was selected in culture for colchicine resistance [10]. Resistant CHO cells expressed large quantities of a 170-kD protein, subsequently named P-gp. Since then, several drugs have been identified as substrates of P-gp [6]. In dogs, ivermectin sensitivity caused by a nonsense mutation of the ABCB1 gene is a well-recognized phenomenon [19]; however, the full sequence of the ABCB1 gene is only available for the wild-type Beagle. Therefore, we first established the ABCB1 coding sequence in wild-type Border Collies and compared that to the wild-type Beagle sequence. We found eight SNPs in all wild-type Border Collies. Taking into account interethnic variation in the human ABCB1 sequence [1,20], our finding suggests that the canine ABCB1 sequence may demonstrate interbreed variation. For this reason, we recommend that the ABCB1 gene sequence be confirmed in each breed of dog prior to molecular investigation for mutations.

Using the wild-type Border Collie sequence examined in this study as a baseline for comparison, we investigated alterations in the sequence of the ABCB1 gene in an ivermectin-sensitive Border Collie that lacked the expected frameshift mutation. While eight SNPs were found in all of the study Border Collies, an insertion mutation was found only in the ivermectin-sensitive animal. The conformational change of P-gp is important for the protein's role as an efflux pump through interaction between the drug-binding and nucleotide-binding domains [15]. In the human ABCB1 gene, several SNPs have been identified that can lead to decreased P-gp function [6]. Furthermore, a silent mutation in the ABCB1 gene has been found to be associated with the level of P-gp expression in humans [8]. Deformation of protein structure caused by the addition or substitution of a single amino acid also induces several diseases in human and animals [2,13]. Malfunction of P-gp has often been noted in Collies and can occur either by decreased expression of a functional gene or by a gene mutation that impairs protein concentration or activity [19]. The eight SNPs identified in this study do not appear to be directly related to the adverse reactions displayed by the ivermectin-sensitive Border Collie, suggesting the possibility that these polymorphisms of the ABCB1 gene are related to the interbreed variation in P-gp function seen in dogs.

Since mitochondrial DNA is inherited exclusively from the mother, it can be used as a tool for tracking maternal lineage [23]. A D-loop-containing region that has a highly variable sequence is commonly used for phylogenetic analysis [11,22,24]. To verify the family relationship of the Border Collies, we sequenced the D-loop region and found that all family members expressed the same sequence except for the father dog. The similarity between the father dog and the rest of the family was 99%. According to the GenBank database, the similarity of the sequence of canine D-loop regions is 98~99%. This suggests that the SNPs of the ABCB1 coding sequence identified in this study are a universal feature of Border Collies.

In conclusion, we found a new insertion mutation of the ABCB1 gene in an ivermectin-sensitive Border Collie. This mutation was not seen in its healthy family members or in the normal Beagle. Additionally, we described eight SNPs in the wild-type Border Collies. This finding suggests the possibility that dogs display interbreed variation in the ABCB1 sequence. The sequence of the ABCB1 gene should be established in each breed of dog prior to molecular investigation of the ABCB1 gene and P-gp.


1. Ameyaw MM, Regateiro F, Li T, Liu X, Tariq M, Mobarek A, Thornton N, Folayan GO, Githang'a J, Indalo A, Ofori-Adjei D, Price-Evans DA, McLeod HL. MDR1 pharmacogenetics: frequency of the C3435T mutation in exon 26 is significantly influenced by ethnicity. Pharmacogenetics. 2001;11:217–221. [PubMed]
2. Bauer JW, Rouan F, Kofler B, Rezniczek GA, Kornacker I, Muss W, Hametner R, Klausegger A, Huber A, Pohla-Gubo G, Wiche G, Uitto J, Hintner H. A compound heterozygous one amino-acid insertion/nonsense mutation in the plectin gene causes epidermolysis bullosa simplex with plectin deficiency. Am J Pathol. 2001;158:617–625. [PubMed]
3. Cascorbi I, Gerloff T, Johne A, Meisel C, Hoffmeyer S, Schwab M, Schaeffeler E, Eichelbaum M, Brinkmann U, Roots I. Frequency of single nucleotide polymorphisms in the P-glycoprotein drug transporter MDR1 gene in white subjects. Clin Pharmacol Ther. 2001;69:169–174. [PubMed]
4. Cordon-Cardo C, O'Brien JP, Casals D, Rittman-Grauer L, Biedler JL, Melamed MR, Bertino JR. Multidrug-resistance gene (P-glycoprotein) is expressed by endothelial cells at blood-brain barrier sites. Proc Natl Acad Sci USA. 1989;86:695–698. [PubMed]
5. Desalle R, Williams AK, George M. Isolation and characterization of animal mitochondrial DNA. Methods Enzymol. 1993;224:176–204. [PubMed]
6. Fromm MF. The influence of MDR1 polymorphisms on P-glycoprotein expression and function in humans. Adv Drug Deliv Rev. 2002;54:1295–1310. [PubMed]
7. Geyer J, Döring B, Godoy JR, Leidorf R, Moritz A, Petzinger E. Frequency of the nt230 (del4) MDR1 mutation in Collies and related dog breeds in Germany. J Vet Pharmacol Ther. 2005;28:545–551. [PubMed]
8. Hoffmeyer S, Burk O, von Richter O, Arnold HP, Brockmöller J, Johne A, Cascorbi I, Gerloff T, Roots I, Eichelbaum M, Brinkmann U. Functional polymorphisms of the human multidrug-resistance gene: multiple sequence variations and correlation of one allele with P-glycoprotein expression and activity in vivo. Proc Natl Acad Sci USA. 2000;97:3473–3478. [PubMed]
9. Hori R, Okamura N, Aiba T, Tanigawara Y. Role of P-glycoprotein in renal tubular secretion of digoxin in the isolated perfused rat kidney. J Pharmacol Exp Ther. 1993;266:1620–1625. [PubMed]
10. Juliano RL, Ling V. A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants. Biochim Biophys Acta. 1976;455:152–162. [PubMed]
11. Kim KI, Lee JH, Li K, Zhang YP, Lee SS, Gongora J, Moran C. Phylogenetic relationships of Asian and European pig breeds determined by mitochondrial DNA D-loop sequence polymorphism. Anim Genet. 2002;33:19–25. [PubMed]
12. Kim RB, Leake BF, Choo EF, Dresser GK, Kubba SV, Schwarz UI, Taylor A, Xie HG, McKinsey J, Zhou S, Lan LB, Schuetz JD, Schuetz EG, Wilkinson GR. Identification of functionally variant MDR1 alleles among European Americans and African Americans. Clin Pharmacol Ther. 2001;70:189–199. [PubMed]
13. Kunieda M, Tsuji T, Abbasi AR, Khalaj M, Ikeda M, Miyadera K, Ogawa H, Kunieda T. An insertion mutation of the bovine Fii gene is responsible for factor XI deficiency in Japanese black cattle. Mamm Genome. 2005;16:383–389. [PubMed]
14. Li M, Hurren R, Zastawny RL, Ling V, Buick RN. Regulation and expression of multidrug resistance (MDR) transcripts in the intestinal epithelium. Br J Cancer. 1999;80:1123–1131. [PMC free article] [PubMed]
15. Litman T, Druley TE, Stein WD, Bates SE. From MDR to MXR: new understanding of multidrug resistance systems, their properties and clinical significance. Cell Mol Life Sci. 2001;58:931–959. [PubMed]
16. Mealey KL. Canine ABCB1 and macrocyclic lactones: Heartworm prevention and pharmacogenetics. Vet Parasitol. 2008;158:215–222. [PubMed]
17. Melaine N, Liénard MO, Dorval I, Le Goascogne C, Lejeune H, Jégou B. Multidrug resistance genes and P-glycoprotein in the testis of the rat, mouse, guinea pig, and human. Biol Reprod. 2002;67:1699–1707. [PubMed]
18. Mickley LA, Lee JS, Weng Z, Zhan Z, Alvarez M, Wilson W, Bates SE, Fojo T. Genetic polymorphism in MDR-1: a tool for examining allelic expression in normal cells, unselected and drug-selected cell lines, and human tumors. Blood. 1998;91:1749–1756. [PubMed]
19. Roulet A, Puel O, Gesta S, Lepage JF, Drag M, Soll M, Alvinerie M, Pineau T. MDR1-deficient genotype in Collie dogs hypersensitive to the P-glycoprotein substrate ivermectin. Eur J Pharmacol. 2003;460:85–91. [PubMed]
20. Schaeffeler E, Eichelbaum M, Brinkmann U, Penger A, Asante-Poku S, Zanger UM, Schwab M. Frequency of C3435T polymorphism of MDR1 gene in African people. Lancet. 2001;358:383–384. [PubMed]
21. Thiebaut F, Tsuruo T, Hamada H, Gottesman MM, Pastan I, Willingham MC. Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues. Proc Natl Acad Sci USA. 1987;84:7735–7738. [PubMed]
22. Vilà C, Savolainen P, Maldonado JE, Amorim IR, Rice JE, Honeycutt RL, Crandall KA, Lundeberg J, Wayne RK. Multiple and ancient origins of the domestic dog. Science. 1997;276:1687–1689. [PubMed]
23. Wong LJ, Boles RG. Mitochondrial DNA analysis in clinical laboratory diagnostics. Clin Chim Acta. 2005;354:1–20. [PubMed]
24. Yamamoto Y, Murata K, Matsuda H, Hosoda T, Tamura K, Furuyama J. Determination of the complete nucleotide sequence and haplotypes in the D-loop region of the mitochondrial genome in the oriental white stork, Ciconia boyciana. Genes Genet Syst. 2000;75:25–32. [PubMed]

Articles from Journal of Veterinary Science are provided here courtesy of The Korean Society of Veterinary Science