High Frequency of a Single Nucleotide Substitution (c.-6-180T>G) of the Canine MDR1/ABCB1 Gene Associated with Phenobarbital-Resistant Idiopathic Epilepsy in Border Collie Dogs
A single nucleotide substitution (c.-6-180T>G) associated with resistance to phenobarbital therapy has been found in the canine MDR1/ABCB1 gene in Border Collies with idiopathic epilepsy. In the present study, a PCR-restriction fragment length polymorphism assay was developed for genotyping this mutation, and a genotyping survey was carried out in a population of 472 Border Collies in Japan to determine the current allele frequency. The survey demonstrated the frequencies of the T/T wild type, T/G heterozygote, and G/G mutant homozygote to be 60.0%, 30.3%, and 9.8%, respectively, indicating that the frequency of the mutant G allele is extremely high (24.9%) in Border Collies. The results suggest that this high mutation frequency of the mutation is likely to cause a high prevalence of phenobarbital-resistant epilepsy in Border Collies. 1. Introduction Recently, a single nucleotide substitution associated with phenobarbital-resistant idiopathic epilepsy was found in Border Collies [1], which frequently present with severe epileptic seizures that are poorly controlled with antiepileptic drugs [2]. This polymorphic mutation is a substitution of thymine for guanine at intron 1 near the 5′-end of the canine MDR1/ABCB1 gene (c.-6-180T>G), where the most important promoter elements are located [3]. The mutation is not directly associated with the pathogenesis of idiopathic epilepsy, but it may create resistance to phenobarbital therapy in epileptic Border Collies [1]. However, the c.-6-180T>G mutation has been shown not to be a fully penetrant polymorphic mutation for phenobarbital nonresponsiveness because phenobarbital-resistant, idiopathically epileptic Border Collies in a previous case control study included a homozygous T/T wild-type dog [1]. On the basis of data from a relatively small cohort of Border Collies, it was hypothesized that the mutation might be related to an upregulation of the gene and an overexpression of P-glycoprotein (P-gp) encoded by the MDR1/ABCB1 gene. Therefore, further studies are required to understand the regulatory effect of the mutation and its potential clinical relevance. For these purposes, an accurate method of genotyping for the mutation should be developed, and the frequency of the mutation should be determined in a large, normal population of dogs. In the present study, a PCR-restriction fragment length polymorphism (RFLP) assay was developed in order to discriminate the genotypes of the mutation, and a genotyping survey was conducted in Japan by using samples from clinically healthy Border Collies in order to determine
References
[1]
L. Alves, V. Hülsmeyer, A. Jaggy, A. Fischer, T. Leeb, and M. Dr?gemüller, “Polymorphisms in the ABCB1 gene in phenobarbital responsive and resistant idiopathic epileptic Border Collies,” Journal of Veterinary Internal Medicine, vol. 25, no. 3, pp. 484–489, 2011.
[2]
V. Hülsmeyer, R. Zimmermann, C. Brauer, C. Sauter-Louis, and A. Fischer, “Epilepsy in Border Collies: clinical manifestation, outcome, and mode of inheritance,” Journal of Veterinary Internal Medicine, vol. 24, no. 1, pp. 171–178, 2010.
[3]
H. Takane, D. Kobayashi, T. Hirota et al., “Haplotype-oriented genetic analysis and functional assessment of promoter variants in the MDR1 (ABCB1) gene,” Journal of Pharmacology and Experimental Therapeutics, vol. 311, no. 3, pp. 1179–1187, 2004.
[4]
K. Mizukami, H.-S. Chang, A. Yabuki et al., “Rapid genotyping assays for the 4-base pair deletion of canine MDR1/ABCB1 gene and low frequency of the mutant allele in Border Collie dogs,” Journal of Veterinary Diagnostic Investigation, vol. 24, no. 1, pp. 127–134, 2012.
[5]
J. A. Johnson, “Pharmacogenetics in clinical practice: how far have we come and where are we going?” Pharmacogenomics, vol. 14, pp. 835–843, 2013.
[6]
H. Bakhouche and O. Slana?, “Pharmacogenetics in clinical practice,” Prague Medical Report, vol. 113, no. 4, pp. 251–261, 2012.
[7]
C. M. Mosher and M. H. Court, “Comparative and veterinary pharmacogenomics,” Handbook of Experimental Pharmacology, vol. 199, pp. 49–77, 2010.
[8]
K. Mizukami, H.-S. Chang, A. Yabuki et al., “Novel rapid genotyping assays for neuronal ceroid lipofuscinosis in Border Collie dogs and high frequency of the mutant allele in Japan,” Journal of Veterinary Diagnostic Investigation, vol. 23, no. 6, pp. 1131–1139, 2011.
[9]
K. Mizukami, A. Yabuk, T. Kawamichi et al., “Real-time PCR genotyping assay for canine trapped neutrophil syndrome and high frequency of the mutant allele in Border Collies,” The Veterinary Journal, vol. 195, pp. 260–261, 2013.
[10]
P. Quignon, L. Herbin, E. Cadieu et al., “Canine population structure: assessment and impact of intra-breed stratification on SNP-based association studies,” PLoS ONE, vol. 2, no. 12, Article ID e1324, 2007.
[11]
H. A. Volk, D. Arabadzisz, J.-M. Fritschy, C. Brandt, K. Bethmann, and W. L?scher, “Antiepileptic drug-resistant rats differ from drug-responsive rats in hippocampal neurodegeneration and GABA A receptor ligand binding in a model of temporal lobe epilepsy,” Neurobiology of Disease, vol. 21, no. 3, pp. 633–646, 2006.
[12]
A. H. Schinkel, “The physiological function of drug-transporting P-glycoproteins,” Seminars in Cancer Biology, vol. 8, no. 3, pp. 161–170, 1997.
[13]
F. Thiebaut, T. Tsuruo, H. Hamada, M. M. Gottesman, I. Pastan, and M. C. Willingham, “Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues,” Proceedings of the National Academy of Sciences of the United States of America, vol. 84, no. 21, pp. 7735–7738, 1987.
[14]
M. F. Fromm, “P-glycoprotein: a defense mechanism limiting oral bioavailability and CNS accumulation of drugs,” International Journal of Clinical Pharmacology and Therapeutics, vol. 38, no. 2, pp. 69–74, 2000.
[15]
K. L. Mealey, S. A. Bentjen, J. M. Gay, and G. H. Cantor, “Ivermectin sensitivity in Collies is associated with a deletion mutation of the mdr1 gene,” Pharmacogenetics, vol. 11, no. 8, pp. 727–733, 2001.