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