Personalised medicine in veterinary practice: the paradigmatic example of canine ABCB1

Pharmacogenetics (PG) is the study of variations in DNA sequence as related to drug response. Close relationships between DNA variants and ADME or drug target genes exist, and may be actionable in the clinic, ultimately resulting in drug phenotype predictions in patients (personalised medicine concept). Variations in drug response (unexpected toxicity or therapeutic failure) occurs also in veterinary species, and most of the available knowledge refers to dogs. However, canine PG lags behind human one, for a number of scientific and economic reasons. Most commonly described canine pharmacogenes are cytochromes P450, drug transporters (e.g., ABCB1), and intended or unintended drug targets (e.g., β1 adrenergic receptor and KIT protooncogene) [1-2]. In the present study, we developed an in-house protocol for canine ABCB1 mutational analysis; in particular, the ABCB1-1Δ four base-pair deletion, resulting in a premature stop codon and non-functional P-glycoprotein (P-gp) [2]. The protocol, an adaptation of a previously published one [3], essentially consists in a quantitative Real time PCR assay and the use of TaqManTM probes. Genomic DNA from whole blood was isolated using the DNeasy Blood & Tissue kit (Qiagen). For the correct interpretation of genotyping data, 15 reference samples (5 ABCB1 WT/WT, 5 WT/mut, and 5 mut/mut), kindly provided by Prof. Joachim Geyer (Justus Liebig University of Giessen, Germany), were used. Moreover, we cloned and sequenced ABCB1 and ABCB1-1Δ from two dogs with a known genotype, in order to obtain the reference sequences for bioinformatic analysis. Finally, the assay was tested in a cohort of 130 blood samples from different dog breeds and coming from different clinical practices. We never observed the ABCB1-1Δ mutation in Border Collie dogs. However, ABCB1 mut/mut (2/4, 50%) and ABCB1 WT/mut (1/4, 25%) genotypes were identified in Rough Collies. In Australian Sheperd dogs, only the heterozygous ABCB1 WT/mut genotype (7/17, 41.18%) was observed. Cross-bred and mixed Collie dogs resulted negative. Interestingly, a case report occurred during the trial. Specifically, a Rough Collie healthy dog, given selamectin spot-on for parasites control, was IM administered acepromazine before induction of inhalatory anesthesia for tartar removal. The animal showed increased depth and duration of sedation. According to the observed clinical symptoms and breed-predisposition, the dog was genotyped and found ABCB1-1Δ mut/mut positive. In conclusion, a PG assay to detect ABCB1-1Δ mutation was set up, validated and proved reliable, rapid, and cheap. At present, we are increasingly using this assay to help the clinician to adopt the best therapeutic approach whenever the use of P-gp substrate drugs is envisaged. On a wider scenario, future advances in canine and veterinary PG will improve the information on drug individual susceptibility (i.e., efficacy or toxicity), further improving animal health and well-being.