Development of an oligonucleotide microarray for simultaneous detection of two canine MDR1 genotypes and association between genotypes and chemotherapy side effects

Abstract

Canine MDR1 gene mutations produce translated P-glycoprotein, an active drug efflux transporter, resulting in dysfunction or over-expression. The 4-base deletion at exon 4 of MDR1 at nucleotide position 230 (nt230[del4]) in exon 4 makes P-glycoprotein lose function, leading to drug accumulation and toxicity. The G allele of the c.-6-180T＞G variation in intron 1 of MDR1 (single nucleotide polymorphism [SNP] 180) causes P-glycoprotein over-expression, making epileptic dogs resistant to phenobarbital treatment. Both of these mutations are reported to be common in collies. This study develops a more efficient method to detect these two mutations simultaneously, and clarifies the genotype association with the side effects of chemotherapy. Genotype distribution in Taiwan was also investigated. An oligonucleotide microarray was successfully developed for the detection of both genotypes and was applied to clinical samples. No 4-base deletion mutant allele was detected in dogs in Taiwan. However, the G allele variation of SNP 180 was spread across all dog breeds, not only in collies. The chemotherapy adverse effect percentages of the SNP 180 T/T, T/G, and G/G genotypes were 16.7%, 6.3%, and 0%, respectively. This study describes an efficient way for MDR1 gene mutation detection, clarifying genotype distribution, and the association with chemotherapy.

Keywords: Canine; Chemotherapy; MDR1 gene; Oligonucleotide microarray; P-glycoprotein.

Fig. 1. Separation of MDR1 genotypes was tested by employing synthesized standard sequences and electrophoresis gels. (A and B) The 4-base deletion genotypes were detected by using 5% agarose gel and 12% polyacrylamide gel electrophoresis gel and 8 h electrophoresis, respectively. Lane M, marker; Lane 1, 4-base no-deletion wild-type genotype (WW genotype); Lane 2, 4-base deletion genotype (MM genotype); Lane 3, hybrid genotype (WM genotype); Lane 4, negative control. (C) The SNP 180 genotypes were detected by using 2% agarose gel and 40-min electrophoresis. Lane M, marker; Lane 1, T/T genotype; Lane 2, G/G genotype; Lane 3, T/G genotype; Lane 4, negative control. (D) Simultaneous detection of the 4-base deletion genotypes and SNP 180 genotypes of MDR1 using multiplex polymerase chain reaction (PCR). Lane M, marker; Lane 1, SNP 180 T/G genotype; Lane 2, 4-base deletion WM genotype; Lane 3, SNP 180 T/G and 4-base deletion WM genotypes; Lane 4, negative control. (E) Lane M, marker; Lanes 1–3, multiplex PCR results from three randomly-selected healthy dogs; Lane 4, negative control. Del 4, a 4-base deletion at exon 4 of the MDR1 gene at nucleotide position 230 (nt230[del4]); SNP, single nucleotide polymorphism.

Fig. 2. (A) Microarray map. VP1, positive control. The designation of each probe is shown in Table 2. Both the 4-base deletion and SNP 180 genotypes of MDR1 were clearly identified using an oligonucleotide microarray system and either the synthesized standard sequences (B) or clinical dog blood samples (C). WW, WM, and MM indicate wild-type homozygote, heterozygote, and homozygote 4-base deletion mutation genotype of MDR1, respectively. T/T, T/G, and G/G indicate allele-specific genotypes of SNP 180.

Fig. 3. The relevance of three SNP 180 genotypes to neutrophil number (A), thrombocyte number (B), and hemoglobin concentration (C) in blood from sampled dogs. SNP, single nucleotide polymorphism.