J. vet. Pharmacol. Therap. 38, 429--433. doi: 10.1111/jvp.12212.

Identification of a nonsense mutation in feline ABCB1 K. L. MEALEY & N. S. BURKE Program in Individualized Medicine, Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, WA, USA

Mealey, K. L., Burke, N. S. Identification of a nonsense mutation in feline ABCB1. J. vet. Pharmacol. Therap. 38, 429–433. The aim of this study was to sequence all exons of the ABCB1 (MDR1) gene in cats that had experienced adverse reactions to P-glycoprotein substrate drugs (phenotyped cats). Eight phenotyped cats were included in the study consisting of eight cats that experienced central nervous system toxicosis after receiving ivermectin (n = 2), a combination product containing moxidectin and imidacloprid (n = 3), a combination product containing praziquantel and emodepside (n = 1) or selamectin (n = 2), and 1 cat that received the product containing praziquantel and emodepside but did not experience toxicity (n = 1). Fifteen exons contained polymorphisms and twelve exons showed no variation from the reference sequence. The most significant finding was a nonsense mutation (ABCB11930_1931del TC) in one of the ivermectin-treated cats. This cat was homozygous for the deletion mutation. All of the other phenotyped cats were homozygous for the wild-type allele. However, 14 missense mutations were identified in one or more phenotyped cats. ABCB11930_1931del TC was also identified in four nonphenotyped cats (one homozygous and three heterozygous for the mutant allele). Cats affected by ABCB11930_1931del TC would be expected to have a similar phenotype as dogs with the previously characterized ABCB1-1D mutation. (Paper received 8 October 2014; accepted for publication 16 January 2015) Katrina L. Mealey, Program in Individualized Medicine, Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, WA 99164-6610, USA. E-mail: [email protected]

INTRODUCTION ATP-binding cassette (ABC) transporters play an integral role in the disposition of substrate drugs. P-glycoprotein (P-gp) is the product of the ABCB1, formerly MDR1, gene and is arguably the most well-characterized ABC drug transporter. P-gp is expressed in a variety of tissues including the intestines, biliary canalicular cells, renal tubular cells, and brain capillary endothelial cells (Van Der Heyden et al., 2009) where it functions to actively extrude substrate drugs (Martinez et al., 2008). In this capacity, P-gp limits oral absorption, enhances biliary and renal excretion, and restricts central nervous system entry of substrate drugs. Much of what is known about P-gp’s role in drug disposition in dogs is a direct result of studies performed in dogs with a genetic defect in ABCB1. The canine MDR1 (ABCB1-1D) mutation consists of a four base pair deletion mutation that generates several premature stop codons within the first 10% of the coding region (Mealey et al., 2001). Dogs that are homozygous for this mutation (MDR1 mutant/ mutant) are P-gp null because they lack the four critical structural elements required for ABC transporters to function (two ATP binding domains and two substrate binding domains). © 2015 John Wiley & Sons Ltd

The critical role that P-gp plays as a component of the blood –brain barrier in dogs is dramatically illustrated by the exquisite neurological toxicity observed in dogs lacking P-gp function (MDR1 mutant/mutant) when treated with routine doses of ivermectin or loperamide (Mealey et al., 2008). P-gp’s protective role at the canine blood–brain barrier has also been demonstrated in experimental animals using the radiolabeled P-gp substrate 99Tc-sestamibi. Nuclear scintigraphy demonstrated significantly greater uptake of 99Tc-sestamibi in brain tissue of MDR1 mutant/mutant dogs compared to MDR1 normal/normal dogs (Mealey et al., 2008). Similarly, P-gp’s role in biliary excretion of substrate drugs in dogs has also been made evident. Biliary excretion of 99Tc-sestamibi was undetectable in MDR1 mutant/mutant dogs at all time points while its excretion in MDR1 normal/normal dogs was detected in the gallbladder within 30 min of injection (Coelho et al., 2009). Biliary excretion of 99Tc-sestamibi continued for several hours in MDR1 normal/normal dogs and was significantly greater than that of MDR1 mutant/mutant dogs at all time points. These studies illustrate that P-gp dysfunction increases the risk for serious adverse reactions to substrate drugs such as macrocyclic lactones, loperamide, vinca alkaloids, and others. 429

430 K. L. Mealey & N. S. Burke

Mutations that affect P-gp function have been identified in dogs (Mealey et al., 2001), mice (Lankas et al., 1997), and humans (Ieri et al., 2004) but have not been described for cats. The aim of the current study was to sequence the coding region of ABCB1 (MDR1) in cats that experienced adverse reactions to P-gp substrate drugs (phenotyped cats). MATERIALS AND METHODS DNA samples from phenotyped and nonphenotyped cats This study protocol was approved by Washington State University’s Institutional Animal Care and Use Committee. DNA was obtained from eight cats (Table 1) that developed central nervous system (CNS) toxicity after receiving ivermectin (n = 2), a combination of moxidectin and imidacloprid (n = 3), a combination of praziquantel and emodepside (n = 1) or selamectin (n = 2) at doses that are not expected to cause adverse reactions. DNA was also collected from one cat treated with the praziquantel-emodepside combination that did not develop CNS toxicity. This cat belonged to the same owner, was treated at the same time, and received the same dose as the cat that did develop CNS toxicity after receiving emodepside. DNA from an additional 105 cats (nonphenotyped) was obtained from the Washington State University College of Veterinary Medicine DNA Bank. Sequencing feline ABCB1 The genomic DNA reference sequence NC_018724 and cDNA reference sequence NM_001171064.2 (NCBI) were used to

design oligonucleotide primers and develop sequencing strategies for this study. Primers were designed to anneal in the flanking intron DNA sequences to amplify each exon. Sequence data were analyzed using Sequencher 5.2 software (Gene Codes Corporation, Ann Arbor, MI, USA). Genomic DNA was extracted from buccal swabs by alkaline lysis prep, or from whole blood using Quick-gDNA MiniPrep (Zymo Research, Irvine, CA, USA), and normalized to 20 ng/mL. Standard PCR amplifications were carried out using 20 ng genomic DNA template, 1.5 mM MgCl2, 0.32 mM each primer, 0.05 U/mL REDTaq Genomic DNA Polymerase (Sigma D2812, St. Louis, MO) in a final volume of 12.5 lL. Reactions were cycled 35 times at 94 °C for 30 sec, 50 °C for 30 sec for primer annealing, and 72 °C for 60 seconds for extension. The annealing temperature was optimized to accommodate individual primer pairs (Table 2). PCR products were visualized on a 1.2% agarose gel before treating the products with ExoSAP-IT reagent (Affymetrix, Cleveland, OH, USA) according to manufacturer’s directions. Alternately, a clean and concentrate-5 purification column (Zymo Research, Irivine, CA, USA) was used to purify PCR products prior to sequencing. Treated PCR products were sequenced with the same primers used for amplification using Big Dye 3.1 reagent mix (Applied Biosystems/Life Technologies, Grand Island, NY, USA). Cloning to confirm ABCB11930_1931del TC genotype After sequencing exon 15 from banked feline DNA samples, three cats appeared to have two sequences present starting at nucleotide 1930, suggesting a heterozygous genotype for ABCB11930_1931del TC. These PCR products were inserted

Table 1. Phenotyped cats including P-glycoprotein substrate drug, approximate dose, clinical signs observed after drug treatment, and potential causative mutation

Cat

Drug (approximate dose) route of dose

Clinical signs

1 2 3

Selamectin (6 mg/kg) applied topically Selamectin (6 mg/kg) applied topically Imidacloprid (10 mg/kg) + moxidectin (1 mg/kg) applied topically Imidacloprid (10 mg/kg) + moxidectin (1 mg/kg) applied topically

Obtunded Seizure Severe ataxia occurred the same day the product was administered Ataxia developed the same day product was administered. Additional signs included hyperesthesia, pupillary dilation and mild tremors Seizure 2 h after product was administered; progressed to severe CNS depression requiring a ventilator Ataxia, mydriasis, lethargy, temporary blindness CNS depression beginning 6 h after injection and lasting several days Ataxia developed the same day the product was administered. None

4

5

Imidacloprid (10 mg/kg) + moxidectin (1 mg/kg) applied topically†

6

Ivermectin (500 lg/kg)

7

Ivermectin (200 lg/kg) injected subcutaneously Emodepside (3 mg/kg) + praziquantel (12 mg/kg) applied topically Emodepside (3 mg/kg) + praziquantel (12 mg/kg) applied topically

8 9

Potential causative mutation (predicted effect on protein)* Exon 14 (Probably Damaging) None None Exon 14 (Probably Damaging)

None

Exon 5 (Possibly Damaging) Deletion Mutation?Stop codon Exon 5 (Possibly Damaging) Exon 5 (Possibly Damaging)

*PolyPhen-2 (http://genetics.bwh.harvard.edu/pph/). Cat may have ingested product applied to another cat in the household.



© 2015 John Wiley & Sons Ltd

Feline ABCB1 mutation 431 Table 2. Primers used to amplify feline ABCB1 Exon

Forward primer

Reverse primer

Product (bp)

1 2 3 4 5 6 7 8 9-11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

GAG GGC AAG CGA GGT GAG C AAA TAC AGG ATC TGT GG GAC CAG GTT GAT ATA GTA GTA CTA G TGC AAC AGC CAA CAG TGA AG TTA AGG ATG GAA AGA TAG CC TGA GAG TGG TTC AGT AGA GT GAG CAC TGG GTG TTG TAT GTA AG TGG AAG TTT TGC TAA ACA TA AGG AAG ATG TAT GGA AA CCT GGT TGG GAA CAG TGG GAA AGA TCA AGA TTC CTT CA CTA GGT GAT TAT CAT ATT TGT GT TTC AGC ATA TTC AGT GGC AA GAT AGC TCT ATT CTT TTA TCC C CCC TTT AGG AAC GAC AGA CG CTC CCC TGG CTA TGC GG ACA ATT AAA ATC ACA TTT GG TTG CTC TGA AGT CAT AGA AAT C CCA ATC TTC CTG CCA GAT CTA A AGG AGC AGT TAA TAC TCC ATC AG CTG CAA AGT CCC ATT ACA AG CTT TCT AGA TCA TGA GG GGA TCA GAA CGT GGG TAT CTT G ACC TGC TGG TTG TAA GAT GAG CAC ACT AGG ATT ATC TC

GGC CGT GGG AAG ATG TAA A AAA TAT AGG CCA AGA AG GTA TCT CTG CCA GCT CTT C CGC TAT ATC CTC CTA GAA AA CCA CTC TCA GAG GAC TTT GT TAT GAA ATC AGG GGA TGA GA GTC ACT ACC CAC CTA AGA CAT TC ACT CAA TAC ATC TAA TTG CC CGA AGA TAT CTC ACA TT GTT GAT GGT GCT AGA GCT TGA A TTA AAA AAT TCC CAA GTA CC GCT AAT GCC TAC TAT GGT GG GGG CAA GAG GAT TTA CAA GT CCT AAG ACA GCA ACC AAC CCC ACT TGG GCA TTC TCT C AGG GGA AAG AAG GAA AGA AA GCA CCC CTA CAA TTT TCT CT TAG TCG TCA GTC TGT GAG CC GCT TTC CAT CAC TTA CCT ACT C CCG AGT TAG GCT TCT TCT CAA A GGG TGG TAT TTC CAT CTC TAG GAT GTC AGA CTT TA AGG GAG AGC TAG TTG GGT AAT GAG CCC AAG TCA GAC ATT TAA C CTT CTA TTA TCT TTC AGC

593 561 535 395 530 609 620 637 874 545 595 584 690 650 513 582 625 561 529 639 596 714 674 748 718

into pCR2.1, TOPO (Life Technologies), grown in competent E coli cells, and plated so that individual colonies (clones) could be isolated. Plasmid DNA was extracted from individual colonies and sequenced to determine whether the cloned feline ABCB1 exon 15 contained the wild-type or deletion allele. The distribution of wild-type (3/6, 5/9 and 2/4) and ABCB11930_1931del TC deletion (3/6, 4/9 and 2/4) alleles in cloned samples from each of these cats was approximately 50% confirming a heterozygous genotype.

RESULTS All 27 exons (3840 bp) of feline ABCB1 from eight phenotyped cats were compared to the reference feline ABCB1 sequence (NC_018724). Twelve exons (1, 2, 4, 8, 9, 10, 17, 18, 21, 22, 25, and 27) showed no variation from the reference sequence. Single nucleotide polymorphisms (SNPs) were detected in 15 exons (3, 5, 6, 7, 11, 12, 13, 14, 15, 16, 19, 20, 23, 24, and 26) compared to the reference sequence, with 16 being synonymous (Table 3) and 14 being nonsynonymous mutations (Table 4). Among the 14 nonsynonymous SNPs identified, 12 were predicted to be ‘Benign’, while one in exon 5 was predicted to be ‘Possibly Damaging’ and another in exon 14 was predicted to be ‘Probably Damaging’ to protein structure and function based on a software toola designed to predict damaging missense mutations (Adzhubei et al., 2010). a

PolyPhen-2. http://genetics.bwh.harvard.edu/pph2/index.shtml.

© 2015 John Wiley & Sons Ltd

Two phenotyped cats were affected by the ‘Probably Damaging’ SNP in exon 14 (Table 1). One of these cats had developed CNS toxicity after treatment with selamectin while the other had developed CNS toxicity after treatment with the combination of imidacloprid and moxidectin. Interestingly, another cat that developed CNS toxicity after treatment with selamectin and the other two cats that developed CNS toxicity after treatment with the combination of imidacloprid and moxidectin did not harbor the ‘Probably Damaging’ SNP or any other mutation in ABCB1 that would explain their adverse reactions (Table 1). Three different phenotyped cats were affected by the ‘Possibly Damaging’ SNP in exon 5 (Table 1). Two of these cats developed CNS toxicity after treatment with either ivermectin or the combination of emodepside and praziquantel. However, another phenotyped cat harboring this SNP did not develop CNS toxicity after treatment with emodepside and praziquantel (Table 1). The most significant ABCB1 mutation identified in this study consisted of a deletion mutation identified in only one phenotyped cat (one that experienced CNS toxicity after treatment with ivermectin). This cat was homozygous for a 2-bp deletion (ABCB11930_1931del TC; GenBank KP269163) within exon 15 (Table 1) with numbering based on the predicted open reading frame. The resulting frameshift generates a series of stop codons immediately downstream from the deletion, producing a severely truncated (~50%) P-gp that would be nonfunctional. To further investigate the frequency of ABCB11930_1931del TC in cats, 100 feline DNA samples from the Washington State

432 K. L. Mealey & N. S. Burke Table 3. Feline ABCB1 synonymous variants Exon

Nucleotide position(cDNA)*

Reference nucleotide

Variant nucleotide

1308 1329 1398 1579 1635 1899 1995 2022 2025 2184 2193 2460 2682 2949 3153 353C

G C T A G T A G A T T C G C A T

A G G C T C G A G C A T C T C C

11 11 12 13 13 15 15 15 15 16 16 19 20 23 24 26

*Numbered based on predicted open reading frame.

University College of Veterinary Medicine DNA Bank were used to amplify and sequence exon 15. Four additional cats harbored ABCB11930_1931del TC, with one being homozygous and three being heterozygous for ABCB11930_1931del TC. DNA from cats that appeared to be heterozygous based on initial sequencing underwent cloning and sequencing to confirm the genotype. ABCB11930_1931del TC was present in a total of 6 of 108 cats genotyped, or 5/100 (5%) of a nonbiased selection of feline DNA samples.

DISCUSSION While sensitivity to macrocyclic lactones in dogs has been well-described, there is no information regarding macrocyclic lactone sensitivity in cats. Sensitivity to macrocyclic lactones

in dogs was initially described in the 1980s primarily in herding breed dogs (Paul et al., 1987). In 2001, the genetic cause of ‘ivermectin sensitivity’ was determined to be a 4-base pair deletion mutation in the ABCB1 gene (Mealey et al., 2001). This mutation generates premature stop codons resulting in a truncated and nonfunctional protein product. The mutation has been identified in herding breed dogs and mixed breeds that have herding breed ancestry (Neff et al., 2004). Dogs with the MDR1 deletion mutation are sensitive not only to macrocyclic lactones but also to other P-gp substrate drugs including loperamide, vinca alkaloids, emodepside, and others (Martinez et al., 2008; Mealey et al., 2008). The Veterinary Clinical Pharmacology Laboratory at Washington State University, which offers MDR1 genotyping for dogs, occasionally receives inquiries regarding MDR1 testing in other species, particularly when an animal has experienced CNS toxicity after treatment with a P-gp substrate drug. This report describes several polymorphisms in feline ABCB1 in cats that had experienced adverse neurological reactions to drugs that are known to be P-gp substrates. Although several SNPs were identified, the majority were predicted to be have a benign effect on P-glycoprotein structure and function. One SNP, in exon 14, was predicted to be ‘Probably Damaging’ and one SNP, in exon 5 was predicted to be ‘Possibly Damaging’. The exon 14 SNP resulted in an amino acid change from arginine (polar; positive charge) to serine (polar; neutral). This SNP occurred in 1 of 2 selamectin-treated cats and 1 of 3 cats treated with the combination of imidacloprid and moxidectin. Whether or not this SNP impacted P-gp expression or function in the affected cats, and therefore caused increased sensitivity to the P-gp substrate moxidectin, is unknown. The exon 5 SNP resulted in an amino acid change from alanine (nonpolar) to glutamate (polar; negatively charged). This SNP occurred in 1 of 2 ivermectin-treated cats and 2 of 2 emodepside-treated cats (one that developed CNS toxicity after treatment with the combination of imidacloprid and moxidectin and one cat that did not experience an adverse

Table 4. Feline ABCB1 nonsynonymous single nucleotide polymorphisms Exon 3 3 5 6 7 12 14 15 15 15 15 15 16 16

Nucleotide position (cDNA)

Reference nucleotide

Variant nucleotide

Amino acid change

Predicted impact on protein structure*

140 266 416 589 727 1465 1762 1891 1935 1936 2000 2045 2072 2194

C C C C G C C A A A C A A T

A A A A A T A G G G T T G C

Phenylananine to Leucine Threonine to Asparagine Alanine to Glutamate Methionine to Leucine Lysine to Glutamate Arginine to Cysteine Arginine to Serine Arginine to Glycine Isoleucine to Methionine Serine to Arginine Serine to Leucine Histidine to Leucine Histidine to Arginine Phenylalanine to Leucine

Benign Benign Possibly Damaging Benign Benign Benign Probably Damaging Benign Benign Benign Benign Benign Benign Benign

*PolyPhen-2 (http://genetics.bwh.harvard.edu/pph/). © 2015 John Wiley & Sons Ltd

Feline ABCB1 mutation 433

reaction). Whether or not this SNP impacted P-gp expression or function in the affected cats, and therefore caused increased sensitivity to the P-gp substrate emodepside, is unknown. This seems unlikely as both cats treated with the combination of emodepside and praziquantel harbored the exon 5 SNP but only one developed CNS toxicity. The most significant finding consisted of a two base pair deletion (ABCB11930_1931del TC) within exon 15 that results in a frameshift generating a series of stop codons immediately downstream from the deletion. The protein product would be severely truncated (~50%) and nonfunctional. Although some ABC transporters can function as half transporters, this has not been shown to be the case with ABCB1. This mutation occurred in a cat that developed progressive CNS depression beginning 6 h after treatment with ivermectin (200 lg/kg subcutaneously) and lasting for several days. Because of the nature of this polymorphism (nonsense mutation) and the fact that the cat was homozygous for the mutation, it is reasonable to predict that it was causative for the affected cat’s apparent sensitivity to ivermectin. None of the other phenotyped cats harbored this polymorphism. However, the frequency of ABCB11930_1931del TC in nonphenotyped cats (n = 100) revealed that five additional cats harbored ABCB11930_1931del TC, with one being homozygous and four being heterozygous for ABCB11930_1931del TC. Thus, ABCB11930_1931del TC was present in a total of 6 of 108 cats genotyped, or 5/100 (5%) of a nonbiased selection of feline DNA samples. Further studies would be necessary to determine the exact nature of ABCB11930_1931del TC on ABCB1 transport function. One reason that not all cats shared a consistent genotype may be that their phenotypes were also not uniform. Substantial evidence exists that ivermectin can cause severe neurological toxicity in animals with defective P-gp at the doses that the cats in this study received. There is less evidence to support this type of cause-and-effect relationship for selamectin, imidacloprid/moxidectin, and emodepside/praziquantel. In fact, dogs with defective P-gp function (MDR1 mutant/mutant) exhibit no neurological toxicity after treatment with the same doses of selamectin and imidacloprid/moxidectin that the cats in this study received. Emodepside, on the other hand, has been shown to cause neurological toxicity in P-gp-deficient mice (Elmshauser et al., 2015). Whether or not the neurological clinical signs displayed by the cat reported in this study were related to P-gp deficiency is not known. Based on data in the literature, the vast majority of dogs that are sensitive to P-gp substrate drugs appear to be affected by a single mutation (the MDR1 mutation or ABCB1-1D). Conversely, there may be multiple causes in cats as a mutation common to all cats in this study that developed CNS toxicity after treatment with ivermectin was not identified. Cats that are affected by ABCB11930_1931del TC would be expected to have a similar phenotype as dogs with the previously characterized ABCB1-1D mutation. Specifically, these cats would be expected to show enhanced susceptibility to adverse effects of P-gp substrate drugs including macrocyclic lactones, vinca © 2015 John Wiley & Sons Ltd

alkaloids, doxorubicin, loperamide, and others. Whether or not cats that are heterozygous for ABCB11930_1931del TC would be expected to have an intermediate drug sensitivity phenotype compared to cats homozygous for the deletion mutation is not known. Further studies investigating the association of ABCB11930_1931del TC with adverse reactions to P-gp substrate drugs in cats are warranted.

ACKNOWLEDGMENTS Funding for this study provided by the Ott Endowment, College of Veterinary Medicine, Washington State University. The Washington State University College of Veterinary Medicine DNA Bank provided samples for this study.

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Identification of a nonsense mutation in feline ABCB1.

The aim of this study was to sequence all exons of the ABCB1 (MDR1) gene in cats that had experienced adverse reactions to P-glycoprotein substrate dr...
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