Haplotype analysis of the MDR1 flanking region in the dog breed Elo

A deletion mutation in the canine multidrug resistance (MDR1) gene provokes drug sensitivity in several dog breeds from the Collie lineage. A haplotype of four microsatellites containing this mdr1-1Delta mutation was conserved among affected breeds. In this study, we analysed the haplotypes of the MDR1 flanking region of 177 dogs of the breed Elo which is composed of several dog breeds including the Old English sheepdog from the Collie lineage. We detected a haplotype in the Elo breed which had previously been associated with the mutant mdr1-1Delta allele in Old English sheepdogs. Using a regression analysis for the probability of the haplotype on the proportion of genes of the founder breeds, we could exclude the Old English sheepdog as origin of this haplotype for the Elo breed. The MDR1 flanking region could be traced back to the Japanese Spitz as one of the founder dog breeds of the Elo and thus, the introgression of the mdr1-1Delta mutation into the dog breed Elo through the Collie lineage is very unlikely.     

Frequency of the nt230 (del4) MDR1 mutation in Collies and related dog breeds in Germany

MDR1 (ABCB1) P-glycoprotein exerts a protective function in the blood–brain barrier thereby limiting the entry of many drugs and other xenobiotics to the central nervous system. A nonsense mutation has been described for Collies and related dog breeds which abolishes this function and is associated with increased susceptibility to neurotoxic side effects of several drugs including ivermectin, moxidectin and loperamide. In order to evaluate the occurrence and frequency of this nt230 (del4) MDR1 mutation in Germany, we screened 1500 dogs. Frequency of the homozygous mutated genotype was highest for Collies (33.0%), followed by Australian Shepherd (6.9%) and Shetland Sheepdog (5.7%). Thirty-seven percent of the Wäller dogs and 12.5% of the Old English Sheepdogs were heterozygous for the mutant MDR1 (−) allele. Considering the predominant role of MDR1 P-glycoprotein in drug disposition and in particular for blood–brain barrier protection, MDR1 genotype-based breeding programs are recommended for improving the safety of drug therapy in these canine breeds.     

Review of prevalence, genetic aspects and adverse effects of the mdr1-1Delta mutation in dogs

A mutation in the canine MDR1 gene causes multiple drug sensitivity in dog breeds of the Collie lineage. Dogs with this genetic defect show severe neurotoxic adverse effects if they are treated with particular drugs. Clinical signs depending on the administered drug and its concentration vary from mild toxicosis with salivation and disorientation to severe effects with coma and finally death of the dog. Drugs which provoke adverse effects are structurally different. Although they are used for many different indications, all of these drugs are substrates of a transporting protein encoded by the MDR1 gene.This P-glycoprotein loses its normal protecting function at the tissue barriers in dogs with the mdrl-1Delta mutation.This article gives a short overview about the present state of analyses regarding the canine MDR1 gene.The genetic background, effects and prevalence in affected dog breeds of the mdrl-1Delta mutation are summarized. On the one hand, the overview might help practical veterinarians to understand the aetiology of drug sensitivity in dogs with the mdrl-1Delta mutation, and on the other hand, it might point out appendages for future research works about the canine MDR1 gene as well as for breeding strategies in affected dog breeds.     

Masitinib reverses doxorubicin resistance in canine lymphoid cells by inhibiting the function of P‐glycoprotein

Overexpression of ABC‐transporters including Pgp, MRP1, and BCRP has been associated with multidrug resistance (MDR) in both human and canine oncology. Therapeutic interventions to reverse MDR are limited, but include multidrug protocols and the temporary concomitant use of inhibitors of ABC‐transporters. Recently, the use of tyrosine kinase inhibitors has been proposed to overcome MDR in human oncology. One of the tyrosine kinase inhibitors, masitinib, is licensed for veterinary use in the treatment of canine mast cell tumors. Therefore, this study aimed to assess the potential of masitinib to revert MDR in canine malignant lymphoma using an in vitro model with canine lymphoid cell lines. Masitinib had a mild antiproliferative effect on lymphoid cells, inhibited Pgp function at concentrations equal to or exceeding 1 μm and was able to reverse doxorubicin resistance. The current findings provide the rationale for a combined use of masitinib with doxorubicin in the treatment of dogs with doxorubicin‐resistant malignant lymphoma but await confirmation in clinical trials.     

Canine mdr1 gene mutation in Japan

Frequency of the 4-bp deletion mutant in canine mdr1 gene was examined in 193 dogs of eight breeds in Japan. The mutant allele was found in Collies, Australian Shepherds, and Shetland Sheepdogs, where its respective frequencies were 58.3%, 33.3%, and 1.2%. The MDR1 protein was detected on peripheral blood mononuclear cells (PBMC) from a MDR1/MDR1 dog, but not on PBMC from a mdr1-1Delta/mdr1-1Delta Collie. Rhodamine 123 was extruded from MDR1/MDR1 lymphocytes. That excretion was inhibited by a MDR1 inhibitor, verapamil. On the other hand, Rh123 excretion was not observed from lymphocytes derived from a mdr1-1Delta/mdr1-1Delta Collie. These results indicated that the mutant mdr1 allele also existed in Collie-breed dogs in Japan at high rates and that mdr1-1Delta /mdr1-1Delta dogs have no functional MDR1.     

Analysis of the canine mdr1-1Delta mutation in the dog breed Elo

A deletion mutation in the canine multidrug resistance gene, MDR1, is associated with drug sensitivity. This was shown for several purebred dog breeds from the Collie lineage such as the Collie (rough-coated and smooth-coated), the Australian Shepherd and the Old English sheepdog. To determine whether the mdr1-1Delta mutation could be found in the newly bred German dog breed Elo which is based amongst other breeds on Old English sheepdogs, 177 blood samples representative for the Elo breed were collected. After DNA extraction, a polymerase chain reaction-based method with subsequent polyacrylamide gel electrophoresis was used for detection of the mdr1-1Delta mutation. The mdr1-1Delta allele was not observed in the Elos investigated. The probability that the mdr1-1Delta allele originated in the Old English sheepdog breed is segregating in the Elo population was estimated at 3.68 x 10(-17).     

Frequency of the mutant MDR1 allele associated with multidrug sensitivity in a sample of herding breed dogs living in Australia

A study was performed to determine the frequency of the mutant MDR1 allele associated with ivermectin sensitivity in a sample of Collies and other herding breeds living in Australia. Buccal swab samples were collected from 33 Collies, 17 Australian Shepherds, 7 Border Collies and 7 Shelties for determination of MDR1 genotype. DNA was extracted and the polymerase chain reaction was performed to amplify a 148 base pair (wildtype MDR1 genotype or 144 base pair (mutant MDR1 genotype) amplicon containing the MDR1 mutation. Sequence analysis was performed to determine the genotype of each dog. Adequate quantities of DNA for unequivocal genotyping were obtained from 61 of 64 samples. The previously described MDR1 mutation was identified in Collies, Australian Shepherds and Shelties living in Australia, but not in Border Collies (although sample numbers were low). Twelve percent (4/33) of the Collies studied were homozygous for the normal allele (normal), 64% (21/33) were heterozygous (carrier) and 24% (8/33) were homozygous for the mutant allele (affected). Results of this study indicate that a high percentage of herding breeds presenting to veterinarians in Australia harbor the MDR1 mutation, thus impacting some therapeutic decisions.     

Increased toxicity of P-glycoprotein-substrate chemotherapeutic agents in a dog with the MDR1 deletion mutation associated with ivermectin sensitivity

Lymphoma was diagnosed in a 4-year-old spayed female Collie, and treatment with a combination chemotherapy protocol incorporating prednisone, L-asparaginase, vincristine, vinblastine, doxorubicin, and cyclophosphamide was initiated. The dog had signs of gastrointestinal tract toxicosis and myelosuppression after treatment with P-glycoprotein-substrate drugs (vincristine, vinblastine, and doxorubicin) even when dosages were reduced, but did not have signs of toxicosis after treatment with cyclophosphamide, a non-P-glycoprotein-substrate drug, even when administered at the full dosage. It was postulated that a deletion mutation in the canine MDR1 gene (deltaMDR1 295-298) could be responsible for the drug toxicoses in this dog. This mutation has been identified as the cause of a functional P-glycoprotein defect in Collies susceptible to the toxic effects of ivermectin, another P-glycoprotein-substrate drug. The MDR1 genotype of this dog consisted of 1 normal and 1 mutant MDR1 allele. Because P-glycoprotein contributes to renal, biliary, and intestinal excretion of P-glycoprotein-substrate drugs, it is possible that drug excretion was delayed in this patient, resulting in clinical signs of toxicosis.     

Collie eye anomaly in Hokkaido dogs: case study

Objective To describe a Hokkaido dog, one of the traditional Japanese breeds that was affected by Collie eye anomaly (CEA), and to report the genotype of this dog and the Hokkaido dog allelic frequency of the CEA‐associated mutation. Case A nine‐month‐old intact female Hokkaido dog without any obvious visual disturbance was diagnosed ophthalmoscopically with CEA. Severe choroidal hypoplasia was observed in the bilateral temporal area adjacent to the optic nerve head, appearing as whitish areas. Therefore, the dog was suspected of possessing the CEA‐associated mutation that was previously reported as an intronic 7.8‐kilo base deletion in the canine NHEJ1 gene. Procedures SYBR Green‐based real‐time PCR with a melting curve analysis, conventional PCR with agarose gel electrophoresis, and direct DNA sequencing were carried out to determine the genotype of the dog. Furthermore, a preliminary genotyping survey was carried out in 17 Hokkaido dogs from three kennels using the real‐time PCR method, and the pedigree relationships were analyzed using their pedigree papers. Results The Hokkaido dog affected by CEA was proven to possess the CEA‐associated mutation. Of these 17 Hokkaido dogs, 12 dogs were heterozygous carriers and five dogs were affected by this mutation. The preliminary genotyping survey and pedigree analysis demonstrated that the allelic frequency of the CEA‐associated mutation is very high in Hokkaido dogs. Conclusion These data suggest that the Hokkaido breed is highly susceptible to CEA because of the known CEA‐associated mutation much like the Collie‐related breeds.     

Development of a PCR-based diagnostic test detecting a nt230(del4) MDR1 mutation in dogs: verification in a moxidectin-sensitive Australian Shepherd

A subpopulation of dogs of the Collie and Australian Shepherd breeds show increased sensitivity to central nervous actions of ivermectin, doramectin, loperamide, and probably several other drugs. The molecular background for this greater sensitivity is a nonsense mutation in the MDR1 efflux pump, which is part of the functional blood-brain barrier and normally limits drug penetration into the brain. This report describes a rapid PCR-based method for detection of this nt230(del4) MDR1 mutation using a small amount of genomic DNA from blood cells. Thereby, homozygous intact, homozygous mutated, and heterozygous mutated MDR1 genotypes can be clearly differentiated by high resolution polyacrylamide gel electrophoresis. Using this diagnostic test two Collies and one Australian Shepherd were screened for the nt230(del4) MDR1 mutation. The Collies had no history of altered drug sensitivity and showed homozygous intact and heterozygous mutated MDR1 alleles, respectively. However, the Australian Shepherd developed clear signs of neurotoxicity including ataxia, crawling, acoustic and tactile hyperexcitability, and miosis after a single dose of moxidectin (400 microg/kg). For this dog two mutated MDR1 alleles were detected. This report describes for the first time moxidectin neurotoxicosis in a dog with a homozygous MDR1 mutation.     

Familial canine pituitary-dependent hyperadrenocorticism in wirehaired Dachshunds

Canine pituitary hyperadrenocorticism (Cushing's disease) caused by neoplasia of the corticotrope cells is one of the most common endocrine diseases especially in smaller dog breeds. Cushing's disease was diagnosed in eleven wire-haired Dachshunds and for further six wire-haired Dachshunds Cushing's disease was suspected on the basis of clinical signs. A joined pedigree could be ascertained for all these 17 dogs. Eleven of these dogs were so closely related to each other, that they were summarized in four nucleus families. Two fullsiblings were examined by means of clinical, laboratory diagnostic and morphological methods. The main lesions consisted of atrophic dermatosis with alopecia, increase of activity of liver enzymes in plasma and bilateral adrenocortical hyperplasia and therefore corresponded to the typical signs of a secondary hyperadrenocorticism. A rather unusual finding was the pituitary carcinoma in one of these dogs. Similarly to human patients affected by hyperadrenocorticism, real-time PCR analysis showed a 2.9-fold increase of expression of the canine MDR1 gene in the liver of one affected wirehaired Dachshund. This study documents the first familial occurrence of pituitary-dependent hyperadrenocorticism in wirehaired Dachshunds, the overexpression of the MDR1 gene in the dog and the third case of familial hyperadrenocorticism in dogs ever described.     

Evaluation of canine heat shock transcription factor 4 (HSF4) as a candidate gene for primary cataracts in the Dachshund and the Entlebucher Mountain dog

OBJECTIVE: Testing of the cataract-causing insertion/deletion mutation in the canine HSF4 gene for its linkage and association with primary cataracts (CAT) in Dachshunds and Entlebucher Mountain dogs. MATERIALS: Exon 9 with flanking intronic regions of the canine HSF4 gene was sequenced in 24 Dachshunds and 20 Entlebucher Mountain dogs. The HSF4 cDNA sequence of lens tissue was analyzed in a CAT-unaffected mixed-breed dog and in three CAT-affected dogs of different breeds, including a Wire-haired Dachshund, a Dachshund-mix and a German Shepherd dog. RESULTS: In all dogs investigated here, the previously reported CAT-causing mutation did not exist. We found a single nucleotide polymorphism (SNP) in intron 9, which was neither associated nor linked with the CAT phenotype in the two dog breeds. CONCLUSION: The CAT phenotype in the two dog breeds investigated here was not caused by the same mutation found to be associated with early-onset CAT in the Staffordshire Bull Terrier and Boston Terrier. The intronic SNP may be useful to test HSF4 for linkage with CAT in further dog breeds.     

Novel genotyping assay for the nt230 (del4) ABCB1 gene mutation and its allele frequency in Border Collie dogs in Mexico

A 4-bp deletion in the ATP-binding cassette subfamily B member 1 (ABCB1) gene, also referred to as the multidrug resistance gene (MDR1), produces stop codons that cause premature termination of P-glycoprotein 1 (P-gp) synthesis. Dogs with the homozygous mutation do not express functional P-gp, which increases their sensitivity markedly to many common veterinary drugs. We detected the nt230 (del4) ABCB1 mutation in Border Collie dogs in western Mexico with a simple and affordable primer-introduced restriction analysis PCR (PIRA-PCR). PIRA-PCR clearly identified all genotypes in our sample of 104 dogs. Genotype frequencies were 0.952 (wild/wild), 0.029 (wild/mut) and 0.019 (mut/mut). Allele frequencies were 0.033 (mutant alleles) and 0.966 (wild-type alleles). In this small subset of the Mexican dog population, we found a higher prevalence of the nt230 (del4) MDR1/ABCB1 gene mutation than reported in other countries.     

Therapeutic implications of the MDR-1 gene

Drug transporters significantly influence drug pharmacokinetics and pharmacodynamics. P-glycoprotein (P-gp), the product of the MDR1 (ABCB1) gene, is among the most well-characterized drug transporters, particularly in veterinary medicine. A number of clinically relevant, structurally and functionally unrelated drugs are substrates for P-gp. P-gp is expressed by a variety of normal tissues including the intestines, renal tubular cells, brain capillary endothelial cells, biliary canalicular cells, and others, where it functions to actively extrude substrate drugs. In this capacity, P-gp limits oral absorption and central nervous system entry of many substrate drugs. A number of MDR1 polymorphisms have been described in human patients, some of which result in altered drug pharmacokinetics and susceptibility to diseases such as Parkinson's disease, inflammatory bowel disease, refractory seizures, and others. An MDR1 polymorphism in herding breed dogs, including collies and Australian shepherds, has been demonstrated to be the cause of ivermectin sensitivity in these breeds. Recent evidence suggests that this polymorphism, a 4-bp deletion mutation, results in increased susceptibility to the toxicity of several drugs in addition to ivermectin. Furthermore, data in rodent models suggest that P-gp may play an important role in regulating the hypothalamic-pituitary-adrenal axis.     

Vincristine-induced central neurotoxicity in a collie homozygous for the ABCB1Δ mutation

A six-year-old, neutered, female collie was presented to an oncology specialty service after developing tetraparesis and self-mutilation that progressively worsened while receiving chemotherapy for lymphoma. Neurologic examination revealed ataxia, paresis and diminished conscious proprioception in all limbs with entire spinal reflexes. Magnetic resonance imaging of the brain and spinal cord was normal. Electromyography of the limbs ruled out a vincristine-induced peripheral neuropathy. Cerebrospinal fluid analysis and cerebrospinal fluid and serum testing for Neospora and Toxoplasma were normal. Results of MDR1 genotyping revealed that the dog was homozygous for the ABCB1-1Δ (MDR1) mutation. This clinical presentation strongly resembled the effects seen from inadvertent intrathecal administration of vincristine in humans. Dogs that are homozygous for the ABCB1-1Δ (MDR1) mutation should not receive standard dosages of chemotherapy drugs known to be eliminated by P-glycoprotein, the gene product of ABCB1. Testing for this mutation is strongly recommended before chemotherapy initiation for at-risk breeds.     

Analysis of a deletion in the nephronophthisis 4 gene in different dog breeds

Cone‐rod dystrophy is a progressive inherited retinal degenerative disorder that occurs in humans and dogs. The deletion in the nephronophthisis 4 (NPHP4) gene was established as a causative mutation in standard wire‐haired Dachshunds. We analyzed all varieties of Dachshunds from the Czech Republic and five other dog breeds and found that the deletion in the NPHP4 (in heterozygous state) is present not only in standard‐, but also in miniature wire‐haired Dachshunds, but not in other varieties of Dachshunds or in other breeds.     

Congenital sensorineural deafness in dogs: a molecular genetic approach toward unravelling the responsible genes

Deafness is often diagnosed in different dog breeds and has been identified as a significant problem for breeders, owners and clinicians. The aetiology can be inherited or acquired, and a distinction must be made between sensorineural and conductive forms of deafness. This paper provides a brief overview of the varieties of findings in different dog breeds and in one breed in particular including prevalence, phenotypic and gender associations, histology, modes of inheritance and the number of contributing genes in congenital sensorineural deafness. We have also described molecular genetic approaches to canine hearing loss and discuss how comparative genomics could help reduce the prevalence of deafness in affected breeds leading to new insights into the molecular mechanisms of auditory function in both dogs and humans.     

Effects of breed, gender, exercise and white-coat effect on markers of endothelial function in dogs

This study examines how systemic biomarkers of endothelial function and nitric oxide metabolism are affected by exercise in dogs. Furthermore, breed variation and white-coat effect have been tested by sampling three different dog breeds both in their home and in a clinical setting. Short-term exercise increased plasma nitrate and nitrite (NOx) and von Willebrand factor (vWf). There was significant difference between Pointers and the small dog breeds Cairn Terriers and Cavalier King Charles Spaniels in the general plasma levels of vWf and asymmetric dimethylarginine (ADMA). NOx and vWf were significantly higher when the sample was taken in the laboratory cf. at home, whereas ADMA and L-arginine were significantly lower. In conclusion, both short-term exercise and white-coat effect influence several plasma markers of endothelial function depending also on the breed and gender of the dogs. These findings should be considered in future studies concerning endothelial function in dogs.     

Breed distribution of the ABCB1-1Delta (multidrug sensitivity) polymorphism among dogs undergoing ABCB1 genotyping

OBJECTIVE: To evaluate the breed distribution of the ABCB1-1Delta polymorphism in a large number of dogs in North America, including dogs of several herding breeds in which this polymorphism has been detected and other breeds in which this polymorphism has not yet been identified. DESIGN: Cross-sectional study. ANIMALS: 5,368 dogs from which buccal swab samples were collected for purposes of ABCB1 genotyping. PROCEDURES: From May 1, 2004, to September 30, 2007, DNA specimens derived from buccal swab samples collected from 5,368 dogs underwent ABCB1 genotyping. These data were reviewed, and results for each dog were recorded in a spreadsheet, along with the dog's breed. The genotypes for each breed were tallied by use of a sorting function. RESULTS: The ABCB1-1Delta allele was identified in 9 breeds of dogs and in many mixed-breed dogs. Breeds that had the ABCB1-1Delta allele included Collie, Longhaired Whippet, Australian Shepherd (standard and miniature), Shetland Sheepdog, Old English Sheepdog, Border Collie, Silken Windhound, and German Shepherd Dog (a breed in which this mutation had not been detected previously). CONCLUSIONS AND CLINICAL RELEVANCE: The ABCB1-1Delta polymorphism is associated with increased susceptibility to many adverse drug reactions and with suppression of the hypothalamic-pituitary-adrenal axis and is present in many herding breeds of dog. Veterinarians should be familiar with the breeds that have the ABCB1-1Delta polymorphism to make appropriate pharmacologic choices for these patients.