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.     

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.     

伊维菌素敏感牧羊犬MDR1基因的克隆与序列分析

本试验旨在探讨利用RT-PCR方法检测牧羊犬MDR1基因突变的可行性及临床意义.从对伊维菌素敏感的苏格兰牧羊犬和不敏感的德国牧羊犬血液里提取RNA,通过PCR获得了目的基因MDR1,连接并转化,增菌培养后,提取阳性质粒进行序列测定.苏格兰牧羊犬的基闪缺少4个碱基,德国牧羊犬的基因则正常.RT-PCR方法鉴定了苏格兰牧羊犬MDR1基因突变,目前是一种简单、方便、快速、准确的基因诊断方法,有助于临床合理地进行药物治疗.     

Motor laterality in 4 breeds of dog

The aim of the current study was designed to explore possible breed differences in a basic behavioral phenotype in dogs. It measured paw use during food-retrieval from a cylindrical, hollow, rubber toy to assess motor laterality (pawedness) in 4 breeds of dogs selected for their morphological differences: 45 greyhounds (males n = 23/females n = 22), 47 whippets (15/32), 46 pugs (15/31), and 45 boxers (17/28). A laterality index was calculated from the use of individual paws to restrain the toy during feeding. An association was found between sex and the laterality index (P = 0.035), reflecting a significant bias for left-paw use by entire male dogs and right-paw use by entire female dogs but no sex difference was found in the mean strength of laterality. No significant association was found between age and the laterality index. No breed difference was found in the laterality index (P = 0.423) or the absolute strength of laterality (i.e., the laterality index without direction, P = 0.259). However, important breed differences in the use of the test device emerged. The frequency of simultaneous use of both paws was lowest in pugs (P < 0.0001). In addition, both brachycephalic breeds (pugs and boxers) took less time than the dolichocephalic breeds (the whippets and greyhounds) to complete the criteria of 100 paw-use scores that was set for the laterality test. The absence of breed differences in the laterality index for paw use suggests that the task is a reliable measure of motor laterality in dogs during a food-retrieval task.     

Estimated frequency of the canine hyperuricosuria mutation in different dog breeds

Background: Hyperuricosuria is a condition that predisposes dogs to urate urolithiasis. A mutation that causes canine hyperuricosuria was previously identified in 3 unrelated dog breeds. The occurrence of the mutation in additional breeds was not determined. Hypothesis/Objectives: Identify additional breeds that have the hyperuricosuria mutation and estimate the mutant allele frequency in those breeds. Animals: Three thousand five hundred and thirty dogs from 127 different breeds were screened for the hyperuricosuria mutation. Methods: DNA samples were genotyped by pyrosequencing and allele‐specific polymerase chain reaction methods. Results: Mutant allele frequencies that range from 0.001 to 0.15 were identified in the American Staffordshire Terrier, Australian Shepherd, German Shepherd Dog, Giant Schnauzer, Parson (Jack) Russell Terrier, Labrador Retriever, Large Munsterlander, Pomeranian, South African Boerboel, and Weimaraner breeds. Conclusions and Clinical Importance: The hyperuricosuria mutation has been identified in several unrelated dog breeds. The mutant allele frequencies vary among breeds and can be used to determine an appropriate breeding plan for each breed. A DNA test is available and may be used by breeders to decrease the mutant allele frequency in breeds that carry the mutation. In addition, veterinarians may use the test as a diagnostic tool to identify the cause of urate urolithiasis.     

Glycogen synthase 1 (GYS1) mutation in diverse breeds with polysaccharide storage myopathy

Background: A missense mutation in the GYS1 gene was recently described in horses with polysaccharide storage myopathy (PSSM).
Objectives: The first objective was to determine the prevalence of the GYS1 mutation in horses with PSSM from diverse breeds. The second objective was to determine if the prevalence of the GYS1 mutation differed between horses diagnosed with PSSM based on grade 1 (typically amylase-sensitive) or grade 2 (typically amylase-resistant) polysaccharide.
Animals: Eight hundred and thirty-one PSSM horses from 36 breeds.
Procedures: Horses with PSSM diagnosed by histopathology of skeletal muscle biopsy samples were identified from the Neuromuscular Disease Laboratory database. Eight hundred and thirty-one cases had blood or tissue that was available for DNA isolation; these 831 cases were genotyped for the GYS1 mutation by restriction fragment length polymorphism.
Results: The PSSM mutation was identified in horses from 17 different breeds. The prevalence of the GYS1 mutation in PSSM horses was high in Draft- (87%) and Quarter Horse-related breeds (72%) and lower in Warmbloods (18%) and other light horse breeds (24%), when diagnosis was based on grade 2 diagnostic criteria. Overall, the PSSM mutation was present in 16% of grade 1 and 70% of grade 2 PSSM horses.
Conclusions and Clinical Importance: GYS1 mutation causes PSSM in diverse breeds and is the predominant form of PSSM in Draft- and Quarter Horse-related breeds. False-positive diagnosis, as well as the possibility of a second glycogenosis in horses with neuromuscular disease (type 2 PSSM), might explain the absence of the GYS1 mutation in horses diagnosed with excessive glycogen accumulation in muscle.     

Frequency of the canine leucocyte adhesion deficiency (CLAD) mutation among Irish red setters in Germany

Canine leucocyte adhesion deficiency (CLAD) is an autosomal recessive hereditary disease occurring among Irish red setters. The genetic defect causative for this disorder was recently identified as a missense mutation in the ITGB2 gene. Irish red setters with one copy of the mutant gene appear normal, while dogs with two copies of the mutant gene manifest the disease. The present report describes the analysis of the single nucleotide polymorphism in 289 Irish red setters by DNA sequencing. The frequency of CLAD carriers in Germany is 11%.     

Frequency of bovine Nramp1 (Slc11a1) alleles in Holstein and Zebu breeds

Natural resistance against brucellosis in cattle is linked to the Nramp1 gene, which encodes a divalent cation transporter that localizes in the phagolysosome membrane in macrophages. Nramp1 gene in mouse plays a critical role in innate immunity favoring bacterial killing by macrophages in addition to its influence on adaptative immunity. Polymorphisms at the bovine Nramp1 3' untranslated region (3'UTR), detectable by Single Strand Conformational Analysis (SSCA), are associated with natural resistance against brucellosis. Such polymorphisms are associated with variation in the number of GT repeats. This study compared the frequency of Nramp1 3'UTR polymorphisms between Zebu and European bovine breeds. Eighty-one Holsteins (Bos taurus taurus) and 167 Zebu (Bos taurus indicus), including the following breeds: Nelore (n=95), Guzerá (n=37), and Gir (n=35), totaling 248 pure breed cattle studied. DNA extraction was performed using the guanidium protocol and genotyping was performed by SSCA. DNA from cattle considered genotypically resistant to brucellosis resulted in a single band (homozygous) with 175bp, corresponding to the 3'UTR with 13 GT pairs (GT13), whereas DNA from genotypically susceptible cattle generated one single band with 177bp (homozygous GT14) or double bands with both 175 and 177bp, or 175 and 179bp (heterozygous GT13/GT14 or GT13/GT15, respectively). A marked difference in the frequency of alleles was detected between the Zebu and Holstein cattle. Holsteins had an extremely homogeneous genotype, with 100% of the individuals with a GT13 genotype. In sharp contrast the Nelore breed had the most heterogeneous genotype with four allelic combinations, namely, homozygous GT13, homozygous GT14, heterozygous GT13/GT14, and heterozygous GT13/GT15. When the Zebu breeds were compared to each other, the only significant difference observed was the frequencies of the genotypes GT13 and GT14 between the Nelore and Guzerá breeds. The knowledge of allelic frequencies in different breeds of cattle may prove to be very useful in the future for planning breeding strategies for selection of resistant cattle.     

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.     

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.     

Presence of the glycogen synthase 1 (GYS1) mutation causing type 1 polysaccharide storage myopathy in continental European draught horse breeds

The purpose of this study was to determine which continental European draught horse breeds harbour a mutation in the glycogen synthase 1 gene (GYS1) that is known to be responsible for type 1 polysaccharide storage myopathy in quarter horses and North American draught horses. Of a non-random selection of continental European draught horses belonging to 13 breeds, 62 per cent (250 of 403) tested were found to carry the mutant allele. The horses were located in Belgium, France, Germany, The Netherlands, Spain and Sweden. The mutation was identified in animals from each of the breeds examined. In the breeds in which more than 15 animals were available for testing, the highest percentages of GYS1-positive horses were found in the Belgian trekpaard (92 per cent; 35 of 38 horses tested), Comtois (80 per cent; 70 of 88), Netherlands trekpaard (74 per cent; 17 of 23), Rheinisch-Deutsches kaltblut (68 per cent; 30 of 44) and Breton (64 per cent; 32 of 51).     

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.     

SNaPshot based genotyping of the RYR1 mutation in Portuguese breeds of pigs

The porcine stress syndrome or malignant hyperthermia is an inherited autosomic recessive disease, which results in neuromuscular disorders leading to death in homozygous individuals and is associated with deterioration of meat quality. The defect in susceptible animals results from modifications in the calcium release channel or Ryanodine Receptor (RYR1), with a mutation leading to a C to T transition in nucleotide 1843 of the gene. The objective of this work was to develop a method based on analysis of SNPs to detect the mutation described in the RYR1 locus in pigs, and study polymorphisms of the gene in four exotic (Large White, Landrace, Duroc and Pietrain) and three native (Bísaro, Alentejano and Malhado de Alcobaça) breeds of pigs in Portugal. The method was successful in identifying the mutation by analysis of SNPs, and results indicate a high incidence of the mutant allele in Pietrain (0.75) and, to a lesser degree, in Malhado de Alcobaça (0.34) and Landrace (0.28); frequencies in Alentejano, Bísaro and Large White ranged between 0.04 and 0.09. These results suggest the need to establish breeding programs aimed at eliminating the susceptibility allele from those populations.     

Isolation of multidrug-resistant (MDR) Mycobacterium bovis from a dog in Korea

A 3-year-old female Miniature Schnauzer dog with a week-long history of generalized intention tremor and progressive weight loss for several months was admitted. Mild anemia, fever, splenomegaly, aseptic cerebral meningitis and systemic lymph nodes enlargement were examined through erythrogram, ultrasonography, computed tomography and magnetic resonance imaging. Mycobacterium bovis was identified via molecular microbiology having the same molecular type as that of isolates from a cattle farm previously identified. However, the dog was raised in a city. The M. bovis had multidrug resistance (MDR)-bearing mutations in both katG and rpoB genes toward first-line antibiotics. To the best of our knowledge, this is the first report describing an MDR M. bovis infection of a dog in Korea.