Mar 18, 2014 - von Willebrand disease in West Highland white terriers and in the Dobermann breed (5). DEVELOPMENT OF A TEST. To analyze the mutation ...
Veterinary Quarterly
ISSN: 0165-2176 (Print) 1875-5941 (Online) Journal homepage: http://www.tandfonline.com/loi/tveq20
Molecular Diagnosis of Copper Toxicosis in Bedlington Terriers G.J. Ubbink, J. Rothuizen, P. van Zon, T.S.G.A.M. van den Ingh & V. YuzbasiyanGurkan To cite this article: G.J. Ubbink, J. Rothuizen, P. van Zon, T.S.G.A.M. van den Ingh & V. Yuzbasiyan-Gurkan (1998) Molecular Diagnosis of Copper Toxicosis in Bedlington Terriers, Veterinary Quarterly, 20:sup1, S91-S92, DOI: 10.1080/01652176.1998.10807437 To link to this article: http://dx.doi.org/10.1080/01652176.1998.10807437
Published online: 18 Mar 2014.
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Research Institute, Milwaukee, WI, USA) did not reveal any abnormalities compared with the Southern blot pattern of dogs of other breeds. We then started to look for smaller mutations by analyzing the von Willebrand mRNA. To this end we have isolated total RNA from a skin biopsy of an affected dog which was euthanized for reasons unrelated to the von Wille brand disease and also from skin of a normal dog. The mRNA was reverse transcribed and it was found that the resulting eDNA of the patient was slightly longer than normal. Such a length difference may be due to a insertion or a splice mutation. Sequence analysis of the polymerase chain reaction (PCR) product revealed that in the affected dog a splice site mutation had occurred, resulting in the presence of some intronic DNA in mature mRNA. At the same time this mutation altered the reading frame in such a way that the codon at the original splice junction was changed into a stop codon. It could be predicted that only about the first 25% of the von Wille brand protein could be produced by the mutant mRNA. This would result in a nonfunctional protein fragment, ex-
plaining the severe and recessive nature of the disease in these kooiker dogs. The mutation is different from that in von Willebrand disease in West Highland white terriers and in the Dobermann breed (5).
DEVELOPMENT OF A TEST To analyze the mutation at the population level a simple PCR test based on genomic DNA was developped. Using whole blood DNA as the template, a PCR product was generated with PCR primers flanking the mutation. Two oligonucleotides specific for either the normal and the mutant sequence were designed and labelled radioactively. Conditions were optimized in such a way that the hybridization of the oligonucleotides was selective, so that normal homozygotes, affected homygotes, and heterozygotes could be distinghuised without any doubt. The gene defect was inherited in the same way as the linked DNA markers described above. With this test eradication of severe von Willebrand's disease in the Dutch kooiker dog breed is within reach.
MOLECULAR DIAGNOSIS OF COPPER TOXICOSIS IN BEDLINGTON TERRIERS G.J. Ubbink1, J. Rothuizen, P. van Zan, T.S.G.A.M. van den lngh, and V. Yuzbasiyan-Gurkan
INTRODUCTION Copper toxicosis is an autosomal recessive disease in Bedlington terriers. Under average circumstances, homozygously affected dogs will show clinical signs at 4-6 years of age. They are born with normal levels of copper in the liver but copper accumulates in the liver as they mature. All affected dogs above one year of age can be detected by measurement of copper in a liver biopsy. The intestinal absorption of copper is normal, as is the clearance of copper from the portal circulation. The basic metabolic defect lies in the excretion of copper from the hepatocytes through the canalicular membrane into the bile canaliculi. Non-excreted copper accumulates and is stored in the lysosomes of the liver cells. The histological or cytological diagnosis depends on staining of these copperpositive granules by, e.g., rubeanic acid. Clinical diagnostic studies do not, however, differentiate heterozygous carriers from unaffected animals. Therefore, it has not yet been possible to set up an absolutely effective breeding programme to reduce the frequency of the disease. A CA-repeat polymorphism linked to the original defect causing copper toxicosis in a family of Bedlington terriers has been discovered by Dr. Yuzbasiyan-Gurkan. It proved to be tightly linked to the disease in this family (1). The I
Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, P.O. Box80154. NL-3508 TD Utrecht, the Netherlands.
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diagnostic value of a DNA marker depends on a number of circumstances. First, the distance in the DNA between the causative gene and the marker determines the chance of recombinations and loss oflinkage. Second, the frequency of the marker allele in the entire population is important. A marker may be closely linked to a disease in affected families, but if the same allele also occurs frequently in nonaffected families the marker becomes less meaningful informative in the analysis of the population. The frequency of alleles may be very different between populations from different countries or continents, and the informativeness of a marker should be investigated for each population. Third, a phenotypically identical disease may be caused by different gene defects. Therefore, a marker identified in one family or closed population should be confirmed when applied in another situation. We report the evaluation in the European population of Bedlington terriers of the CA-repeat marker discovered in the USA.
METHODS AND RESULTS We have examined a sample of 98 Bedlington terriers from the Netherlands, Germany, and Belgium. The dogs shared many familial interrelations. In all dogs the diagnosis of copper toxicosis was confirmed or excluded by examination of liver tissue, either histological sections or cytological smears. Tissue was stained with rubeanic acid, which stains copper dark blue or black. We have previously shown that this stain has the same diagnostic accuracy as quantitative
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were genetically free or carrier, 37 were found to be homozygous for the 163 bp allele and 8 were heterozygous. Thus, the results of the PCR test were in agreement with the genetic status as determined independently by clinical findings and pedigree data in all of the cases. This means that in the animals studied there had been no crossing over, by which linkage would be lost. The defect is the same defect as that in the American population, as expected. The test seems to be reliable enough for use for genetic counselling in the European population since all of the animals were diagnosed correctly. However, on the basis of this number of cases (n=53) the total accuracy of the PCR test had a 95% confidence interval of94-1 00%. This is the first example of the use of the genetic microsatellite marker map of the canine genome by which an inherited disease has been mapped successfully. The availability of a reliable DNA marker for copper toxicosis implies that it is possible to differentiate between unaffected dogs and nonsymptomatic carriers, and that henceforth the birth of affected dogs can be prevented.
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measurement of copper in liver by activation analysis (2). Only dogs older than one year were included. Blood anticoagulated with EDTA was collected at the same time. DNA was isolated and the microsatellite alleles were amplified by the polymerase chain reaction (PCR). The length differences between the alleles were determined. The two alleles found in this population were 167 and 163 base pairs in length. The 167 bp allele was the marker allele present in dogs with copper accumulation. Copper toxicosis was found by rubeanic acid staining in 17 of the 98 dogs. All of these dogs were homozygous for the 167 bp allele. By comparing and linking of pedigrees of diseased and non-diseased dogs, and because of the autosomal recessive nature of the disease, many of the other 81 dogs without copper toxicosis could be characterized as heterozygous carriers. Thus 36 ofthe 81 non-diseased dogs had either an affected parent or affected offspring. These were determined to be obligate carriers. All of the obligate carriers were heterozygous, having one 163 bp and one 167 bp allele. Of the 45 dogs that were characterized as being free of copper toxicosis but of which it was uncertain whether they
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