Copper Toxicosis In Bedlington Terriers Research Articles

Untangling the genetic traits underlying the development of copper toxicosis in Bedlington terriers.

Untangling the genetic traits underlying the development of copper toxicosis in Bedlington terriers.

Copper toxicosis in Bedlington terriers is associated with multiple independent genetic variants.

Bedlington terrier copper toxicosis (CT) is due to a homozygous exon deletion in COMMD1. CT also occurs in Bedlingtons lacking this deletion. An association with two ABCA12 single nuceotide polymorphism (SNP) splice variants was reported. Labrador retriever CT is associated with a missense mutation in ATP7B, and with a protective mutation in ATP7A. Liver and DNA samples from 24 affected and 10 unaffected Bedlingtons were assessed for copper and genetic variants. Allelic frequencies were compared. The ATP7B mutation frequency was investigated in 144 dogs of other breeds. The ABCA12 SNPs showed no differences between groups. The COMMD1 deletion was less frequent in unaffected than in affected dogs and in affected dogs post-2001 than pre-2001. The ATP7B mutation was more frequent in affected than unaffected Bedlingtons. Thirty-five of 144 dogs of other breeds were homo- or heterozygous for the ATP7B mutation. The ATP7A mutation was absent from Bedlingtons. Clinical information and qualitative copper measurements were unavailable for most dogs. The COMMD1 deletion remains present in Bedlington terriers but is no longer the primary cause of CT. ABCA12 SNPs were not associated with CT. The ATP7B:c.4358G>A mutation was significantly associated with Bedlington CT and was more common in dogs of this breed than in the 144 dogs of other breeds.

Evaluation of COMMD1 in copper toxicosis in Labrador retrievers and Dobermans

Copper toxicosis is a major cause of hepatitis in dogs. We have shown that variants in ATP7A and ATP7B modulate hepatic copper levels in Labrador retrievers and Dobermans. However, these variants cannot fully explain the observed variation in hepatic copper levels in these dog breeds. Homozygous deletion of exon 2 of COMMD1 causes copper toxicosis in Bedlington terriers. We investigated the possible involvement of COMMD1 in the multifactorial aetiology of copper toxicosis in Labrador retrievers and Dobermans. Thirty dogs of each breed with known hepatic copper status were selected for DNA sequence analysis of the three exons and flanking intronic regions of COMMD1. The observed variants were tested for association with hepatic copper levels by linear model analysis. Several variants were observed in the DNA sequence of COMMD1 in both Labrador retrievers (nine variants) and Dobermans (11 variants) but none of these was associated with variations of hepatic copper concentrations. We conclude that COMMD1 did not play a major role in the aetiology of copper associated hepatitis in Labrador retrievers and Dobermans.

Preclinical models of Wilson's disease, why dogs are catchy alternatives.

Copper toxicosis is frequently encountered in various dog breeds. A number of differences and similarities occur between Wilson disease and copper toxicosis in Bedlington terriers, caused by a mutation in the COMMD1 gene, and copper toxicosis in Labrador retrievers, caused by mutations in both ATP7A and ATP7B gene. First the specific population structure of dog breeds is explained with reference to its applicability for genetic investigations. The relatively large body size (variable from less than 1 kg to over 50 kg) and life-span (over 10 years) of dogs facilitates preclinical studies on safety on long-term effects of novel procedures. Then copper toxicosis in the two dog breeds is described in detail with an emphasis on the functions of the causative proteins. Some of the advantages of this species for preclinical studies are described with an example of liver stem cell transplantations in COMMD1 deficient dogs. Since the genetic background of copper toxicosis in other dogs' breeds has not yet been elucidated, it is conceivable that novel copper-related gene products or modifier genes will be discovered. About a century after the Novel prize was awarded to the research on dogs (Pavlov), dogs are in spotlight again as important preclinical model animals.

Prevalence and Clinical Relevance of Exon 2 Deletion of COMMD1 in Bedlington Terriers in Korea.

BackgroundDeletion of exon 2 of copper metabolism domain containing 1 (COMMD1) results in copper toxicosis in Bedlington terriers (CT‐BT).ObjectivesThis study was conducted to identify the prevalence and clinical relevance of the COMMD1 mutation in Bedlington terriers in Korea.AnimalsA total of 105 purebred Bedlington terriers (50 males, 55 females) from the kennels and pet dog clubs in Korea were examined during the period 2008–2013.MethodsA multiplex PCR was carried out to detect exon 2 deletion of COMMD1. Clinical analysis was performed on each genetic group, and clinical status of the dogs was followed up to estimate survival probability.ResultsOf the 105 samples, 52 (49%) were wild‐type homozygote, 47 (45%) were heterozygote, and 6 (6%) were mutant‐type homozygote. Plasma alanine aminotransferase (ALT) activity was increased in the mutant‐type homozygous group >2 years of age (P < .0001). The survival probability of 6 mutant‐type homozygotes surviving 2.5 years was 0.67, and 4 years was 0.5.Conclusions and Clinical ImportanceResults show the prevalence and clinical relevance of exon 2 deletion of COMMD1 and could help establish a structured selective breeding program to prevent CT‐BT in Korea.

Inherited disease in boxer dogs: a cautionary tale for molecular geneticists

IN a study summarised on p 492 of this issue of Veterinary Record , Bruce Cattanach and collaborators from four institutions remind us of the importance of old-fashioned pedigree analysis...

The Exon 2 Deletion of the COMMD1 Causing Copper Toxicosis in Bedlington Terriers in Korea

This study was performed to survey prevalence of Copper metabolism domain containing 1 (COMMD1) mutation using molecular diagnostic method in a population of Bedlington terriers in Korea. COMMD1 gene (formerly MURR1) functions as a regulator of sodium transport and copper metabolism. The deletion of exon 2 of the COMMD1 gene causes copper toxicosis in Bedlington terriers. Bedlington terriers with this autosomal recessive disorder were shown to have the elevated liver copper levels due to genetic derangement in the biliary copper excretion pathway. DNA samples were extracted from whole blood collected from 257 Bedlington terriers (109 males, 148 females) of pet dog clubs in Korea. A multiplex PCR was carried out to detect of exon 2 deletion of COMMD1 gene. In this study, it was possible to know the existence and prevalence of exon 2 deletion of COMMD1 in Bedlington terriers in Korea. Of the 257 samples, 131 (51%) were wild type homozygous for the normal COMMD1 gene, 108 (42%) were heterozygous, having both normal and mutated copy of the COMMD1 gene. The eighteen (7%) were mutant type homozygous. The results of genetic analysis could help establish proper management strategy and selective breeding program to prevent COMMD1 mutation in Bedlington terriers in Korea.

D-penicillamine treatment of copper-associated hepatitis in Labrador retrievers

d-penicillamine is effectively used in the lifelong treatment of copper toxicosis in Bedlington terriers and Wilson’s disease in humans. A complex form of copper-associated hepatitis has recently been characterized in the Labrador retriever. The aims of this study were to evaluate the effectiveness of d-penicillamine treatment for copper-associated hepatitis in this breed, to study the effects on hepatic copper, iron and zinc concentrations, and to evaluate parameters to predict optimal duration of treatment. Forty-three client owned Labrador retrievers that were diagnosed with increased hepatic copper were treated with d-penicillamine and underwent at least one follow-up examination including a liver biopsy for histopathological scoring of inflammatory lesions. Hepatic copper, iron and zinc concentrations were determined in the initial and follow-up biopsies by instrumental neutron activation analysis. The influence of initial hepatic copper concentration, sex, age, d-penicillamine formulation and the occurrence of side effects were investigated for their influence on hepatic copper concentration after a certain period of treatment by generalized mixed modelling.d-penicillamine proved to be effective in reducing hepatic copper concentration and associated inflammatory lesions. Parameters derived from the model can be used to estimate the necessary duration of d-penicillamine treatment for Labrador retrievers with increased hepatic copper concentration. Continuous, lifelong d-penicillamine treatment is not recommended in this breed, as there may be a risk for hepatic copper and zinc deficiency.

Liver-Specific Commd1 Knockout Mice Are Susceptible to Hepatic Copper Accumulation

Canine copper toxicosis is an autosomal recessive disorder characterized by hepatic copper accumulation resulting in liver fibrosis and eventually cirrhosis. We have identified COMMD1 as the gene underlying copper toxicosis in Bedlington terriers. Although recent studies suggest that COMMD1 regulates hepatic copper export via an interaction with the Wilson disease protein ATP7B, its importance in hepatic copper homeostasis is ill-defined. In this study, we aimed to assess the effect of Commd1 deficiency on hepatic copper metabolism in mice. Liver-specific Commd1 knockout mice (Commd1 Δhep) were generated and fed either a standard or a copper-enriched diet. Copper homeostasis and liver function were determined in Commd1 Δhep mice by biochemical and histological analyses, and compared to wild-type littermates. Commd1 Δhep mice were viable and did not develop an overt phenotype. At six weeks, the liver copper contents was increased up to a 3-fold upon Commd1 deficiency, but declined with age to concentrations similar to those seen in controls. Interestingly, Commd1 Δhep mice fed a copper-enriched diet progressively accumulated copper in the liver up to a 20-fold increase compared to controls. These copper levels did not result in significant induction of the copper-responsive genes metallothionein I and II, neither was there evidence of biochemical liver injury nor overt liver pathology. The biosynthesis of ceruloplasmin was clearly augmented with age in Commd1 Δhep mice. Although COMMD1 expression is associated with changes in ATP7B protein stability, no clear correlation between Atp7b levels and copper accumulation in Commd1 Δhep mice could be detected. Despite the absence of hepatocellular toxicity in Commd1 Δhep mice, the changes in liver copper displayed several parallels with copper toxicosis in Bedlington terriers. Thus, these results provide the first genetic evidence for COMMD1 to play an essential role in hepatic copper homeostasis and present a valuable mouse model for further understanding of the molecular mechanisms underlying hepatic copper homeostasis.

Prevalence of the exon 2 deletion of the COMMD1 gene in Australian Bedlington terriers

Inherited copper toxicosis in Bedlington terriers is a chronic progressive hepatic disease caused by an abnormal accumulation of copper due to genetic derangement in the biliary copper excretion pathway. In this study, we screened a group of Australian Bedlington terriers for a previously identified mutation, an exon 2 deletion in the COMMD1 gene. The DNA samples used in this study were collected from 149 Bedlington terriers (60 males and 89 females), aged between four weeks to 13 years from eight different pedigrees obtained over a period of three years. We found 60 dogs (40%) were homozygous wild type, 42 dogs (28%) were homozygous mutant, and 47 dogs (32%) were heterozygous for the deletion of exon 2 in the COMMD1 gene. Not only this reports the first large scale genetic screening of the known COMMD1 mutation in Bedlington terriers, but our results also indicate a high prevalence of the COMMD1 mutation in Bedlington terriers that might also be useful as a marker of copper toxicosis. Inherited copper toxicosis was first identified in Bedlington terrier in USA, in 1975 (Hardy et al. 1975) and has since been reported in other countries including Australia (Eriksson 1983; Robertson et al. 1983; Kelly et al. 1984). This disease is an inherited autosomal recessive disorder characterized by a progressive accumulation of copper, predominantly in the liver (Hardy 1984; Johnson et al. 1980). The main pathogenesis of the copper toxicosis in Bedlington terrier appears to be related to the reduced biliary excretion of stored copper resulting from a genetic derangement in copper metabolism (Hardy 1984; Hyun and Filippich 2004). A mutation resulting in the deletion of exon 2 in the COMMD1 (formerly known asMurr1) gene was found to be responsible for copper toxicosis in a subpopulation of Bedlington terriers (Van de Sluis et al. 2002)

Increased activity of hypoxia-inducible factor 1 is associated with early embryonic lethality in Commd1 null mice.

COMMD1 (previously known as MURR1) belongs to a novel family of proteins termed the copper metabolism gene MURR1 domain (COMMD) family. The 10 COMMD family members are well conserved between vertebrates, but the functions of most of the COMMD proteins are unknown. We recently established that COMMD1 is associated with the hepatic copper overload disorder copper toxicosis in Bedlington terriers. Recent in vitro studies indicate that COMMD1 has multiple functions, including sodium transport and NF-kappaB signaling. To elucidate the function of Commd1 in vivo, we generated homozygous Commd1 null (Commd1(-/-)) mice. Commd1(-/-) embryos died in utero between 9.5 and 10.5 days postcoitum (dpc), their development was generally retarded, and placenta vascularization was absent. Microarray analysis identified transcriptional upregulation of hypoxia-inducible factor 1 (HIF-1) target genes in 9.5-dpc Commd1(-/-) embryos compared to normal embryos, a feature that was associated with increased Hif-1alpha stability. Consistent with these observations, COMMD1 physically associates with HIF-1alpha and inhibits HIF-1alpha stability and HIF-1 transactivation in vitro. Thus, this study identifies COMMD1 as a novel regulator of HIF-1 activity and shows that Commd1 deficiency in mice leads to embryonic lethality associated with dysregulated placenta vascularization.

Characterization and Copper Binding Properties of Human COMMD1 (MURR1)

COMMD1 (copper metabolism gene MURR1 (mouse U2af1-rs1 region1) domain) belongs to a family of multifunctional proteins that inhibit nuclear factor NF-kappaB. COMMD1 was implicated as a regulator of copper metabolism by the discovery that a deletion of exon 2 of COMMD1 causes copper toxicosis in Bedlington terriers. Here, we report the detailed characterization and specific copper binding properties of purified recombinant human COMMD1 as well as that of the exon 2 product, COMMD(61-154). By using various techniques including native-PAGE, EPR, UV-visible electronic absorption, intrinsic fluorescence spectroscopies as well as DEPC modification of histidines, we demonstrate that COMMD1 specifically binds copper as Cu(II) in 1:1 stoichiometry and does not bind other divalent metals. Moreover, the exon 2 product, COMMD(61-154), alone was able to bind Cu(II) as well as the wild type protein, with a stoichiometry of 1 mol of Cu(II) per protein monomer. The protection of DEPC modification of COMMD1 by Cu(II) implied that Cu(II) binding involves His residues. Further investigation by DEPC modification of COMMD(61-154) and subsequent MALDI MS mapping and MS/MS sequencing identified the protection of His101 and His134 residues in the presence of Cu(II). Fluorescence studies of single point mutants of the full-length protein revealed the involvement of M110 in addition to H134 in direct Cu(II) binding. Taken together, the data provide insight into the function of COMMD1 and especially COMMD(61-154), a product of exon 2 that is deleted in terriers affected by copper toxicosis, as a regulator of copper homeostasis.

Evaluation of haplotypes associated with copper toxicosis in Bedlington Terriers in Australia

To evaluate the haplotype distribution associated with the copper toxicosis gene and the segregation of the mutated allele in a Bedlington Terrier population in Australia. 131 Bedlington Terriers. Samples of DNA and RNA were obtained from each dog. Genetic status of each dog was evaluated by use of the DNA markers C04107; single nucleotide polymorphism (SNP), which was adjacent to exon 2 of Murr1; and a deletion marker for exon 2. A subgroup of the study population was evaluated by use of biochemical and histologic techniques to elucidate the correlation between genotype and phenotype. We identified a recombination between the C04107 marker and Murr1 and a variation in a nucleotide in the splice site of exon 2 in our Bedlington Terrier cohort. Furthermore, we identified a novel haplotype associated with copper toxicosis in this cohort. Our findings indicate that the deletion of exon 2 was not the sole cause of copper toxicosis, although only exon 2 deletion of Murr1 has been responsible for copper toxicosis in Bedlington Terriers. Although we failed to find a novel mutation in our cohort, we identified an affected dog family with an intact exon 2. Furthermore, we found that an SNP in the 5' splicing site of exon 2 may or may not be associated with a novel mutation of the Murr1 gene or other genes. Loss of linkage between the C04107 marker and the Murr1 gene was also identified in a certain family of dogs.

Inherited copper toxicosis with emphasis on copper toxicosis in Bedlington terriers

Canine copper toxicosis is an important inherited disease in Bedlington terriers, because of its high prevalence rate and similarity to human copper storage disease. It can lead to chronic liver disease and occasional haemolytic anaemia due to impaired copper excretion. The responsible gene for copper toxicosis in Bedlington terriers has been recently identified and was found not to be related to human Wilson’s disease gene ATP7B. Although our understanding of copper metabolism in mammals has improved through genetic molecular technology, the diversity of gene mutation related to copper metabolism in animals will help identify the responsible genes for non-Wilsonian copper toxicoses in human. This review paper discusses our knowledge of normal copper metabolism and the pathogenesis, molecular genetics and current research into copper toxicosis in Bedlington terriers, other animals and humans.

Inherited canine copper toxicosis in Australian Bedlington Terriers

Inherited copper toxicosis in Bedlington Terriers (CTBT) is a copper associated hepatopathy caused by an autosomal recessive genetic defect of gene involving copper metabolism. To compare clinical and histopathological findings with previous reports and to expand our knowledge for future genetic studies, 18 terriers were clinically and histopathologically examined in this study. Pedigree information and dietary history were obtained from the owners before a thorough clinical examination was undertaken. Following the examination, a blood sample was collected for haematology, biochemistry and genetic analysis and a urine sample for urinalysis. Seven dogs were also liver biopsied for histopathology, histochemistry and electron microscopy. In this study, plasma alanine transaminase (ALT) activity was highly concordant with DNA marker test results and was the most reliable and sensitive biochemical test measured. Also clinical and biochemical copper toxicosisaffected states were noticed in a genotyped carrier dog. Histopathological and electron microscopy findings showed that the severity of the lesion was more closely correlated to the presence of clinical signs than to hepatic copper concentration. In addition, the involvement of apoptosis and p53 gene was observed in electron microscopy. The general findings related to CT-BT in this study was similar to those previously reported except few differences in histopathology and electron microscopy.

The Copper Toxicosis Gene Product Murr1 Directly Interacts with the Wilson Disease Protein

Copper toxicosis in Bedlington terriers is an autosomal recessive disorder characterized by excessive hepatic copper accumulation in association with a marked decrease in biliary copper excretion. Recent genetic data have revealed that MURR1, a single copy gene on dog chromosome 10q26, is mutated in this disorder. This gene encodes a 190-amino acid open reading frame of unknown function that is highly conserved in vertebrate species. The Wilson disease protein is a copper transporting ATPase shown to play a critical role in biliary copper excretion. Here we demonstrate that the Wilson disease protein directly interacts with the human homologue of Murr1 in vitro and in vivo and that this interaction is mediated via the copper binding, amino terminus of this ATPase. Importantly, this interaction is specific for this copper transporter, a finding consistent with the observation that impaired copper homeostasis in affected terriers is confined to the liver. Our findings reveal involvement of Murr1 in the defined pathway of hepatic biliary copper excretion, suggest a potential mechanism for Murr1 function in this process, and provide biochemical evidence in support of the proposed role of the MURR1 gene in hepatic copper toxicosis.

Indirect molecular diagnosis of copper toxicosis in Bedlington terriers is complicated by haplotype diversity.

Positional cloning recently identified the mutation causing copper toxicosis (CT) in Bedlington terriers. Isolation of the MURR1 gene will be of great value in developing a reliable diagnostic test for the breeding of a copper toxicosis-free stock. It will replace the current diagnostic test using the CT-linked marker, C04107, which is located in intron 1 of the MURR1 gene with a distance of approximately 8 kb from the exon 2 deletion. Despite the short distance between C04107 and the CT mutation, possible recombinant dogs have been reported with C04107. Although these dogs have a normal phenotype, they carry the C04107 allele 2, which is associated with CT. To study the origin of this possible recombination event we collected a pedigree consisting of two unaffected American Bedlington terriers and their litter of four pups, which were all homozygous for the C04107 2,2 genotype. Mutation analysis showed that two dogs were heterozygous for the CT exon 2 deletion mutation, whereas four dogs were homozygous for the wild-type (WT) allele. Haplotype analysis was performed using two DNA markers in the MURR1 gene and four DNA markers flanking the gene and spanning a region of approximately 600 kb. Surprisingly, we identified a new haplotype (haplotype C) that contains allele 2 of marker C04107 in combination with the WT MURR1 allele. Analysis of the flanking markers suggests there are different genetic backgrounds in the Bedlington terrier population.

Animal models of copper-associated liver disease

Recent advances in molecular biology have made possible the identification of genetic defects responsible for Wilson's disease, Indian childhood cirrhosis and copper toxicosis in Long Evans Cinnamon rats, toxic milk mice, and Bedlington terriers. The Wilson's disease gene is localized on human chromosome 13 and codes for ATP7B, a copper transporting P-type ATPase. A genetic defect similar to that of Wilson's disease occurs in Long Evans Cinnamon rats and toxic milk mice. Familial copper storage disorders in Bedlington and West Highland white terriers are associated with early subclinical disease, and copper accumulation with subsequent liver injury culminating in cirrhosis. The canine copper toxicosis locus in Bedlington terriers has been mapped to canine chromosome region CFA 10q26. Recently, a mutated MURR1 gene was discovered in Bedlington terriers affected with the disease. Idiopathic childhood cirrhosis is biochemically similar to copper toxicosis in Bedlington terriers, but clinically much more severe. Both conditions are characterized by the absence of neurologic damage and Kayser-Fleisher rings, and normal ceruloplasmin levels. A recent study added North Ronaldsay sheep to the list of promising animal models to study Indian childhood cirrhosis. Morphologic similarities between the two conditions include periportal to panlobular copper retention and liver changes varying from active hepatitis to panlobular pericellular fibrosis, and cirrhosis. Certain copper-associated disorders, such as chronic active hepatitis in Doberman pinschers and Skye terrier hepatitis are characterized by copper retention secondary to the underlying disease, thus resembling primary biliary cirrhosis in humans. Copper-associated liver disease has increasingly being recognized in Dalmatians. Copper-associated liver diseases in Dalmatians and Long Evans Cinnamom rats share many morphologic features. Fulminant hepatic failure in Dalmatians is characterized by high serum activities of alanine aminotransferase and aspartate aminotransferase, and severe necrosis of centrilobular areas (periacinar, zone 3) hepatocytes. Macrophages and surviving hepatocytes contain copper-positive material. Liver disease associated with periacinar copper accumulation has also been described in Siamese cats. Many questions regarding copper metabolism in mammals, genetic background, pathogenesis and treatment of copper-associated liver diseases remain to be answered. This review describes the similarities between the clinico-pathological features of spontaneous copper-associated diseases in humans and domestic animals.

Identification of a new copper metabolism gene by positional cloning in a purebred dog population.

Domesticated animal species such as dogs and cats, with their many different characteristics and breed-specific diseases, and their close relationship and shared environment with humans, are a potentially rich source for the identification of the genetic contribution to human biology and disease. Copper toxicosis in Bedlington terriers is a genetic disease occurring with a high prevalence worldwide and is unique to this breed. Copper homeostasis appears to be well regulated in mammals. Two copper carrier proteins have been identified in man and rodents which, when dysfunctional, cause either copper deficiency (Menkes disease) or copper accumulation in various tissues (Wilson disease). However, these proteins are not primarily involved in the biliary excretion of copper. Bedlington terriers have a high prevalence of copper toxicosis and it is well documented that their biliary excretion of copper is impaired. This disease is of direct relevance for the understanding of copper metabolism in mammals. Previously, we mapped the copper toxicosis gene to dog chromosome region 10q26. Based on DNA samples obtained from privately owned dogs, we were able to confine the localization of the copper toxicosis gene to a region of <500 kb by linkage disequilibrium mapping. While screening genes and expressed sequence tags in this region for mutations we found that exon 2 of the MURR1 gene is deleted in both alleles of all affected Bedlington terriers and in single alleles in obligate carriers. Although the function of the MURR1 gene is still unknown, the discovery of a mutated MURR1 gene in Bedlington terriers with copper toxicosis provides a new lead to disentangling the complexities of copper metabolism in mammals.

Population Dynamics of Inherited Copper Toxicosis in Dutch Bedlington Terriers (1977–1997)

Inherited copper toxicosis in Bedlington terriers was 1st reported in 1975 and the entire Dutch population was examined from 1976 until the present for presence of the disease. To examine the effect on the prevalence of the disease of excluding affected dogs from breeding we have compared 2 time cohorts, the 1st consisting of dogs born from January 1, 1976, to January 1, 1986 (n = 155), and the 2nd of dogs born from January 1, 1990, to January 1, 1997 (n = 195). The diagnosis was made in the 1st cohort by evaluating liver biopsies, and in the 2nd cohort with a DNA marker. The population was also resolved into clusters of related dogs to analyze the familial distribution of the disease in the population and to search for ancient founders of the disease among the ancestors of sick dogs. Forty‐six percent of dogs examined between 1976 and 1986 had copper toxicosis. Eleven percent of dogs examined in the 2nd cohort had evidence of disease. This reduction was achieved while maintaining the already limited genetic heterogeneity of the population: the number of clusters and the mean relatedness between the clusters were similar in both time cohorts. The disease was evenly distributed over the clusters of related dogs in both cohorts. All ancestors had contributed to the distribution of copper toxicosis and no specific founders could be identified. This indicates that when the breed was established in The Netherlands, the disease was already highly prevalent in the founding dogs.