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.     

New haplotypes in the Bedlington terrier indicate complexity in copper toxicosis

Copper toxicosis (CT) is an autosomal recessive disorder common in Bedlington terriers. Previously, the CT locus was mapped to canine Chromosome (Chr) 10q26 through linkage to marker C04107. Diagnosis, traditionally based on liver biopsy, has recently shifted to interpretation of the C04107 microsatellite alleles where allele 2 segregates with the disease with 90–95% accuracy. Recently, CT has been attributed to a deletion of exon 2 in the MURR1 gene. We also identified a deletion of exon 2 of MURR1 in our collection of 2-2 homozygous affected terriers. However, our collection also included affected 1-1 homozygotes and 1-2 heterozygotes, and these dogs did not have the homozygous deletion. In addition to C04107, we analyzed an adjacent microsatellite (C04107B), and two novel SNPs, all within intron 1 of MURR1, and sequenced all exons and their intronic boundaries. Pedigree analysis indicates that there are two typical haplotypes, one normal and one affected, maintaining complete linkage disequilibrium between C04107 allele 2 and the deletion in most pedigrees. Most importantly, we identified a recombinant haplotype present in a North American pedigree, where allele 2 is not linked with the deletion, and a fourth haplotype containing a splice site variant. Although the splice site alteration appears to be a normal variant, it is present in two affected dogs, which do not carry homozygous deletions of MURR1.     

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.     

Copper toxicosis in non-COMMD1 Bedlington terriers is associated with metal transport gene ABCA12

Wilson’s disease, caused by a mutation in the ATP-ase 7B gene, is the only genetically characterised human disease with inhibition of biliary copper excretion and toxic copper accumulation in liver and occasionally brain. A similar copper toxicosis occurs in Bedlington terriers (CT) with liver damage only. Although CT has been associated with a defect in the COMMD1 gene (COMMD1 ^del/del), Bedlington terriers with CT and lacking this mutation are also recognised (non-COMMD1 ^del/del).A study was designed to identify any other gene polymorphisms associated with copper toxicity in Bedlington terriers employing genome wide association studies (GWAS) followed by deep sequencing of the candidate region. Blood for DNA analysis and liver for confirmation of the diagnosis was obtained from 30 non-COMMD1 ^del/del Bedlington terriers comprising equal numbers of CT-affected dogs and controls. DNA was initially subjected to GWAS screening and then further sequencing to target the putative mutant gene.The study has identified a significant disease association with a region on chromosome 37 containing identified SNP’s which are highly significantly associated with non-COMMD1 ^del/del Bedlington terrier CT. This region contains the ABCA12 gene which bears a close functional relationship to ATP-ase 7B responsible for Wilson’s disease in man.     

Quantitative PCR method to detect a 13-kb deletion in the MURR1 gene associated with copper toxicosis and HIV-1 replication

The recently discovered locus for copper toxicosis (CT) in Bedlington terriers (BT) has a 13-kb deletion enveloping the 187-bp exon-2 of the MURR1 gene. This MURR1 gene is not only involved with biliary copper excretion but also associated with HIV-1 replication. The microsatellite C04107 lying in an intron of the MURR1 gene is highly associated with the disease but shows haplotype diversity. The only solid molecular test for the disease is by showing the deletion in exon-2 in cDNA in liver tissue; this test is not robust on RNA from peripheral leukocytes because of their low MURR1 expression level. Because of these drawbacks, we developed a new quantitative PCR (Q-PCR) protocol. Here we show that the MURR1 exon-2/exon-3 ratio measured by Q-PCR on genomic DNA correlates perfectly with the microsatellite marker and with RT-PCR data from blood samples, buccal swabs, and liver biopsies. In view of the important role of MURR1 in cells of many tissues, this new test has a wide range of applications in comparative biomedical research. Furthermore, Q-PCR on DNA may be a new tool in general to analyze mutations that cannot be approached by standard methods.Robert P. Favier and Bart Spee contributed equally to this work.     

The copper chaperone Atox1 in canine copper toxicosis in Bedlington terriers

Copper toxicosis, resulting in liver disease, commonly occurs in Bedlington terriers. This recessively inherited disorder, similar in many respects to Wilson disease, is of particular interest because the canine Atp7b gene, homologous to ATP7B defective in Wilson disease, is not responsible for canine copper toxicosis as has been expected. Atox1, a copper chaperone delivering copper to Atp7b, therefore became a potential candidate. We cloned canine Atox1, which shows conserved motifs of the copper-binding domain (MTCXXC) and of the lysine-rich region (KTGK), and showed 88, 80, and 41% amino acid sequence identity with the orthologous mouse, human, and yeast proteins. No gross deletions of Atox1 could be identified in the affected Bedlington terriers by Southern blot analysis of genomic DNA. The canine Atox1 gene spans about 4 kb, with a 204-bp open reading frame cDNA contained within two exons. Sequence analysis of the coding regions, including intron/exon boundaries, showed no mutations in Atox1 from genomic DNA of an affected dog. We have also identified an apparently nontranscribed canine Atox1 pseudogene, with 12 sequence changes and no intron. Mapping of Atox1 and a marker closely linked to the canine copper toxicosis locus indicated lack of synteny. Atox1 is therefore excluded as a candidate gene for canine copper toxicosis, indicating that some other unidentified gene must be responsible for this copper storage disease in dogs and also suggesting the possibility of a similar gene responsible for a copper storage disease in humans.     

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.     

Diagnostic value of a microsatellite DNA marker for copper toxicosis in West-European Bedlington terriers and incidence of the disease

Recently, linkage of a DNA microsatellite marker to inherited copper toxicosis has been reported in American Bedlington terrier families. Due to the fact that there is little exchange of breeding stock between the USA and Europe, it remains to be investigated whether in Europe the marker is informative and is linked with the disease. We have therefore examined the diagnostic value of the microsatellite marker in the European Bedlington. In 130 dogs at least one year of age (62 from The Netherlands, 35 from Belgium, and 33 from Germany) histo- or cytochemical staining of copper was done in liver biopsies. Based on liver histo- or cytochemistry, 51 dogs were obligate carriers, and 25 dogs had copper toxicosis. The inferred genotypes of these 76 dogs were compared with the marker genotypes. All dogs with the disease were homozygous for the 167 bp marker allele. All obligate carriers were heterozygotes with the 167 bp and a 163-bp alleles. All phenotypically healthy dogs were either homozygous for the 163 bp allele or heterozygous. Thus, the marker was in complete linkage disequilibrium with the putative copper toxicosis gene with the 167 bp allele in phase with the disease allele. The frequencies of the 167 bp and the 163 bp allele, respectively, were 0.33 and 0.67 in Dutch dogs, 0.31 and 0.69 in German dogs, and 0.57 and 0.43 in Belgian dogs. We have confirmed the utility of this marker for diagnosis of inherited copper toxicosis in European Bedlington terriers.     

Canine models of copper toxicosis for understanding mammalian copper metabolism

Hereditary forms of copper toxicosis exist in man and dogs. In man, Wilson's disease is the best studied disorder of copper overload, resulting from mutations in the gene coding for the copper transporter ATP7B. Forms of copper toxicosis for which no causal gene is known yet are recognized as well, often in young children. Although advances have been made in unraveling the genetic background of disorders of copper metabolism in man, many questions regarding disease mechanisms and copper homeostasis remain unanswered. Genetic studies in the Bedlington terrier, a dog breed affected with copper toxicosis, identified COMMD1, a gene that was previously unknown to be involved in copper metabolism. Besides the Bedlington terrier, a number of other dog breeds suffer from hereditary copper toxicosis and show similar phenotypes to humans with copper storage disorders. Unlike the heterogeneity of most human populations, the genetic structure within a purebred dog population is homogeneous, which is advantageous for unraveling the molecular genetics of complex diseases. This article reviews the work that has been done on the Bedlington terrier, summarizes what was learned from studies into COMMD1 function, describes hereditary copper toxicosis phenotypes in other dog breeds, and discusses the opportunities for genome-wide association studies on copper toxicosis in the dog to contribute to the understanding of mammalian copper metabolism and copper metabolism disorders in man.     

Functional consequences of RNA interference targeting COMMD1 in a canine hepatic cell line in relation to copper toxicosis

A deletion in the copper metabolism (Murr1) domain containing 1 (COMMD1) gene is associated with hepatic copper toxicosis in dogs, yet evidence of copper retention in COMMD1-depleted hepatic cells has not been shown. In a dog hepatic cell line, we analysed the copper metabolic functions after an 80% (mRNA and protein) COMMD1 reduction with COMMD1-targeting siRNAs. Exposure to 64Cu resulted in a significant increase in copper retention in COMMD1-depleted cells. COMMD1-depleted cells were almost three times more sensitive to high extracellular copper concentrations. Copper-mediated regulation of metallothionein gene expression was enhanced in COMMD1-depleted cells. Based on the increased copper accumulation and enhanced cellular copper responses upon COMMD1 reduction, we conclude that COMMD1 has a major regulatory function for intracellular copper levels in hepatic cells.     

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.     

COMMD1, a multi-potent intracellular protein involved in copper homeostasis,protein trafficking,inflammation, and cancer

Copper is a trace element indispensable for life, but at the same time it is implicated in reactive oxygen species formation. Several inherited copper storage diseases are described of which Wilson disease (copper overload, mutations in ATP7B gene) and Menkes disease (copper deficiency, mutations in ATP7A gene) are the most prominent ones. After the discovery in 2002 of a novel gene product (i.e. COMMD1) involved in hepatic copper handling in Bedlington terriers, studies on the mechanism of action of COMMD1 revealed numerous non-copper related functions. Effects on hepatic copper handling are likely mediated via interactions with ATP7B. In addition, COMMD1 has many more interacting partners which guide their routing to either the plasma membrane or, often in an ubiquitination-dependent fashion, trigger their proteolysis via the S26 proteasome. By stimulating NF-κB ubiquitination, COMMD1 dampens an inflammatory reaction. Finally, targeting COMMD1 function can be a novel approach in the treatment of tumors.     

Evaluation of the present breeding programme against copper toxicosis in Danish Bedlington terriers

A breeding programme to eradicate copper toxicosis in Danish Bedlington terriers has been established based on a DNA marker test. Genotyping of both parents is compulsory and after 1 January 2000, only homozygous non-carriers are used for breeding. In this study, two groups of Bedlington terriers were genotyped at 18 microsatellite loci. One group represented the original population of Bedlington terriers before introducing the breeding programme (n = 23); the other represented a group of homozygous non-carriers (n = 24) available for breeding after year 2000. Allele numbers, allele frequencies, observed heterozygosities (Ho), expected heterozygosities (He), locus-specific coefficients of inbreeding (Fl) and Nei's genetic distance (D) was calculated. Individual coefficients of inbreeding (Fi) were calculated from the pedigrees and an assignment test was performed. Four rare alleles were lost in the group of homozygous non-carriers. No significant differences were observed between the mean values of allele numbers, Ho, He, Fl and Fi of the two populations of dogs. Nei's genetic distance between the two populations was 0.06 and 88% of the homozygous non-carriers were assigned correctly in the assignment test. The overall diversity of the breed was low (Ho = 0.41) and the breeders were advised to include the heterozygous carriers again.     

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.     

COMMD1 upregulation is involved in copper efflux from ischemic hearts

Copper depletion is associated with myocardial ischemic infarction, in which copper metabolism MURR domain 1 (COMMD1) is increased. The present study was undertaken to test the hypothesis that the elevated COMMD1 is responsible for copper loss from the ischemic myocardium, thus worsening myocardial ischemic injury. Mice (C57BL/6J) were subjected to left anterior descending coronary artery permanent ligation to induce myocardial ischemic infarction. In the ischemic myocardium, copper reduction was associated with a significant increase in the protein level of COMMD1. A tamoxifen-inducible, cardiomyocyte -specific Commd1 knockout mouse (C57BL/6J) model (COMMD1^CMC▲/▲) was generated using the Cre-LoxP recombination system. COMMD1^CMC▲/▲ and wild-type littermates were subjected to the same permanent ligation of left anterior descending coronary artery. At the 7th day after ischemic insult, COMMD1 deficiency suppressed copper loss in the heart, along with preservation of vascular endothelial growth factor and vascular endothelial growth factor receptor 1 expression and the integrity of the vascular system in the ischemic myocardium. Corresponding to this change, infarct size of ischemic heart was reduced and myocardial contractile function was well preserved in COMMD1^CMC▲/▲ mice. These results thus demonstrate that upregulation of COMMD1 is at least partially responsible for copper efflux from the ischemic heart. Cardiomyocyte-specific deletion of COMMD1 helps preserve the availability of copper for angiogenesis, thus suppressing myocardial ischemic dysfunction.     

ATP6H, a subunit of vacuolar ATPase involved in metal transport: evaluation in canine copper toxicosis

Copper toxicosis (CT), resulting in liver disease, occurs commonly in Bedlington terriers. Canine CT is of particular interest because identification of the causative gene may lead to the discovery of another important gene in the copper transport pathway possibly related to human copper diseases not yet identified. Homologs of the copper transporting ATPase ATP7B, defective in Wilson disease, and the copper chaperone ATOX1 were potential candidates, but both have been excluded. The CT locus in Bedlington terriers has been mapped to canine chromosome region CFA10q26, which has a syntenic human chromosome region, HAS2p13-21. The gene ATP6H, for human vacuolar proton-ATPase subunit M9.2, is associated with copper and iron transport in yeast and has been mapped to HAS2p21 and suggested as a candidate gene for CT. We cloned canine ATP6H, which encodes a predicted protein with 99% amino acid sequence identity to the orthologous human protein. Canine ATP6H shows a conserved potential metal binding site, CSVCC, and a glycosylation site, NET. The canine ATP6H is organized into four exons, with a 246-bp open reading frame. Sequence analysis of the coding regions showed no mutations in ATP6H from genomic DNA of an affected dog. We have also identified two, apparently non-transcribed canine ATP6H pseudogenes. Mapping of the true ATP6H gene and a marker closely linked to the CT locus on a canine radiation hybrid panel indicted lack of close physical association. We have therefore excluded canine ATP6H as a candidate gene for canine copper toxicosis, indicating that some other unidentified gene is responsible for this copper storage disease. Received: 8 February 2001 / Accepted: 12 April 2001     

Canine hepatic lysosomal copper protein: identification as metallothionein

We studied the amino acid sequence of canine hepatic lysosomal copper protein obtained from Bedlington terriers affected by inherited copper toxicosis. The primary structure was determined by manual Edman degradations and carboxypeptidase Y digestions of peptides generated by cleavage of the S-carboxyamidomethylated and S-aminoethylated protein with trypsin. Although the amino terminus was blocked and heterogeneous, the protein showed extensive sequence homology to mammalian metallothioneins. In particular, all cysteinyl residues were conserved, in agreement with their function as metal ligands. The microheterogeneity observed in the amino-terminal part of the molecule indicated the presence of two isoforms in canine liver like those found in most other mammals studied so far.     

The puzzle posed by COMMD1, a newly discovered protein binding Cu(II)

Copper is critically important for cellular metabolism. It plays essential roles in developmental processes, including angiogenesis. The liver is central to mammalian copper homeostasis: biliary excretion is the major route of excretion for ingested copper and serves to regulate the total amount of copper in the organism. An extensive network of proteins manipulates copper disposition in hepatocytes, but comparatively little is known about this protein system. Copper exists in two oxidation states: most extracellular copper is Cu(II) and most, if not all, intracellular copper is Cu(I). Typical intracellular copper-binding proteins, such as the Cu-transporting P-type ATPases ATP7B (Wilson ATPase) and ATP7A (Menkes ATPase), bind copper as Cu(I). Accordingly, the recent discovery that the ubiquitous protein COMMD1 binds Cu(II) exclusively raises the question as to what role Cu(II) may play in intracellular processes. This issue is particularly important in the liver and brain. In humans, Wilson’s disease, due to mutations in ATP7B, exhibits progressive liver damage from copper accumulation; in some Bedlington terriers, mutations in COMMD1 are associated with chronic copper-overloaded liver disease, clinically distinct from Wilson’s disease. It seems unlikely that Cu(II), which generates reactive oxygen species through the Fenton reaction, has a physiological role intracellularly; however, Cu(II) might be the preferred state of copper for elimination from the cell, such as by biliary excretion. We argue that COMMD1 participates in the normal disposition of copper within the hepatocyte and we speculate about that role. COMMD1 may contribute to the mechanism of biliary excretion of copper by virtue of binding Cu(II). Additionally, or alternatively, COMMD1 may be an important component of an intracellular system for utilizing Cu(II), or for detecting and detoxifying it.     

A study of the role of metallothionein in the inherited copper toxicosis of dogs.

The role of metallothionein (MT) was assessed in the copper-loading disease prevalent in Bedlington terriers. Fractionation of tissue supernatants over Sephadex G-75 showed that most of the additional cytosolic copper present in liver tissue of these dogs was bound to MT, and that substantially more MT-bound copper could be solubilized by detergent plus mercaptoethanol. Zinc contents were only slightly raised, although most of the extra zinc was associated with a 4000-Mr ligand. Ion-exchange chromatography revealed two isoproteins, MT1 and MT2, in all the dog liver samples examined. In Bedlington terrier liver, copper associated with both isoproteins was increased, although the increase for MT2 was greater than for MT1. The content of MT protein was also raised, although cell-free translations and RNA blots of total liver RNA showed that this increase was not associated with a rise in MT mRNA. The significance of these results to the mechanism of copper accumulation in the Bedlington terrier disorder is discussed.