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 of the COMMD1 (MURR1) mutation causing copper toxicosis in Bedlington terriers

Copper toxicosis is an autosomal recessive disorder affecting Bedlington terriers, characterized by elevated liver copper levels and early death of affected dogs. Genetic linkage mapping studies initially identified linkage between the disease and the microsatellite marker C04107. Subsequently, the deletion of exon 2 of the copper metabolism domain containing 1 (COMMD1) gene (formerly MURR1) was shown to be the major cause of copper toxicosis, although the deletion breakpoints were not defined. In this investigation, polymerase chain reaction (PCR)-based techniques and sequencing were used to isolate the deletion breakpoints, utilizing the newly available dog genome sequence. The breakpoints were positioned at 65.3091 and 65.3489 Mb of dog chromosome 10, in intron 1 and intron 2 of COMMD1 respectively, a deletion of 39.7 kb. The two breakpoints share sequence homology suggesting that homologous recombination may have been responsible for the deletion. Using this information, a genomic diagnostic test for the COMMD1 deletion was developed and compared with microsatellite C04107 genotypes of 40 Bedlington terriers. Results from the 40 samples showed allele 2 of C04107 to be in linkage disequilibrium with the COMMD1 deletion.     

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.     

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.     

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.     

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.     

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.     

CYP21/C4 gene organisation in Italian 21-hydroxylase deficiency families

Summary A total of 33 Italian 21-hydroxylase (21-OH) deficiency families were investigated using a combination of short and long range restriction mapping of the CYP21/C4 gene cluster. The analyses revealed that large-scale length polymorphism in this gene cluster strictly conformed to a compound variable number of tandem repeats (VNTR) plus insertion system with between one and four CYP21 + C4 units and seven BssHII restriction fragment length polymorphisms (RFLPs) (75kb, 80kb, 105kb, 110kb, 135kb, 140kb and 180kb). A total of 9/66 disease haplotypes, but only 1/61 nondisease haplotypes, showed evidence of gene addition by exhibiting three or more CYP21 + C4 repeat units. Of these, two were identified in one 21-OH deficiency patient who has a total of eight CYP21 + C4 units, being homozygous for the HLA haplotype DR2 DQ2 B5 A28. This haplotype carries four CYP21 + C4 units, three of which contain CYP21A-like genes and one of which contains a CYP21B-like gene that presumably carries a pathological point mutation. Of the other gene addition haplotypes associated with 21-OH deficiency, four show three CYP21 + C4 units flanked by HLA-DR1 and HLA-B14 markers. Although such haplotypes have commonly been associated with non-classical 21-OH deficiency, three examples in the present study are unexpectedly found in two salt-wasting patients, who are respectively homozygous or heterozygous for this haplotype. Only 7/66 disease haplotypes showed evidence of a CYP21B gene deletion.     

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.     

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.     

A one base pair deletion in the canine ATP13A2 gene causes exon skipping and late-onset neuronal ceroid lipofuscinosis in the Tibetan terrier

Neuronal ceroid lipofuscinosis (NCL) is a progressive neurodegenerative disease characterized by brain and retinal atrophy and the intracellular accumulation of autofluorescent lysosomal storage bodies resembling lipofuscin in neurons and other cells. Tibetan terriers show a late-onset lethal form of NCL manifesting first visible signs at 5-7 years of age. Genome-wide association analyses for 12 Tibetan-terrier-NCL-cases and 7 Tibetan-terrier controls using the 127K canine Affymetrix SNP chip and mixed model analysis mapped NCL to dog chromosome (CFA) 2 at 83.71-84.72 Mb. Multipoint linkage and association analyses in 376 Tibetan terriers confirmed this genomic region on CFA2. A mutation analysis for 14 positional candidate genes in two NCL-cases and one control revealed a strongly associated single nucleotide polymorphism (SNP) in the MAPK PM20/PM21 gene and a perfectly with NCL associated single base pair deletion (c.1620delG) within exon 16 of the ATP13A2 gene. The c.1620delG mutation in ATP13A2 causes skipping of exon 16 presumably due to a broken exonic splicing enhancer motif. As a result of this mutation, ATP13A2 lacks 69 amino acids. All known 24 NCL cases were homozygous for this deletion and all obligate 35 NCL-carriers were heterozygous. In a sample of 144 dogs from eleven other breeds, the c.1620delG mutation could not be found. Knowledge of the causative mutation for late-onset NCL in Tibetan terrier allows genetic testing of these dogs to avoid matings of carrier animals. ATP13A2 mutations have been described in familial Parkinson syndrome (PARK9). Tibetan terriers with these mutations provide a valuable model for a PARK9-linked disease and possibly for manganese toxicity in synucleinopathies.     

Common deletion of SMAD4 in juvenile polyposis is a mutational hotspot

Juvenile polyposis (JP) is an autosomal dominant syndrome in which affected patients develop upper- and/or lower-gastrointestinal (GI) polyps. A subset of families with JP have germline mutations in the SMAD4 (MADH4) gene and are at increased risk of GI cancers. To date, six families with JP have been described as having the same SMAD4 deletion (1244-1247delAGAC). The objective of the present study is to determine whether this deletion is a common ancestral mutation or a mutational hotspot. DNA from members of four families with JP, from Iowa, Mississippi, Texas, and Finland, that had this 4-bp deletion was used to genotype 15 simple tandem repeat polymorphism (STRP) markers flanking the SMAD4 gene, including 2 new STRPs within 6.3 and 70.9 kb of the deletion. Haplotypes cosegregating with JP in each family were constructed, and the distances of the closest markers were determined from the draft sequence of the human genome. No common haplotype was observed in these four families with JP. A 14-bp region containing the deletion had four direct repeats and one inverted repeat. Because no common ancestor was suggested by haplotype analysis and the sequence flanking the deletion contains repeats frequently associated with microdeletions, this common SMAD4 deletion in JP most likely represents a mutational hotspot.     

Expression of a myelin basic protein gene in transgenic shiverer mice: correction of the dysmyelinating phenotype

Mice homozygous for the autosomal recessive mutation shiverer (shi) lack myelin basic protein (MBP) and exhibit a distinct behavioral pattern including tremors (shivering), convulsions, and early death. We have previously demonstrated that shiverer mice have a partial deletion in the gene encoding MBP. We now have introduced the wild-type MBP gene into the germ line of shiverer mice by microinjection into fertilized eggs. Transgenic shiverer mice homozygous for the introduced gene have MBP mRNA and protein levels that are approximately 25% of normal, and produce compacted myelin with major dense lines. Correct temporal and spatial expression of the MBP gene is achieved with a genomic MBP cosmid clone containing 4 kb of 5' flanking sequence and 1 kb of 3' flanking sequence. Moreover, the four different forms of MBP produced by alternative patterns of RNA splicing are present. These homozygous transgenic shiverer mice no longer shiver nor die prematurely.     

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.     

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.     

Two Independent Mutations in ADAMTS17 Are Associated with Primary Open Angle Glaucoma in the Basset Hound and Basset Fauve de Bretagne Breeds of Dog

PurposeMutations in ADAMTS10 (CFA20) have previously been associated with primary open angle glaucoma (POAG) in the Beagle and Norwegian Elkhound. The closely related gene, ADAMTS17, has also been associated with several different ocular phenotypes in multiple breeds of dog, including primary lens luxation and POAG. We investigated ADAMTS17 as a candidate gene for POAG in the Basset Hound and Basset Fauve de Bretagne dog breeds.MethodsWe performed ADAMTS17 exon resequencing in three Basset Hounds and three Basset Fauve de Bretagne dogs with POAG. Identified variants were genotyped in additional sample cohorts of both breeds and dogs of other breeds to confirm their association with disease.ResultsAll affected Basset Hounds were homozygous for a 19 bp deletion in exon 2 that alters the reading frame and is predicted to lead to a truncated protein. Fifty clinically unaffected Basset Hounds were genotyped for this mutation and all were either heterozygous or homozygous for the wild type allele. Genotyping of 223 Basset Hounds recruited for a different study revealed a mutation frequency of 0.081 and predicted frequency of affected dogs in the population to be 0.007. Based on the entire genotyping dataset the association statistic for the POAG-associated deletion was p = 1.26 x 10⁻¹⁰. All affected Basset Fauve de Bretagne dogs were homozygous for a missense mutation in exon 11 causing a glycine to serine amino acid substitution (G519S) in the disintegrin-like domain of ADAMTS17 which is predicted to alter protein function. Unaffected Basset Fauve de Bretagne dogs were either heterozygous for the mutation (5/24) or homozygous for the wild type allele (19/24). Based on the entire genotyping dataset the association statistic for the POAG-associated deletion was p = 2.80 x 10⁻⁷. Genotyping of 85 dogs of unrelated breeds and 90 dogs of related breeds for this variant was negative.ConclusionThis report documents strong associations between two independent ADAMTS17 mutations and POAG in two different dog breeds.     

WT1 exon 1 deletion/insertion mutations in Wilms tumor patients, associated with di- and trinucleotide repeats and deletion hotspot consensus sequences.

The WT1 gene is known to play a role in at least some cases of Wilms tumor (WT). The first exon of the gene is highly GC rich and contains many short tandem di- and trinucleotide repeats, interrupted direct repeats, and CCTG (CAGG) motifs that have been identified as hotspots for DNA deletions. We have analyzed 80 WT patient samples for mutations in the first exon of WT1, either by SSCP analysis of the first 131 bp of the coding portion of WT1 exon 1 or by size analysis of a PCR product encompassing the coding region of exon 1 in addition to flanking noncoding regions. We report here the occurrence of somatic and germ-line deletion and insertion mutations in this portion of the gene in four WT patients. The mutations are flanked by short direct repeats, and the breakpoints are within 5 nt of a CCTG (CAGG) sequence. These data suggest that a distinctive mutational mechanism, previously unrecognized for this gene, is important for the generation of DNA mutations at the WT1 locus.     

Evolution of Microsatellite Alleles in Four Species of Mice (Genus Apodemus)

Microsatellite length variation was investigated at a highly variable microsatellite locus in four species of Apodemus. Information obtained from microsatellite allele sequences was contrasted with allele sizes, which included 18 electromorphs. Additional analysis of a 400-bp unique sequence in the flanking region identified 26 different haplotype sequences or ``true' alleles in the sample. Three molecular mechanisms, namely, (1) addition/deletion of repeats, (2) substitutions and indels in the flanking region, and (3) mutations interrupting the repeat, contributed to the generation of allelic variation. Size homoplasy can be inferred for alleles within populations, from different populations of the same species, and from different species. We propose that microsatellite flanking sequences may be informative markers for investigating mutation processes in microsatellite repeats as well as phylogenetic relationships among alleles, populations, and species. Received: 3 November 1999 / Accepted: 2 May 2000