Journal of Heredity 2003:94(3)
© 2003 The American Genetic Association 94:256-259
Indirect Molecular Diagnosis of Copper Toxicosis in Bedlington Terriers Is Complicated by Haplotype Diversity
From the Department of Medical Genetics, University Medical Center Utrecht (van de Sluis and Wijmenga) and the Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Purdue University, West Lafayette, Indiana (Peter).
Address correspondence to Cisca Wijmenga, Department of Medical Genetics, KC04.084.2, University Medical Center Utrecht, WKZ, Lundlaan 6, 3584 EA Utrecht, The Netherlands, or e-mail: t.n.wijmenga{at}med.uu.nl.
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Abstract |
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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.
Linkage disequilibrium (LD) is the nonrandom association of alleles at linked loci (Jorde 2000) and has been successfully used in localizing Mendelian disease genes. One of the tools that make use of the presence of LD around a disease gene is homozygosity mapping. It is applied to families in which autosomal recessive diseases segregate, and it is based on the identification of patients sharing chromosomal segments in the homozygous state inherited from a common ancestor (Lander and Botstein 1987). As a consequence, homozygosity mapping has been successfully applied in consanguineous families (Neufeld et al. 1997; Wang et al. 1997; Wijmenga et al. 1998) and in isolated human populations, such as the Finns (Peltonen et al. 1999). Purebred dogs, which often have breed-specific diseases, should also offer a unique source of pedigrees for identifying disease loci by LD mapping, since it has been suggested that each dog breed represents an isolated inbred population (Ostrander and Giniger 1997; Ostrander and Kruglyak 2000; Ostrander et al. 2000). LD mapping in genetically homogeneous dog populations has been successfully reported (Acland et al. 1998, 1999; Jonasdottir et al. 2000; Lin et al. 1999; Lingaas et al. 1998).
The copper accumulation disorder, copper toxicosis (CT), is mainly seen in the Bedlington terrier population, where it segregates as an autosomal recessive disease (Hardy et al. 1975; Johnson et al. 1980; Owen and Ludwig 1982). The prevalence of CT in Bedlington terriers is extremely high due to the intense selection during breeding of Bedlington terriers in different countries (Rothuizen et al. 1999). It was anticipated that all affected Bedlington terriers carry an identical CT mutation in the homozygous state, inherited from a common ancestor, due to the extremely high inbreeding. This hypothesis is further substantiated by the observation that all affected Bedlington terriers show the 2,2 genotype for the CT-linked marker C04107 (Rothuizen et al. 1999). We have successfully applied this homozygosity approach to Bedlington terriers with CT and have identified the responsible gene mutation.
The haplotype shared by all affected dogs in the homozygous state could be reduced to a region of approximately 500 kb. Mutation analysis of genes in this 500 kb region eventually led to the identification of the CT gene MURR1; we showed that exon 2 of the MURR1 gene was deleted in all Bedlington terriers with CT (van de Sluis et al. 2002). The discovery of the CT mutation is important for discriminating affected dogs from carriers and healthy Bedlington terriers in order to breed a CT-free stock.
Of interest is that the closely linked C04107 marker is located within intron 1 of MURR1. This close linkage between the C04104 marker and the CT mutation and the fact that affected dogs are homozygous for C04107 allele 2 would indicate that the C04107 marker is a good predictive marker for CT. However, recombination events between the CT mutation and C04107 have also been reported (Haywood et al. 2001; Holmes et al. 1998).
To investigate the origin of a suggestive recombination event between the C04107 marker and the MURR1 exon 2 deletion mutation in Bedlington terriers, blood from a family of six Bedlington terriers in the United States was collected. The parents of the Bedlington terrier litter were healthy and homozygous for the 2,2 genotype of the C04107 marker. Mutation analysis showed that the sire was homozygous for the wild-type (WT) allele and the dam was heterozygous for the CT mutation. Haplotype analysis using five polymorphic markers and a single nucleotide polymorphism (SNP) covering a genomic region of approximately 600 kb revealed a new haplotype containing the allele 2 of marker C04107 in combination with a WT MURR1 allele.
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Materials and Methods |
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Animals and Samples for Diagnostics
Blood was collected from a family of six Bedlington terriers (sire, bitch, and four pups) in the United States. DNA was isolated from peripheral blood according to Miller et al. (1988). The diagnosis of CT was established in dogs I:1 and I:2 based on histological examination and atomic absorption analysis of a wedge liver biopsy. The hepatic copper concentrations were 235 parts per million per gram dry weight (ppm/gdw) for dog I:1 and 220 ppm/gdw for dog I:2. The diagnosis of CT in the pups (II:1, II:2, II:3, and II:4) could not be determined because they were younger than 1 year of age. (It is generally accepted that the diagnosis of CT in Bedlington terriers is not reliable at an age of less than 1 year [Rothuizen et al. 1999]). The litter was included in the molecular analysis for determining the phase of the genotypes in establishing haplotypes.
Haplotype Analysis
Dog DNA was amplified for the microsatellite markers CF10B18, CF10B17, C04107, CF10B23, and CF10B11 and analyzed as described by van de Sluis et al. (2002). In addition, a SNP in intron 2 of MURR1 was detected by restriction fragment length polymorphism (RFLP) analysis. Six micrograms of genomic DNA were digested with BamHI and hybridized with exon 3 of the MURR1 gene, showing the presence (allele 1) or absence (allele 2) of the BamHI restriction site. Mutation analysis on MURR1 was performed by Southern blot analysis [described previously by van de Sluis et al. (2002)]. In cases where no deletion of exon 2 was discovered, the entire MURR1 gene was subjected to sequence analysis. The haplotypes for the six markers and the MURR1 exon 2 deletion were constructed manually using the pedigree information.
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Results |
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In this study we collected a pedigree consisting of six Bedlington terriers originating from the United States. The parents, dogs I:1 and I:2, were diagnosed as healthy, since the histological examination showed a normal liver architecture and their hepatic copper concentrations were within in the normal range of less than 400 ppm on a dry weight basis (Johnson et al. 1984; Thornburg 2000). Mutation analysis demonstrated that I:1 dog was homozygous for the WT allele, showing two copies of a 1.5 EcoRI fragment, and I:2 was heterozygous for the MURR1 exon 2 deletion, showing only one copy of a 1.5 EcoRI fragment (Figure 1).
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Five polymorphic markers and a SNP were used to construct the haplotypes of the CT region depicted in Figure 2A. All six dogs were homozygous for allele 2 of the C04107 marker and for allele 1 of the CF10B17 marker. Two dogs, I:2 and II:4, were heterozygous for the CT haplotype B and a novel haplotype C (Figure 2A); haplotype B was recently shown to contain the MURR1 exon 2 deletion (van de Sluis et al. 2002). Three pups were homozygous for haplotype C and the fourth pup was heterozygous for haplotype B and haplotype C. The alleles, 7, 4, and 5, for the markers CF10B18, CF10B23, and CF10B11, respectively, have not previously been identified in our cohort of Bedlington terriers, and they may be unique to the U.S. breed. The different haplotypes that we can distinguish using markers within the MURR1 gene (C04107, the exon 2 deletion and SNP-B) are depicted in Figure 2B; these three markers span a genomic region of less then 100 kb. Haplotypes A and B have already been published (van de Sluis et al. 2002).
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Discussion |
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We recently identified a mutation in the MURR1 gene suggesting that MURR1 is the underlying gene in CT in Bedlington terriers. We found that exon 2 of the MURR1 gene was deleted by a large genomic deletion resulting in a truncated protein of 94 amino acids, which suggested a loss of function of the protein. Given the high degree of inbreeding, it is likely that all Bedlington terriers suffering from CT have exactly the same mutation inherited from a common ancestor.
During characterization of the genomic structure of the canine MURR1 gene we found that the C04107 marker was localized in intron 1 of the MURR1 gene. All affected dogs in our study were homozygous for both the exon 2 deletion and allele 2 of the C04107 marker. The distance between the MURR1 deletion and the C04107 marker is estimated to be approximately 8 kb, which is extremely close and makes C04107 an excellent predictive marker for CT. However, several groups have recently published recombinants between the C04107 marker and the CT mutation (Haywood et al. 2001; Holmes et al. 1998). To investigate the origin of a possible recombination event between C04107 and exon 2 of the MURR1 gene, we constructed haplotypes from Bedlington terriers from the United States that were homozygous for allele 2 of the C04107 marker. Haplotype analysis revealed a new haplotype (haplotype C) in 10 of 12 chromosomes tested. Although haplotype C contains allele 2 of marker C04107, it does not contain a MURR1 mutation and is therefore not associated with an increased risk for CT. Haplotype C has never been observed in the Bedlington terrier population from Belgium or the United Kingdom (van de Sluis et al. 2002). Nevertheless, this observation has immediate implications for molecular diagnostics using the C04107 marker in this pedigree. Since allele 2 of the C04107 marker is unlinked with CT in this pedigree, the only reliable diagnostic test is mutation analysis for the exon 2 deletion of the MURR1 gene.
It is interesting to speculate about the origin of haplotype C. There are at least two possibilities: either haplotypes B and C are related to each other, or haplotypes B and C have a different genetic origin, with the latter seeming more likely. If haplotypes B and C are related to each other, we have to assume either a double recombination event between C04107 and the exon 2 deletion, and between the exon 2 deletion and SNP-B, or two sequential recombination events. Double recombinants over a region of approximately 100 kb are extremely rare. Two sequential events are also unlikely because the diversity of haplotypes would have been larger than currently observed. Since the flanking markers for haplotypes B and C are quite different, these recombination events would have to have occurred a long time ago and there would have been enough time for fixation of the different haplotypes. But a much more likely explanation is the presence of a different genetic background for the American Bedlington terrier population, suggesting that the origin of the present Bedlington terrier was selected from one or more different breed lineages or is caused by outcrossing with other breeds into the American Bedlington terrier.
Although the pedigree reported here in no way represents the entire Bedlington terrier population in the United States, it is possible that our findings in this pedigree explain the uncoupling of allele 2 for the C04107 marker and the CT mutation in the pedigrees studied by Yuzbasiyan-Gurkan et al. (1997). They used seven Bedlington terrier pedigrees for the initial identification of the CT-linked C04107 marker. In these seven pedigrees, 21 unaffected Bedlington terriers were reported that were 2,2 for the C04107 marker. It would be interesting to study these seven pedigrees using extensive haplotype analysis to determine the origin of this C04107-allele 2 chromosome.
The results of our study show that the C04107 marker is not predictive for CT in our American pedigree, while the predictive value in other American pedigrees needs to be further evaluated. As long as detailed insight into the CT-linked haplotype is lacking, analysis for the exon 2 deletion of the MURR1 gene is the only reliable diagnostic test.
Limited genetic diversity was hypothesized in the Bedlington terrier population (Rothuizen et al. 1999), as assumed for other purebred dogs, which are therefore attractive models for LD mapping (Ostrander and Kruglyak 2000). Identification of the CT gene by LD mapping confirms this hypothesis. In retrospect, however, it seems that the Bedlington terrier is not as genetically homogeneous as first expected. The identification of the MURR1 gene causing CT was so successful because one large and genetically homogenous pedigree was used in which the C04107 marker was 100% predictive. Thus these data once more stress the importance of using a genetically homogeneous population for LD mapping.
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Acknowledgments |
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This paper was delivered at the Advances in Canine and Feline Genomics symposium, St. Louis, MO, May 16–19, 2002. We thank the breeder for providing DNA samples of Bedlington terriers, and Jackie Senior for improving the manuscript. We also thank the British Bedlington terrier club for its financial support. This work also received financial support from the International Copper Association (TPT0551-98) and The Netherlands Organization for Scientific Research (NWO 902-23-254).
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Footnotes |
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Corresponding Editor: Urs Giger
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