Refined genetic and comparative physical mapping of the canine
copper toxicosis locus
Bart van de Sluis,
Monique van Wolferen,
Nigel G. Holmes,
Peter L. Pearson,
Bernard A. van Oost,
Department of Medical Genetics, KC04.084.2, University Medical Center Utrecht, WKZ, Lundlaan 6, 3584 EA Utrecht, the Netherlands
Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, the Netherlands
Centre for Preventive Medicine, Animal Health Trust, Suffolk, UK
Received: 16 December 1999 / Accepted: 23 February 2000
Abstract. Recently, the copper toxicosis (CT) locus in Bedlington
terriers was assigned to canine chromosome region CFA10q26,
which is homologous to human chromosome region HSA2p13-21.
A comparative map between CFA10q21-26 and HSA2p13-21 was
constructed by using genes already localized to HSA2p13-21. A
high-resolution radiation map of CFA10q21-26 was constructed to
facilitate positional cloning of the CT gene. For this map, seven
Type I and eleven Type II markers were mapped. Using homozy-
gosity mapping, the CT locus could be confined to a 42.3 cR
region, between the FH2523 and C10.602 markers. On the basis of
a partial BAC contig, it was estimated that 1-cR
to approximately 210 kb, implying that the CT candidate region is
therefore estimated to be about 9 Mb.
Copper toxicosis (CT) in Bedlington terriers is an autosomal re-
cessive disorder (Johnson et al. 1980; Owen and Ludwig 1982)
characterized by an inefficient excretion of copper via the bile (Su
et al. 1982), resulting in accumulation of copper in the liver, lead-
ing to chronic hepatitis and, ultimately, cirrhosis (Twedt et al.
1979). Although CT is phenotypically very similar to Wilson dis-
ease (WD; Su et al. 1982), a human copper storage disorder, the
WD gene ATP7B (Bull et al. 1993) was recently excluded as a
candidate for CT on the basis of mapping criteria (Dagenais et al.
1999; van de Sluis et al. 1999). In addition, the proposed mam-
malian copper transport genes CTR1 and CTR2 (Zhou and
Gitschier 1997) and the copper chaperone ATOX1 (Klomp et al.
1997) have also been excluded as candidate genes for CT (Dage-
nais et al. 1999; van de Sluis et al. 1999).
The CT locus has been assigned to canine Chromosome (Chr)
10, region q26 (CFA10q26) (van de Sluis et al. 1999), based on the
cytogenetic localization of the CT-linked microsatellite marker
C04107 (Yuzbasiyan-Gurkan et al. 1997). This canine chromo-
somal region was found to be homologous to human Chr 2, region
p13-21 (van de Sluis et al. 1999). As yet, no possible CT candidate
genes with a role in copper homeostasis have been identified in
Owing to intense selection in the breeding of Bedlington ter-
riers, the prevalence of CT in these dogs in different countries
(Rothuizen et al. 1999) is extremely high. It is likely that all
affected Bedlington terriers carry an identical CT mutation in the
homozygous state, which is inherited from a common ancestor
originating from England. All affected Bedlington terriers show
the genotype 2,2 for marker C04107 (Rothuizen et al. 1999;
Yuzbasiyan-Gurkan et al. 1997). So far, only one affected Bedling-
ton terrier with the C04107 genotype 1,2 has been reported
(Holmes et al. 1998). Hence, it should be feasible to delineate the
CT region by homozygosity mapping, in a way similar to that
performed in a variety of human diseases (Neufeld et al. 1997;
Wang et al. 1997; Wijmenga et al. 1998). Homozygosity mapping
is based on the inheritance of two identical copies of the disease
locus and the surrounding chromosomal region from a common
ancestor (Lander and Botstein 1987).
Although resources for genetic mapping studies in dogs are
becoming better, a high-resolution genetic map of CFA10q26 is
not yet available. Therefore, we set out to construct a high-
resolution radiation hybrid (RH) map of CFA10q26. Such a high-
resolution map of CFA10q21-26 will facilitate the mapping of
candidate genes from the comparative genetic regions in human
and mouse. The progress being made in sequencing the human
genome will certainly lead to the identification of all genes present
in the target area in the near future (Collins et al. 1998). Moreover,
construction of this high-resolution map will be instrumental in
developing an increased density of polymorphic markers that will
facilitate the localization of the CT gene by establishing the region
of homozygosity in our panel of affected dogs.
Mapping of Type I (ESTs and genes) and Type II markers
(microsatellites) was performed on the canine T72 RH panel (Re-
search Genetics, Huntsville, Ala., USA), and the human Gene-
bridge 4 RH panel (Gyapay et al. 1996).
Homozygosity mapping refined the CT region to a 42.3 cR
area, between markers FH2523 and C10.602. This CT-candidate
region is homologous to the region in the human genome between
the genes SPTBN1 and SLC1A4, a region of approximately 30
in the dog genome was found to be
equivalent to 210 kb, resulting in a CT-candidate region of ap-
proximately 9 Mb.
Materials and methods
Animals and samples for DNA isolation.
Blood was collected from 23
related Bedlington terriers of Belgian origin (9 affected, 12 carriers, 2
unaffected; Fig. 1) and from 11 unrelated affected Bedlington terriers.
DNA was isolated according to Miller et al. (1988). Diagnostic criteria
were described, and the results of the copper analysis were published by
Rothuizen et al. (1999). In addition, DNA from 10 English Bedlington
terriers (4 affected, 3 carriers, 1 unaffected, 2 unknown; Fig. 2, Table 2)
was collected by NGH; their affected status was defined as described by
Holmes et al. (1998).
Identification and characterization of Traced Orthologous Ampli-
fied Sequence Tags (TOASTs) and gene-specific primers.
ers for the TOAST corresponding to ATP6H (R07157) and a new forward
Correspondence to: C. Wijmenga; E-mail: email@example.com
Mammalian Genome 11, 455–460 (2000).
© Springer-Verlag New York Inc. 2000