WO2003033734A2

WO2003033734A2 - Diagnostic tests for the diagnosis of copper storage disease

Info

Publication number: WO2003033734A2
Application number: PCT/GB2002/004687
Authority: WO
Inventors: Albertus Johannes Alphons Van De Sluis; Cisca Wijmenga; Bernard Anton Van Oost; Jan Rothuizen
Original assignee: Mars Uk Limited
Priority date: 2001-10-19
Filing date: 2002-10-18
Publication date: 2003-04-24
Also published as: WO2003033734A3; GB0125179D0; GB0224326D0; AU2917502A; GB2382077A; AU2002334179A1

Abstract

Methods of diagnosis of copper storage diseases are described. The methods comprise determining whether a subject has a genetic abnormality in the MURR1 gene. The methods are of particular use for the diagnosis of copper toxicosis in Bedlington terriers. Methods of providing non human animals which are neither affected by, nor carriers of, a copper storage disease are also described.

Description

Diagnostic Tests

This invention relates to the diagnosis of mammalian copper storage diseases, particularly in canines and humans.

Canine copper toxicosis (CT) is an autosomal recessive disease which is characterized by inefficient excretion of copper via the bile (II), resulting in accumulation of copper in the liver, and leading to chronic hepatitis and, finally, cirrhosis (13). It is found in several dog breeds. Unless treatment is instituted, most affected dogs die at three to seven years of age. CT is a severe problem in Bedlington Terriers. The frequency of the CT gene in Bedlingtons is estimated to be as high as 50% in the US and England. This means that more than 25% of Bedlingtons are affected by CT, and another 50% are carriers of the disease.

If CT is to be eradicated from Bedlington Terriers and other dog breeds, it is necessary to have a reliable test which can identify both affected animals (particularly those too young to show symptoms of the disease) and carriers of the disease so that these animals are not used for breeding. A genetic linkage test for CT in Bedlington Terriers is known (NetGen) which can identify carriers of the disease with 95% accuracy, and affected terriers with 72% accuracy. However, because such tests are not 100% accurate, breeders cannot guarantee to produce non affected, non carrier terriers.

Copper storage diseases are also known in other mammals. Examples of human copper storage diseases are Wilson's disease, Indian-childhood cirrhosis (ICC), non-Indian childhood cirrhosis (ΝICC), and idiopathic copper toxicosis (ICT). Reliable diagnosis of these diseases is important in order that appropriate treatment can be administered as soon as possible. In particular, diagnosis before symptoms become evident would allow preventative treatment to be administered.

In order to provide reliable diagnostic tests, it is important to understand the molecular basis of the diseases. The molecular basis of some copper storage diseases is starting to be understood. Two copper carrier proteins have been identified in man and rodents which, when dysfunctional, cause either copper deficiency (Menkes disease MIM 277900) or copper accumulation in various tissues (Wilson disease MIM 309400). However, the genetic basis of copper toxicosis is not known. An anonymous microsatellite marker, C04107 (14), was shown to be genetically linked very close to the CT locus in Bedlington terriers. Although this marker has been available since 1995, the molecular basis of the disease has not yet been reported. We have previously reduced the putative CT region to a minimal region of homozygosity of approximately 9Mb (16). However, the gene(s) involved was not identified.

According to the invention there is provided a method of diagnosing whether a subject has, is at risk of developing, or is a carrier of a copper storage disease which comprises determining from a biological sample obtained from the subject whether the subject has a genetic abnormality in the MURRl gene, or an abnormality in an expression product of the MURRl gene.

Sequence of the canine MURRl gene, and separate sequence for each exon is shown below (SEQ ID Nos 1-7). The sequence of the human gene is known and is in the NCBI database (accession number AX 060277). The function of the human MURRl gene has not previously been assigned.

Methods of the invention relate particularly to diagnosis of canine CT. However, it is expected that the methods may be used for the diagnosis of other mammalian copper storage diseases, in particular for non-Indian childhood cirrhosis (NICC) in humans. NICC is an hereditary and fatal form of infantile liver cirrhosis found with high frequency in the Tyrol. Other human copper storage disorders include Wilson's disease Indian-childhood cirrhosis (ICC), and idiopathic copper toxicosis (ICT). The term "copper storage disease" is used herein to mean any disease resulting from an abnormal level of copper in tissue. In general, the abnormal level will be an accumulation of copper, but diseases in which there is an abnormally low level of copper are also within the scope of this term.

The term "MURRl gene" as used herein is not restricted to the protein coding sequence of this gene.

The genetic abnormality may be a deletion in the MURRl gene. In particular, the deletion may comprise exon 2. We have identified a 1.5Kb EcσRI fragment spanning exon 2 of the MURRl gene (this fragment includes exon 2, and intron sequence upstream and downstream of this exon, as shown in Figure 3) which is deleted in all affected Bedlington terriers in the study described in example 1 below. Sequence around the proximal breakpoint is shown in Figure 4, and sequence around the distal breakpoint is shown in Figure 5.

We expect that other deletions or mutations in the MURRl gene may be associated with other mammalian copper storage diseases. In some forms of copper storage disease, it is possible that abnormal expression of a normal MURRl gene could be involved. Such forms may be diagnosed by determining from the biological sample whether the subject has an abnormal expression product (nucleic acid or protein) of the MURRl gene.

Methods by which genetic abnormalities in the MURRl gene may be determined will be apparent to those of ordinary skill in the art. Preferred methods include amplifying nucleic acid of the biological sample (for example, by PCR) and determining from the amplified product whether the subject has a genetic abnormality in the MURRl gene. However, other methods may involve analysis of nucleic acid of the biological sample without amplification. A preferred example is Southern blot analysis of restriction enzyme digested genomic DNA.

Where the genetic abnormality is a deletion of a 1.5Kb EcoRI fragment (or corresponding fragment) spanning exon 2 of the MURRl gene, a probe capable of hybridizing to either strand of this fragment can be used to determine whether nucleic acid of the sample, or whether a product amplified from such nucleic acid, comprises nucleic acid corresponding to the fragment.

Preferred methods of the invention can distinguish between homozygous and heterozygous abnormalities of the MURRl gene. This allows identification both of subjects suffering from a copper storage disease (or likely to develop such a disease) and subjects who are carriers of the disease. This is a particular advantage for non human copper storage diseases, especially canine copper storage diseases, because homozygous normal animals can be identified and used for breeding. For example, methods of the invention should allow eradication of CT from Bedlington terriers.

Where the genetic abnormality is a deletion (for example of a 1.5Kb EcoRI fragment or corresponding fragment spanning exon 2) in the MURRl gene, identification of homozygous and heterozygous abnormalities can be achieved, for example, by amplifying nucleic acid of the MURRl gene, or nucleic acid expressed from the MURRl gene, using first and second oligonucleotide primers capable of hybridizing to regions of the wild-type MURRl gene flanking the deletion. The length of the amplified product will then depend on whether the deletion is present or absent. Genomic nucleic acid, primary transcript, mRNA, or cDNA synthesized from mRNA may be amplified. Amplification of genomic DNA is preferred. It is less preferable to amplify mRNA, or cDNA because this will involve isolation of mRNA from the biological sample which increases the cost and complexity of diagnosis.

Where genomic nucleic acid or primary transcript of the MURRl gene is amplified from the biological sample, and the genetic abnormality is a deletion comprising exon 2, preferably the first oligonucleotide primer is capable of hybridizing to intron sequence upstream of the deletion and the second oligonucleotide primer is capable of hybridizing to intron sequence downstream of the deletion. Where the deletion is a 1.5 Kb EcoRI or corresponding fragment, preferably the first primer is capable of hybridizing to a region of intron 1 of the MURRl gene upstream of the deletion, and the second primer is capable of hybridizing to a region of intron 2 of the MURRl gene downstream of the deletion. Nucleotide sequence upstream of the deletion (i.e. upstream of the proximal breakpoint) is shown in Figure 4, and nucleotide sequence downstream of the deletion (i.e. downstream of the distal breakpoint) is shown in Figure 5. Thus, primers capable of hybridizing (under stringent conditions) to these upstream and downstream sequences are provided for use in a method of diagnosis of a copper storage disease.

Where mRNA (or cDNA synthesized from mRNA) of the MURRl gene is amplified from the biological sample, preferably the first oligonucleotide primer is capable of hybridizing to exon 1 of the MURRl gene, and the second oligonucleotide primer is capable of hybridizing to exon 3 of the MURRl gene. An example of such a test is described in Example 2 below.

Other techniques known to those of ordinary skill in the art could be used to determine from nucleic acid of the biological sample, or from a product amplified from such nucleic acid, whether the subject has a deletion (or other genetic abnormality) in the MURRl gene. Such techniques include primer extension analysis and restriction enzyme analysis. A particularly preferred technique is Southern blot analysis of restriction enzyme digested genomic DNA. An example of such a test is described in Example 3 below.

In other methods, a genetic abnormality of the MURRl gene could be identified by establishing whether the biological sample contains a protein expression product of the gene only found when the genetic abnormality is present. For example, deletion of the 1.5Kb EcøRI fragment is expected to result in expression of a truncated protein from the mutant gene. An antibody capable of binding to the truncated protein, but not to the wild-type protein, could be used to determine whether or not the truncated protein is present in the biological sample, and thus whether the subject has the genetic abnormality.

It is possible that the level of an expression product of a gene other than MURRl correlates with the level of expression of wild-type or an abnormal MURRl gene. In this case, a method of diagnosis would determine the level of such an expression product in the biological sample to establish the level of wild-type expression product from the MURRl gene or of abnormal expression product in the subject.

In some mammalian copper storage diseases it may be that the MURRl gene is normal, but that the level of the wild-type expression product is reduced. Such diseases may be diagnosed by determining the level of an expression product of the MURRl gene in a biological sample obtained from the subject.

There is also provided according to the invention a kit for the diagnosis of a mammalian copper storage disease which comprises means for determining from a biological sample obtained from a subject whether the subject has an abnormality in the MURRl gene.

Preferably the determining means comprise a pair of oligonucleotide primers capable of amplifying nucleic acid of a wild-type MURRl gene (or wild-type nucleic acid expression product of the MURRl gene), but which are not capable of amplifying nucleic acid of a MURRl gene which comprises a genetic abnormality (or of an abnormal nucleic acid expression product of the MURRl gene), or which are capable of amplifying such nucleic acid but the amplified product is of different length to the amplified product obtained from the wild-type gene (or wild-type nucleic acid expression product).

Alternatively or additionally, the determining means may comprise a first antibody

(or an antibody fragment or derivative) capable of binding abnormal expression product of the MURRl gene but not (or with higher affinity than) wild-type expression product of the

MURRl gene, and a second antibody (or an antibody fragment or derivative) capable of binding wild-type expression product of the MURRl gene but not (or with higher affinity than) abnormal expression product of the MURRl gene. Instead of antibodies, binding partners for the expression products may be used. An abnormal expression product includes an expression product of a mutant MURRl gene.

Kits of the invention may further comprise any reagent required to visualize amplified product, or antibody (or other binding partner) bound to expression product in order to obtain the result of a diagnosis performed using the test.

The invention also provides a chip comprising a nucleic acid capable of hybridizing under stringent conditions to either strand of a deleted region of the MURRl gene, the deleted region being a genetic abnormality responsible for a mammalian copper storage disease. The chip may be used in a method of diagnosis of the invention and can allow rapid screening of biological samples.

Identification of the role of the MURRl gene in mammalian copper storage diseases allows the possibility of prevention, treatment, or amelioration of a copper storage disease in a subject suffering from, or at risk of developing, a copper storage disease by providing the subject with an expression product of a wild-type MURRl gene, or a functional derivative thereof.

This could be achieved by administering to the subject a nucleic acid encoding a wild- type MURRl gene, or a functional derivative thereof, which is capable of directing expression of the gene or derivative in the subject. In other forms of gene therapy nucleic acid could be administered to a subject who is not producing wild-type expression product of the MURRl gene or normal levels of wild-type expression product in one or more tissues, the nucleic acid being capable of inserting into genomic nucleic acid of the subject, to provide the subject with a wild-type MURRl gene, or a functional derivative thereof, which is capable of expressing wild-type expression product or a functional derivative thereof in the tissue or tissues. The nucleic acid for administering to the subject may be encoded by a vector.

The invention also provides a protein or nucleic acid which is or corresponds to a wild-type expression product of the MURRl gene, or a functional derivative thereof, for use in the prevention, treatment, or amelioration of a mammalian copper storage disease.

The invention also provides use of a protein or nucleic acid which is or corresponds to a wild-type expression product of the MURRl gene, or a functional derivative thereof, in the manufacture of a medicament for the prevention treatment, or amelioration of a mammalian copper storage disease.

A protein which is or corresponds to wild-type MURRl gene expression product, preferably canine expression product, or a functional derivative thereof, may be used in a diet or as a dietary supplement to prevent, treat or ameliorate a copper storage disease.

The invention further provides a wild-type MURRl gene, or a functional derivative thereof, for use in the prevention, treatment, or amelioration of a mammalian copper storage disease. There is also provided use of a wild-type MURRl gene, or a functional derivative thereof, in the manufacture of a medicament for the prevention treatment, or amelioration of a mammalian copper storage disease.

There is also provided according to the invention a method of preventing, treating, or ameliorating a copper storage disease which comprises diagnosing the subject as having the disease using a method of diagnosis of the invention, and then administering appropriate treatment to the subject. The appropriate treatment may be a conventional treatment or a treatment of the present invention.

The invention also allows breeding of non human animals who are not affected by, or carriers of, a mammalian copper storage disease. This can be achieved by identifying individuals of opposite sex who are homozygous normal for the MURRl gene, and breeding from the identified individuals to produce a homozygous normal individual. Because copper toxicosis is an autosomal recessive disease, it will be appreciated that homozygous normal individuals for the MURRl gene can also be provided by identifying individuals of opposite sex who are each heterozygous normal for the MURRl gene, or identifying a homozygous normal individual and a heterozygous normal individual for the MURRl gene of opposite sex, breeding from the identified individuals to produce an individual and determining whether the individual is homozygous normal for the MURRl gene. Such methods are, however, less preferred than methods in which only homozygous normal parents are used because there is a 50% chance that the offspring will be carriers of the disease. The individuals may be identified using a method of diagnosis of the invention. Such methods are particularly suitable for canines, especially Bedlington terriers. It will be appreciated that the invention now allows eradication of CT from Bedlington terriers.

There is provided a protein in substantially isolated form comprising an amino acid sequence corresponding to SEQ ID No 1, 3, or 6. A protein in substantially isolated form having at least 88% homology to SEQ ID No 1 is also provided.

Identification of the role of the MURRl gene in mammalian copper storage diseases also allows development of more accurate linkage tests than are currently available. A linkage test could be developed simply by identifying a DNA marker which is more closely linked to a genetic abnormality of the MURRl gene than the known linked marker C04107 (Yuzbasiyan-Gurkan et al, 1997, Am J Hum Genet 63, 803-809). A person skilled in the art will readily understand how this may be achieved. More than one marker may be used in the linkage tests.

An expression product of the MURRl gene, or a binding partner of an expression product of the MURRl gene, can be used as a target for drug discovery to identify compounds for the prevention, treatment, or amelioration of a copper storage disease.

Embodiments of the invention are now described, by way of example only, with reference to the accompanying drawings in which:

Figure 1 shows genetic and physical mapping of the CT locus in Bedlington terriers. (A)

Haplotypes of a selected number of affected, carriers and unaffected Bedlington terriers (17) showing that the minimal region of homozygosity shared by affected Bedlington terriers is flanked by markers CF10B18 and CF10B23. The probes ys77hl0.slpr, SHGC-34371pr,

FLJ13305ρr, nh35cl2.sl and cct4pr correspond to the human ESTs ys77hl0.sl (H93765),

SHGC-34371 (G28276), FLJ13305 (accession number AK023367), nh35cl2.sl (AA524967) and the CCT4 gene (accession number AF026291), respectively. These probes were obtained by PCR amplification from human genomic DNA or human placenta cDNA. Primers

(sequence information can be found at: http://humgen.med.uu.nl/research/copper/vdsluis2001) were selected from the coding regions

(19). (B) Physical map of the CT region. Mapped genes, EST in and putative transcripts in the CT region are shown upper part, the BAC clones (20) in the middle part and the microsatellite markers (24) below. The refined CT candidate region of approximately 500Kb is indicated with an arrow.

Figure 2 shows mutation analysis of the Murrl gene at the cDNA level in Bedlington terriers with copper toxicosis. (A) Reverse transcription polymerase chain reaction (RT-PCR) of liver mRNA from unaffected (U), affected (A), carrier (C) and H20 as a negative control for the

RT-PCR. Locations of the primers used (F and R) are indicated in Fig.3B. PCR products were separated on a 1,5% agarose gel. The size marker indicated by M is a 50 bp ladder and the expected sizes of the normal and deleted Murrl PCR fragment are given on the right. (B)

Deletion of exon 2 in the Murrl mRNA from a affected Bedlington terrier as found by direct

DNA sequencing of Murrl cDNA obtained in A.

Figure 3 shows Murrl mutation analysis at the genomic level in Bedlington terriers with copper toxicosis. (A) Southern blot analysis of genomic dog DNA digested with EcoRI (E) or BamΗΪ (B) and hybridized with Murrl exon 2 or Murrl exon 3. Equal amount of genomic dog DNA (Unaffected (U), affected (A), carrier (C)) was loaded onto the gels (data not shown). (B) Genomic organization of the Murrl gene. The restriction EcoRI (Ε) and BamHT (B) fragments present in genomic DNA are indicated with lines. A dotted line indicates that the exact location of the restriction site is yet unknown.

Figure 4 shows nucleotide sequence in the region of the proximal breakpoint of the MURRl gene deletion associated with canine copper toxicosis. The whole sequence is designated SΕQ ID NO: 11. The sequence in which the proximal breakpoint occurs is underlined. The sequence preceding this sequence is SΕQ ID NO: 12. The "n"s indicate unsequenced nucleotides. The number of "n"s is an estimate of the length of the unsequenced region; and Figure 5 shows nucleotide sequence in the region of the distal breakpoint of the MURRl gene deletion associated with canine copper toxicosis.

Example 1

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 potential rich source for the identification of the genetic contribution to human biology and disease. Copper toxicosis in Bedlington terriers is of direct relevance for the understanding of copper metabolism in mammals as it is caused by the inability to excrete copper, an hitherto undefined pathway. Based on DNA samples obtained from privately owned dogs we have positionally cloned the disease gene, MURRl, and thus provide a new lead to disentangle the complexities of copper metabolism in mammals.

Naturally occurring canine genetic diseases have been useful models for the study of the pathophysiology, genetics, and treatment of the homologous diseases in humans (1). In general, genetic diseases in dogs more faithfully resemble human disease than do their rodent counter parts. This is not surprising given the closer evolutionary relationship and higher degree of DNA sequence identity between humans and dogs than between humans and rodents. Dogs receive a very high degree of medical scrutiny, nearly comparable to that applied in human medicine, and the same diagnostic procedures used in humans may be applied to dogs. The positional cloning of canine homologues of human disease genes has largely depended on the establishment and maintenance of breeding colonies (2-6). However, these genetic disease models represent only a small portion of the canine disease models that might be utilized to advance our knowledge of mammalian genetic diseases. Purebred dogs, propagated by dog breeders, offer a unique source of pedigrees to elucidate the molecular basis of simple and complex genetic diseases and traits. The unusual and useful feature of dog breeds that make them particularly amenable to genetic analysis is the fact that each breed represents an isolated inbred population (7-9). We show here the first example of the exploitation of the over 300 existing canine populations worldwide to discover a new gene in the strictly regulated copper homeostasis in mammals (10).

In 1999 we reported the localization of the CT locus to canine chromosome region CFA10q26 (15), a region homologous to human chromosome region HSA2pl3-21. By this localisation any candidate gene known at that time could be ruled out. More recently, we refined the CT region by homozygosity mapping to a region of 42.3 cR₃₀₀o that corresponds to a region of approximately 9 Mb (16). Starting from a canine BAC clone containing the linked C04107 marker, we built a BAC contig in both directions (Fig. IB). Overlapping BAC clones were isolated by screening a total canine BAC library (21) by colony hybridization with [α-³²P]dATP- and [ -³²P]dCTP-labeled oligos (22,23) based on BAC end-clones and

human genes and ESTs that mapped to HSA 2pl3-21. Eleven new microsatellite markers were isolated from BAC clones and for each canine microsatellite marker we determined whether the dogs were heterozygous or homozygous. When additional family members were available, haplotypes were constructed to determine identity by descent (IBD) of the mutant chromosome. The haplotypes of all typed dogs are available as an online resource at the following website address: humgen.med.uu.nl/research/copper/vdsluis2001. Using these new markers we were able to reduce the putative CT region to a minimal region of homozygosity of approximately 500 kb shared by all 22 affected Bedlington terriers used in our study (16) (some dogs are shown in Fig. 1A).

The CT candidate region flanked by the markers CF10B18 and CF10B23 was completely covered by five BAC clones (Fig. IB). Human genes and ESTs that mapped to

HSA2pl3-21 were used for constructing a transcription map of the canine CT region. In addition, the BAC contig was subjected to sample sequencing. Fifteen putative transcripts were identified in the CT region, six of which represented known genes or ESTs. Full- length canine mRNA sequences were obtained by 5'- and 3 '-RACE PCR on liver mRNA derived from an unaffected Beagle. We subjected the sequences for CRM1 (accession number XM_002691), cDNA FLJ13305 (accession number AK023367), CCT4 (accession number AF026291), MURRl (accession number D85433) and EST nh35cl2.sl (accession number AA524967) to mutation analysis using liver mRNA from Bedlington terriers with CT. A homozygous 282 bp deletion in the MURRl mRNA was found in all the 22 affected Bedlington terriers (Fig. 2A and 2B). The canine full-length mRNA sequence of MURRl (accession number AY047597; see the sequences below) spans 1,518 bp including an open reading frame of 564 bp that encodes a predicted protein consisting of 188 amino-acid residues. The 282 bp deletion was present in the coding region of the MURRl gene, resulting in an in-frame deletion and the production of a predicted truncated protein of 94 amino-acid residues.

Direct sequencing of canine BAC clone N21-27 an d E6-166 (Fig. IB) revealed that the normal canine MURRl gene consists of three exons in total (accession numbers AY047598, AY047599, AY047600; see the sequences below). Southern blot analysis of genomic DNA from affected Bedlington terriers indicated that exon 2 was deleted: all 22 affected dogs showed a homozygous deletion of a 1.5 kb EcoRI fragment and obligate carriers only had one copy of the 1.5 kb EcoRI fragment (Fig. 3 A). The location of the EcoRI sites was confirmed by sequencing the flanking intronic sequences of exon 2 (accession number AY047599). In addition, hybridization with exon 3 showed a BamϊTI junction fragment of 6.7 kb in affected dogs and a 5.5 kb BamϊTT fragment in unaffected dogs, indicating a genomic deletion of at least 10 kb (Fig. 3B).

The canine MURRl protein shows high homology with the human (accession number

CAC24864) and mouse MURRl proteins (accession number CAC24865) with 87% and 86% amino acid similarities, respectively. The accession number of the human gene in the NCBI database is AX060277. In the mouse the Murrl gene harbors an imprinted gene, U2afl-rsl, which is not present in the human gene (27). Database searches also identified MURRl orthologs in pig (accession number BF703083), cow (accession number BE682977) and rat (accession number BF404829), but no detectable homologs were present in Saccharomyces cerevisiae, Drosophila melanogaster, Caenorhabditis elegans, Fugu rubripe or Danio rerio implying that this gene is restricted to vertebrates and perhaps even to mammals. Moreover, no homology with any other protein, identifiable motifs, domains or structural features could be identified to indicate the putative function of the protein. Ubiquitous expression of MURRl is seen by RT-PCR (data not shown).

The presence of a deletion of exon 2 of the MURRl gene in all affected Bedlington terriers in a homozygous state strongly suggests that MURRl is the disease causing gene in copper toxicosis in Bedlington terriers. Our study shows for the first time the identification of a mutated gene by positional cloning using a particular dog breed with a relatively common hereditary disorder that was not specifically bred for the purpose of gene mapping and cloning. Dogs kept as experimental animals are expensive and are difficult to justify on ethical grounds. The "proof of principle" shown here encourages investigators to exploit the unique diversity of domesticated canines by analyzing these animals spread out over all continents.

The inefficient copper excretion seen in Bedlington terriers with CT leads us to assume that MURRl acts downstream of the Wilson disease protein, ATP7B, leading to a significant extension of this pathway. Alternatively, MURRl may unravel an entirely new pathway in copper homeostasis. Recently, knowledge about copper uptake into the cell and cellular copper transport into different proteins has increased substantially (10). However, knowledge about copper excretion into the bile is limited. MURRl is a good candidate gene for the human copper storage disorders that still need to be elucidated, including Indian- childhood cirrhosis (28) and non-Indian childhood cirrhosis (29) as well as the copper toxicosis disorders seen in other dog breeds and in sheep (30,31).

Example 2

Diagnostic test for canine copper toxicosis

The primers sequences are: for: 5 -CCCAGGAAGCTTTCCACGG-3' (SEQ ID No 8) rev: 5'-CCCAGGAAGCTTTCCACGG-3' (SEQ ID No 9)

PCR conditions are:

PCR reactions were performed in a "GeneAmp" PCR system 9700 (Perkin Elmer) in a 20 ml volume containing 50 ng cDNA, 50 ng of each oligonucleotide primer, 200 mM dNTP and

0.5 units Amplitaq Gold (Perkin Elmer), in lx PCR buffer II with 2.5 mM MgCl₂ (Perkin

Elmer). DNA was initially denaturated at 95°C for 10 min and was then subjected to 35 cycles of 95°C for 30 s, annealing of 58°C for 30 s and at 72°C for 1 min, followed by a final extension step of 4 min at 72°C.

Expected DNA fragments:

469 bp in healthy dogs, 187 bp in affected dogs, 187 bp and 469 bp in obligate carriers.

In other diagnostic tests, the primers are capable of hybridizing to nucleic acid flanking the 1.5 Kb EcoRI deletion fragment so that genomic nucleic acid of the MURRl gene can be amplified. Such tests are advantageous because it is not necessary to prepare cDNA template from the biological sample.

Example 3

Diagnostic test for canine copper toxicosis

SEQ ID NO: 10 (see below) is used as a probe for Southern blot analysis of EcoRI digested genomic DNA from Bedlington terriers. In normal Bedlingtons a 5Kb product is seen, while in affected dogs only a 2.7Kb fragment is seen. In carrier dogs both the 5Kb and the 2.7Kb fragments are observed. The probe may be used in routine testing for carriers of copper toxicosis in the Bedlington terrier.

Southern blot probe (SEQ ID NO: 10): tctagcatgtgtcagactctgtataggtgttggggatatatcagtgaataacatagatgagtccttta tcttatggagctgacagaaaaagaaaaatagtagtaattagtgtaaaaaagtatatcagaaactgaag aatcttaccaaagctcagaagagggaatagccctctttacccaggaagttgaggaaggcagtttggag caggtaacatttgcatttgatttctctgacaacttcagctctcctattcagagctgagagtcatcact aagaaaagttggtacaggcagtacagattctttagagtttatgaacatatcttttcatttaacagata ggtgcgacctttggtagggtttagtacaggttcctgatgattggcatttctttttttaccacttccta catgttggttttccagtttctcaacccacaggcagtaagaaggtagcaagaagtaaattggcagaaaa gttattgcaagcaaaagcaaga tcatttagtgcagtggtctcagcatcactgaatatcaga

References and Notes

1 D.F. Patterson, Canine Genetic Disease Information System: A computerized Knowledgebase of Genetic Diseases in Dogs (Mosby-Harcourt, St. Louis), in press.

2 F. Lingaas et al., Animal Genet. 29, 371 (1998).

3 G.M. Ackland et al., Proc. Natl. Acad. Set USA 95, 3048 (1998).

4 G.M. Ackland et al., Genomics 59, 134 (1999).

5 L. Lin et al., Cell 98, 365 (1999).

6 T.J. Jonasdottir et al., Proc. Natl. Acad. Sci. USA 91, 4132 (2000).

7 E.A. Ostrander, E. Giniger, Am J Human Genet 61, 475 (1997).

8 E.A Ostrander, F. Galibert, D.F. Patterson, Trends Genet 16, 117 (2000).

9 E.A. Ostrander, L. Kruglyak, Genome Research 10, 1271 (2000).

10 M. M. Pena, J. Lee, D.J. Thiele, J. Nutr. 129, 1251(1999).

11 Hardy, R.M., Stevens, J.B. & Stowe, CM. Minn. Vet. 15, 13-24 (1975).

12 G.F. Johnson, I. Sternlieb, D.C.Twedt, P.S. Grushoff, I. Scheinberg, I. Am. J. Vet. Res. 41, 1865 (1980).

13 D.C. Twedt, I. Sternlieb, S.R. Gilbertson, J. Am. Vet. Med. Assoc. 175, 269 (1979).

14 V. Yuzbasiyan-Gurkan, et al., Am. J. Vet. Res. 58, 23 (1997).

15 B.J. van de Sluis et al., Hum. Mol. Genet. 8, 501-7 (1999).

16 B. van de Sluis et al., Mamm. Genome 11, 455-60. (2000).

17 Blood was collected from 23 related Bedlington terriers of Belgian origin (9 affected,

12 carriers, 2 unaffected) and 11 unrelated affected Bedlington terriers (described in

(10) in which also the pedigree of the Belgian Bedlington terriers has been depicted).

Diagnostic criteria and the results of the copper analysis were described previously

(18). In addition, DNA from 3 affected English Bedlington terriers was kindly provided by Dr. Nigel Holmes (Centre for Preventive Medicine, Animal Health Trust, Suffolk, UK). J. Rothuizen et al, Animal Genet 30, 190 (1999). PCR reactions were performed in a Gene Amp® PCR system 9700 (Perkin Elmer) in

a 20 μl volume containing 50 ng template DNA or cDNA, 50 ng of each

oligonucleotide primer, 200 mM dNTP and 0.5 units Amplitaq Gold (Perkin Elmer), in lx PCR buffer II with 2.5 mM MgCl₂ (Perkin Elmer). DNA was initially denaturated at 94°C for 10 min and was then subjected to 35 cycles of 94°C for 30 s,

annealing for 30 s and at 72°C for 1 min, followed by a final extension step of 4 min

at 72°C. The identity of all probes was confirmed by sequence analysis.

BAC DNA was isolated by the alkaline lysis method as described on the BacPac website: bacpac.med.buffalo.edu. BAC DNAs were digested with EcoRI, BamϊTT, and EcoRTJBam S., separated on a 0.7% agarose gel, transferred to Hybond N⁺ (Amersham), and hybridised at 65°C with ys77hl0.slpr, SHGC-34371ρr, FLJ13305pr, nh35cl2.sl and cct4pr probes. R. Li et al, Genomics 58, 9 (1999). C.S. Han et al, Genome Res. 10, 714 (2000).

The overgo 's were generated from BAC end sequences determined as described previously (16). The verification of the overlapping BAC clones were determined by PCR using PCR primers generated from the BAC end sequences. A microsatellite enriched-library of BAC clones was constructed as follows. BAC DNA from the BAC clones comprising the CT-contig was isolated by the alkaline lysis method as described on the BacPac website: bacpac.med.buffalo.edu. Five hundred ng BAC DNA was digested with S w3AI, adapters were ligated to the end of the restriction fragments and the restriction fragments were amplified using adaptors specific primers as described before (25). Enrichment of the CA- and GAAA-repeats was performed as described previously (26) using 3'biotinylated [CA]₂₂ and 3'biotinylated [GAAA] oligos. The enriched fragments were amplified by PCR using the adaptor specific primers and the PCR products were cloned into the pCR 2.1 cloning vector using the TA-cloning kit (Invitrogen). Positive clones were identified by colony hybridization and their identity was determined by sequence analysis using BigDye Terminator cycle sequencing (Perkin Elmer), followed by separation on an ABI Prism 3700DNA Analyzer (Perkin-Elmer). Thereafter, specific primers were generated to amplify the microsatellite repeat sequences. Primer sequences are listed at the website address: humgen.med.uu.nl/research/copper/vdsluis2001. The approximate localization and order of the microsatellite markers was determined by PCR analysis of the individual BAC clones comprising the CT-contig. P.C. Groot, B.A. van Oost, Nucl. Acids. Res. 26, 4476 (1998). R. Korstanje et al., Animal Genet, in press. A. Nabetani, I. Hatada, H. Morisaki, M. Oshimura, T. Mukai, Mol. Cell. Biol. 17, 789

(1997) . M.S. Tanner, Am. J. Clin. Nutr. 67, 1074S (1998). T. Muller, H. Feichtinger, H. Berger, W. Muller, Lancet 2> 1, 877 (1996). L.P Thornburg, E Vet. Diagn. Invest. 12, 101 (2000). R.J. Sutherland, J Am Vet MedAssoc 180, 984 (1982). Sequences

DEFINITION Canis familiaris beagle Murrl (Murrl) mRNA, complete cds. LOCUS AY047597 1518 bp mRNA ACCESSION AY047597 VERSION AY047597 KEYWORDS . SOURCE dog. ORGANISM Canis familiaris

Eukaryota; Metazoa; Chordata; Craniata; Nertebrata; Euteleostomi; Mammalia; Eutheria; Carnivora; Fissipedia; Canidae; Canis. FEATURES Location/Qualifiers source 1..1518

/organism- 'Canis familiaris" /db_xref="taxon:9615" /chromosome=" 10" /map="10q26" /tissue_type="liver" /note- 'breed: beagle" gene <1..1518

/gene="Murrl" 5UTR <1..18

/gene="Murrl" CDS 19..585

/gene="Murrl" /codon_start=l /product="Murrl" /protein_id="AAK98638"

/translation="MAAELEGSKALGGLLSGLAQEAFHGHHGITEELLRSQLYPEVSLEEFRP FLAKMRGIUKSIASADMDFΝQLEAFLTAQTKKQGGITSDQAANISKFWKΝHKTKIRE SLMΝQSRWDSGLRGLSWRNDGKSQSRHSAQIHTPVAIMELEIGKSGQESEFLCLEFD ENKNSQLLKKLSENEESISTLMQPA" (SEQ ID No 1) 3TJTR 586..1518

/gene="MunT" ρolyA_signal 1462..1467

/gene="Murrl" polyA_site 1488

/gene="Murrl" BASE COUNT 459 a 309 c 356 g 3941 ORIGIN

1 gcggggctgc tggccagcat ggcggccgag ctcgagggct ccaaggcgct gggcgggctg 61 ctgagcggcc tggcccagga agctttccac gggcaccacg gcatcacgga ggagctgctg 121 cggagccagc tctatccgga ggtgtccctc gaggagttcc gcccctttct ggcgaagatg 181 aggggcatcc ttaagtcgat tgcatctgca gacatggatt tcaaccagct ggaggcattc 241 ttgactgctc aaaccaaaaa gcaaggtggg atcacatctg accaagctgc tgtcatttcc 301 aaattttgga agaaccataa gacaaaaatt cgagagagcc tcatgaacca gagccgttgg 361 gacagtgggc ttcggggcct gagttggaga gttgacggca aatcacagtc aaggcattca 421 gctcaaatac atacccctgt tgccataatg gagctggaaa taggaaaaag tggacaggaa 481 tcagaatttc tgtgtttgga atttgatgaa gtcaaggtca gtcaactcct aaagaagctc 541 tcagaggtag aagaaagtat cagcacactg atgcagccag cctagctgaa gatggagttg 601 ttgaagcaaa ggtgttcatg atccctcccc agtgacctgc gatttttttt ttttaaatct 661 tattcaccca ttttattaaa tccccaaatt caaatctgtt tgtctcactt gctgagattt 721 cttttgtctt tctctttcat tcattcttac agttgtacta cttgtagagg ttctaaaact 781 ttagcatgca gagtgctcat aaaagcacct tgagatcaag agtcacctgc ttcactgagt 841 aagccagccc ggttccccat tataaaagca ttttaaaaag gcttaagggc catatcccta 901 gacacttgaa ttcagtagga ctaggtggag tcaacttcag gaacccttac tatggtggtc 961 ggttcctgga gtaggattcc ataaatcatg gaagaattag aaaaggggga acaaaacatt 1021 ttattcatgc aaatacaagg ctgaccaaaa gccatgtgtc ttgagctgag gcttagaaag 1081 cctgcctaac ctacaaaaag tttactgatc ctggtaaaac ctgtgatgct cctggaactg 1141 attcagacat ttggattcat tctcactaaa tatgaggggt ctggtttgat ctgaactact 1201 gagaaagttg ggcttttctg gaacctagaa ctaaacggtc cttgtcacaa agggactgac 1261 tccctttata cttacttcaa gtcagagttg tatgaaagga aaaatgtcta ctgagctgat 1321 gtgaggtctt ttacatcaga aaattttact tgggtcatca aataaaacct tttgaagaaa 1381 actaataaag gggtacctgg ctgggtcagt tggtataaca tgtgactctt gatcttgggg 1441 ttgagggcat agagattact taataaataa atacaaatta aaagaaagaa aaaaaaaaaa 1501 aaaaaaaaaa aaaaaaaa (SEQ ID No 2) // DEFINITION Canis familiaris Doberman pinscher Murrl (Murrl) gene, exon 1, partial sequence, and partial cds. LOCUS AY047598 215 bp DNA ACCESSION AY047598 VERSION AY047598 KEYWORDS . SOURCE dog. ORGANISM Canis familiaris

Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Carnivora; Fissipedia; Canidae; Canis. FEATURES Location/Qualifiers source 1..215

/organism="Canis familiaris" /db_xref="taxon:9615" /clone="E6-166"

/note="breed: Doberman pinscher" mRNA <1„>282

/gene="Murrl" exon <1..282

/gene="Murrl" /number=l gene <1..>331

/gene="Murrl" CDS 106..>282

/gene="Murrl" /codon_start=l /product="MunT " /protein_id="AAK98639"

/translation="MAAELEGSKALGGLLSGLAQEAFHGHHGITEELLRSQLYPENSLEEFRP FLAKMRGILK" (SEQ ID No 3)

BASE COUNT 31 a 69 c 78 g 37 t ORIGIN gtcgcctccgtccgcccccgcgccttcgcccttggcctttgggcccctcccggctgccgtagcgggggccgcgctcggctgcgggg ggcggggctgctggccagcatggcggccgagctcgagggctccaaggcgctgggcgggctgctgagcggcctggcccaggaag ctttccacgggcaccacggcatcacggaggagctgctgcggagccagctctatccggaggtgtccctcgaggagttccgcccctttct ggcgaagatgaggggcatccttaaggtaccggtcctcccttgccgctgcggccgcagagcccccaccccttccc (SEQ TD No

4)

//

DEFINITION Canis familiaris Murrl (Murrl) gene, exon 2.

LOCUS AY047599S1 2942 bp DNA

ACCESSION AY047599

VERSION AY047599

KEYWORDS .

SEGMENT l of 2

SOURCE dog. ORGANISM Canis familiaris

Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Carnivora; Fissipedia; Canidae; Canis.

FEATURES Location/Qualifiers source 1..2942

/organism- 'Canis familiaris" /db_xref="taxon:9615" /clone="N21-27" exon 1324..1605

/gene="Murrl" /number=2

BASE COUNT 915 a 493 c 509 g 1022 1 3 others

ORIGIN

1 tggcaggatt ccagtaaatg tttattgaaa tataaagtga aatatcttta ccagaaggtt 61 gttatattta tcatacagtt ttgtttttac attaatggaa gaatgaaagg cagtatgttt 121 gtctgtctgc cccagtccta cttcctctac ctatactcta cctatacagt cttccttaca 181 ctcctctctt ctcactgcta ctgcccaagt ttagtcagga ctattggttc taaataatag 241 gcatatagct taaactatct tgaatagtaa attgagtttt ttggtatctg aaggtggaaa 301 aaatgctgaa atagcataca caattgaaga aaggtttaca ggaacaggtt tacagggcct 361 tggagattat agagcaagga attcactccc ttagaaccca cttctcttgt cttcattctt 421 tttattgcag gtaggctttt gccaaaagat caaagagagc agtctgcatc ctagtcatat 481 attcttctga tacttatgac cccaagaggc aagactgttt cttctcctac tctaaattgg 541 gagattgatt tggatcagtt gttgtgtcca gagagaggag gatactatgc tttgtctggt 601 ttggagctca taccagtccc agtggccagg agaggataag gatataccaa agaagaggca 661 aggaaaatct tgagaccaaa ccattaatag tcactgtcct ctctaaggct ccattgcgac 721 agacatttga tgattttctg cttcagttta tatagcagct cactgttctc aaaacagcaa 781 tggcaattaa attttaaaaa tttatggaga aacagaacta ttattgtgaa tgagccattt 841 ggctaaagat ggacttaaac aactgacata atacattcct ttaacagaat ttacttagtg 901 cttctatgga caaggcatta tacaattata gtatatcaaa ccaagcctgg ctataagcca 961 ttgtgaaatt atgtaagatt tcctcagagg atgaattaat ttatattctt gttttattat 1021 ttagtttata aattttttan aattattttt tcgccatgga atttaatatt tcagaattag 1081 attctagtta ttgatacaga atttgagtga catggaattt ttaacaggta gcttttctga 1141 tactagatac ttgtaaaaag aatatttttt ttcttttaaa gagcttattc tcattactgt 1201 cattgatgac ccttcttgaa agcttattca gtgattaaga atcattcaaa atatgacctg 1261 cagttaagaa gctgggtttt tttctagtta tttattactt tattactttt tctgtcattc

1321 tagtcgattg catctgcaga catggatttc aaccagctgg aggcattctt gactgctcaa 1381 accaaaaagc aaggtgggat cacatctgac caagctgctg tcatttccaa attttggaag 1441 aaccataaga caaaaattcg agagagcctc atgaaccaga gccgttggga cagtgggctt 1501 cggggcctga gttggagagt tgacggcaaa tcacagtcaa ggcattcagc tcaaatacat 1561 acccctgttg ccataatgga gctggaaata ggaaaaagtg gacaggtaaa tcaaatttca 1621 ctttcctttt gtaaactctg tttcccatta catgttagca atatgtccag attgatttta 1681 tgtaactgtc ttttttgcaa gataataata ggaaaaactc ttttcctaga atcccttttc 1741 agagatagaa tcatctgaaa atttggatat tcccagttgt ggatagaatc tggaaaccaa 1801 cctcatggtc ctggcatcta tctcagaaga ataaaataga tcattgaggt gattgaacag 1861 aagttggcca ctactccgtt ttggttagat ctcagaattc ttggcatttg gtcgaatctc 1921 caaagcaatg atagaaatac taaataaaga ccaagaattg agattaaaca acacagagta 1981 aaactgtctc acccagtcat ctttattatt tgtgctgctg aagcgagcac acccagttat 2041 ctttattggc agtgtatctt aattgtagta atatatttgt tcctagagac cttaaatgtg 2101 caatctctgt ccttaaggct acaaataatt tttaatttga tcttaattac ttacaaagta 2161 gttccttttt tttcttgaag tttccatagt cattatagcg gataattttg gcatctacat 2221 ttgtttttct tgaagtcatt cagtttgtgc ttcagatgat tgccagatca gacttttaat 2281 aacctggggt nngccttttg ggaaaaaggg ggaaaaaaaa aaaagcagca gccataatta 2341 ggaagagaaa acccagatca ctgaattagc taaccagatt tattttcaga taaataatca 2401 aataagtgct tctattacat cgtataatag aaattaattt ttcttctaga gatgagagca 2461 tttttttttt agttgtccat ttccctcaca tattccttaa gtttcttagg agttttcagt 2521 gtttatagtt ttataatgac actagtttta atttaaaagg taattccttt aaaaaaataa 2581 ttttttttca aaatacattt ggagaatctt ttatacagtt attgtaaaat agtgctattg 2641 cctttattga ctttggtttg tctgacccta aagtccattc tcttaaccat tgtcttacat 2701 ggccttcctt ttactgttcc tagaccaaaa atctgacaga taagaaaggt gtgataacat 2761 gtagatatct aagtttcact aaccatttgt ataggctatt cattttttat ttggcaatag 2821 tgattatttc atttttttgt tttttagtat agaacaattt tgtcaatctg taaatctaaa 2881 catcactaca aataatttgt tgactagctt cagggacatt ttatgaccaa ttagaacaag 2941 tc (SEQ ID No 5)

//

DEFINITION Canis familiaris Murrl (Murrl) gene, exon 3 and partial cds.

LOCUS AY047599S2 2142 bp DNA

ACCESSION AY047600

VERSION AY047600

KEYWORDS .

SEGMENT 2 of 2

SOURCE dog. ORGANISM Canis familiaris

FEATURES Location/Qualifiers source 1..2142

/organism="Canis familiaris" /db_xref="taxon:9615" /clone="N21-27" gene order(AY047599:<L.2942,1..1954)

/gene="Murrl" mRNA join(AY047599:<1324..1605,944..1954)

/gene="Murrl" /product- 'Murrl" CDS join(AY047599:<1324..1605,944..1051)

/gene- 'Murrl" /codon_start=l /product="Murrl" /protein_id="AAK98637"

/translation="SIASADMDFNQLEAFLTAQTKKQGGITSDQAAVISKFWKNHKTKIRESL

MNQSRWDSGLRGLSWRVDGKSQSRHSAQIHTPVAIMELEIGKSGQESEFLCLEFDEV

KVSQLLKKLSEVEESISTLMQPA" (SE ID No 6) exon 944..1954

/gene="Murrl" /number=3 polyA_signal 1928..1933 /gene="Murrl"

BASE COUNT 623 a 413 c 446 g 651 1 9 others

ORIGIN

1 aaatttctgt cnntacactt cagaggcctt nttcttttat ttctccntgg tctagggttg 61 actgactgat cctaacagta tttncagaga atgtgtttga tggaaatcta ggagagttgc 121 cttatagcaa gtggaagggc ctcagaaatg tttaaggcta tttcaagatc tggttggtta 181 tctttcagtc atctggttga tgtttcccaa gcttcttggt cacaaccggt cacaatttct 241 aatataactg ctaaatccaa gggagctcaa ttaccagcta acacnnnntg cttatactat 301 gtggagtgct acggagacaa gtagggactt tggagtcagc cagaactggg agtttacagt 361 ctgactctgc ctttttctga ttttgttata taggttaacc tgtctgtacc tcccgcgtca 421 tcatctatat aacagcaata cttatgtact tagcttaaaa attgttatag ggagacacct 481 ggatggttca gtagtggagc atctgccttt ggctcaagtc atgatactgg agtcccagga 541 tcgagtccca catcgggctc ccttcacaga gcctgcttct tcctctgcct gtgtctctgc 601 ctctctgtgt gtctctcatg aataaataca taaaatcttt ttaaaaattg ttatgggatc 661 aaatgtggtg acagacaaaa acagcttctc acgtgcctca tcatgagaac acccaacatt 721 tgtgtacctc tttaccgttt atgaaacacc tcattgaagt agttttagtt agggaactaa 781 atatgtggat ttgctttgtt ggctaatata tcatttaggt attgagacct gagtcaggtt 841 tgctaggtaa ggggccctgg atcttcggaa ctgggtcagg ccaggtccag aaccttggct 901 atcttgaagg tctcttataa aaacaccctt ttatgttttc caggaatcag aatttctgtg 961 tttggaattt gatgaagtca aggtcagtca actcctaaag aagctctcag aggtagaaga 1021 aagtatcagc acactgatgc agccagccta gctgaagatg gagttgttga agcaaaggtg 1081 ttcatgatcc ctccccagtg acctgcgatt tttttttttt aaatcttatt cacccatttt 1141 attaaatccc caaattcaaa tctgtttgtc tcacttgctg agatttcttt tgtctttctc 1201 tttcattcat tcttacagtt gtactacttg tagaggttct aaaactttag catgcagagt 1261 gctcataaaa gcaccttgag atcaagagtc acctgcttca ctgagtaagc cagcccggtt 1321 ccccattata aaagcatttt aaaaaggctt aagggccata tccctagaca cttgaattca 1381 gtaggactag gtggagtcaa cttcaggaac ccttactatg gtggtcggtt cctggagtag 1441 gattccataa atcatggaag aattagaaaa gggggaacaa aacattttat tcatgcaaat 1501 acaaggctga ccaaaagcca tgtgtcttga gctgaggctt agaaagcctg cctaacctac 1561 aaaaagttta ctgatcctgg taaaacctgt gatgctcctg gaactgattc agacatttgg 1621 attcattctc actaaatatg aggggtctgg tttgatctga actactgaga aagttgggct 1681 tttctggaac ctagaactaa acggtccttg tcacaaaggg actgactccc tttatactta 1741 cttcaagtca gagttgtatg aaaggaaaaa tgtctactga gctgatgtga ggtcttttac 1801 atcagaaaat tttacttggg tcatcaaata aaaccttttg aagaaaacta ataaaggggt 1861 acctggctgg gtcagttggt ataacatgtg actcttgatc ttggggttga gggcatagag 1921 attacttaat aaataaatac aaattaaaag aaagaaaaaa aaaacaaatt aaaagaaaga 1981 aagaaggcta gtgtaaaagc taggtcctca tggagaagtt ataccaaaca ccaagaatac 2041 tctaaaagga ggaagctgcc aaccagtata ttgaggctat ggccttaact ggtatttatc 2101 atttggtact gataacattt agaaacccca ggtgcctttc ag (SEQ ID No 7)

Claims

1. A method of diagnosing whether a subject has, is at risk of developing, or is a carrier of a copper storage disease which comprises determining from a biological sample obtained from the subject whether the subject has a genetic abnormality in the MURRl gene or an abnormality in an expression product of the MURRl gene.

2. A method according to claim 1 in which the genetic abnormality is a deletion in the MURRl gene.

3. A method according to claim 2 in which the deletion comprises exon 2.

4. A method according to any preceding claim which includes amplifying nucleic acid of the biological sample and determining from the amplified product whether the subject has a genetic abnormality in the MURRl gene.

5. A method according to claim 4, when dependant from claim 2 or 3, in which the nucleic acid is amplified using first and second oligonucleotide primers which are capable of hybridizing to nucleic acid of the wild-type MURRl gene flanking the deletion.

ft

6. A method according to claim 5 in which the first oligonucleotide primer is capable of hybridizing to intron sequence upstream of the deletion and the second oligonucleotide primer is capable of hybridizing to intron sequence downstream of the deletion.

7. A method according to claim 5 in which the first oligonucleotide primer is capable of hybridizing to exon 1 and the second oligonucleotide primer is capable of hybridizing to exon 3 of the MURRl gene.

8. A method according to any of claims 4 to 7 in which the nucleic acid of the biological sample comprises genomic DNA.

9. A method according to claim 4, 5, or 7 in which the nucleic acid of the biological sample comprises mRNA.

10. A method according to claim 2 or 3 which comprises probing nucleic acid of the biological sample, or a nucleic acid product amplified from nucleic acid of the biological sample, with a probe capable of hybridizing to either strand of the deleted region of the MURRl gene.

11. A method according to any of claims 1 to 3 which includes Southern blot analysis of restriction enzyme digested genomic DNA.

12. A method according to claim 1, 2, or 3 which comprises detecting for the presence of a protein expression product of a MURRl gene having the genetic abnormality.

13. A method according to any preceding claim in which the copper storage disease is a human or a canine copper storage disease.

14. Use of a nucleic acid capable of hybridizing under stringent conditions to either strand of a MURRl gene or to a nucleic acid expression product of a MURRl gene in an in vitro method of diagnosis of a copper storage disease.

15. Use according to claim 14 of a nucleic acid which is capable of hybridizing to either strand of the 1.5 Kb EcoRI fragment comprising exon 2 of the MURRl gene.

16. Use according to claim 14 of a nucleic acid which is capable of hybridizing to either strand of the MURRl gene upstream of the 1.5 Kb EcoRI fragment comprising exon 2 of the gene.

17 Use according to claim 14 of a nucleic acid which is capable of hybridizing to either strand of the MURRl gene downstream of the 1.5 Kb EcoRI fragment comprising exon 2 of the gene.

18. Use according to claim 16 or 17, wherein the nucleic acid is capable of hybridizing to either strand of intron sequence of the MURRl gene.

19. Use according to claim 16 or 17, wherein the nucleic acid is capable of hybridizing to either strand of exon 1 or exon 3 of the MURRl gene.

20. A kit for the diagnosis of a copper storage disease which comprises means for determining from a biological sample obtained from a subject whether the subject has a genetic abnormality in the MURRl gene.

21. A kit according to claim 20 comprising a nucleic acid as specified in claim 16 and a nucleic acid as specified in claim 17, wherein the nucleic acids are together capable of amplifying nucleic acid of the wild-type and mutant MURRl genes, or of wild-type and mutant expression products of the MURRl gene.

22. Use of a nucleic acid defined in any of claims 16 to 19 to amplify nucleic acid of a wild-type or mutant MURRl gene, or nucleic acid of an expression product of a wild-type or mutant MURRl gene.

23. Use of a nucleic acid defined in claim 15 to probe for the presence of the 1.5 Kb EcoRI fragment in a sample comprising nucleic acid.

24. Use according to claim 22 or 23 in a method of diagnosis of a copper storage disease.

25. Use of a kit according to claim 20 or 21 in a method of diagnosis of a copper storage disease.

26. A method of prevention, treatment, or amelioration of a copper storage disease which comprises providing a subject suffering from, or at risk of developing, a copper storage disease with wild-type MURRl gene expression product, or a functional derivative thereof.

27. A method of preventing, treating, or ameliorating a subject with a mammalian copper storage disease which comprises diagnosing the subject as having the disease using a method according to any of claims 1 to 13, and then administering appropriate treatment to the subject.

28. A diet comprising a wild-type MURRl protein, or a functional derivative thereof.

29. Use of a wild-type MURRl protein, or a functional derivative thereof, as dietary supplement.

30. A protein or nucleic acid which is or corresponds to a wild-type expression product of the MURRl gene, or a functional derivative thereof, for use in the prevention, treatment, or amelioration of a mammalian copper storage disease.

31. Use of a protein or nucleic acid which is or corresponds to a wild-type expression product of the MURRl gene, or a functional derivative thereof, in the manufacture of a medicament for the prevention treatment, or amelioration of a mammalian copper storage disease.

32. A method of providing a non human animal who is neither affected by nor a carrier of a mammalian copper storage disease, which comprises identifying individuals of opposite sex who are homozygous normal for the MURRl gene, and breeding from the identified individuals to produce a homozygous normal individual.

33. A method of providing a non human animal who is neither affected by nor a carrier of a mammalian copper storage disease, which comprises identifying individuals of opposite sex who are each heterozygous normal for the MURRl gene, or a homozygous normal individual and a heterozygous normal individual for the MURRl gene of opposite sex, breeding from the identified individuals to produce an individual and determining whether the individual is homozygous normal for the MURRl gene.

34. A method according to claim 32 or 33 in which the individuals are identified as being homozygous normal for the MURRl gene using a method according to any of claims 1 to 11

35. A method according to any of claims 32 to 34 in which the non human animal is a canine.

36. A method according to claim 35 in which the canine is a Bedlington terrier.

37. Use of an expression product of the MURRl gene, or a binding partner of an expression product of the MURRl gene, as a target for drug discovery.

38. A linkage test for diagnosing whether a subject has, is at risk of developing, or is a carrier of a copper storage disease which comprises using a DNA marker(s) for a genetic abnormality of the MURRl gene which is more closely linked to the MURRl gene than the C04107 marker to determine whether there is a high likelihood that the subject has the genetic abnormality.

39. Nucleic acid capable of hybridizing under stringent conditions to nucleic acid of SEQ ID NO: 11, 12, 15, or 16, or to nucleic acid which is complementary to SEQ ID NO: 11, 12, 15, or 16.

40. Nucleic acid according to claim 39 which is less than lkb in length.

41. Nucleic acid according to claim 39 which is less than lOObp in length.

42. Use of nucleic acid according to any of claims 39 to 41 in a method of diagnosis of a copper storage disease.

43. Use of nucleic acid capable of hybridizing under stringent conditions to nucleic acid of SEQ ED NO: 13 or 14, or to nucleic acid which is complementary to SEQ ID NO: 13 or 14 in a method of diagnosis of a copper storage disease.