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WO1995008641A1 - Gene de la maladie de wilson - Google Patents

Gene de la maladie de wilson Download PDF

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Publication number
WO1995008641A1
WO1995008641A1 PCT/CA1994/000519 CA9400519W WO9508641A1 WO 1995008641 A1 WO1995008641 A1 WO 1995008641A1 CA 9400519 W CA9400519 W CA 9400519W WO 9508641 A1 WO9508641 A1 WO 9508641A1
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gene
wilson disease
dna
copper
disease
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PCT/CA1994/000519
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English (en)
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Diane W. Cox
Peter Bull
Gordon Thomas
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Hsc Research And Development Limited Partnership
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Priority claimed from CA 2106602 external-priority patent/CA2106602A1/fr
Application filed by Hsc Research And Development Limited Partnership filed Critical Hsc Research And Development Limited Partnership
Publication of WO1995008641A1 publication Critical patent/WO1995008641A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes

Definitions

  • Copper is an essential trace metal for prokaryotes and eukaryotes, and is a required component for a variety of enzymes, including cytochrome oxidase and other electron transport proteins. Dietary intake of copper generally far exceeds the trace amounts required, and organisms have evolved effective means for the elimination of the excess. Toxicity of copper is believed to act predominantly through the formation of highly reactive hydroxide radicals, which can damage cell membranes, mitochondria, proteins and
  • Copper homeostasis requires appropriate mechanisms for copper absorption, cellular transport, incorporation into protein, storage, and excretion.
  • various proteins or peptides have been recognized for these functions (2): albumin (and copper histidine) for copper transport in the blood, ceruloplasmin as a possible copper donor to tissues and enzymes (3), and metallothionein for intracellular copper storage (4).
  • the mechanism of copper efflux from tissues has remained an enigma.
  • Menkes disease and Wilson disease are both caused by a disruption in copper transport (see review (5)). However, these two diseases affect different tissues. In X-linked Menkes disease, copper export is defective in many tissues (5) but is normal in the liver (6). Copper enters into the intestinal cells, but is not transported further, resulting in severe copper deficiency. In contrast, Wilson disease is characterized by failure to incorporate copper into ceruloplasmin in the liver, and failure to excrete copper from the liver into bile. This results in toxic accumulation of copper particularly in the liver, and also in kidney, brain, and cornea. The resulting liver cirrhosis and/or progressive neurological damage has an age of onset from childhood to early adulthood. Consequently, there is a real need to identify the gene responsible for Wilson disease in order to develop new diagnostic and therapeutic strategies useful in the detection and treatment of the disease.
  • Wilson disease gene has been assigned to a single locus by genetic linkage first to esterase D (7), then to a cluster of polymorphic markers on chromosome 13 band q14.3 (8, 9, 10). Multipoint linkage analysis indicates that WND is flanked proximally by marker D13S31 and distally by D13S59 at distances of 0.4 cM and 1.2 cM respectively (11). Marker D13S31 is sufficiently close to show allelic association with the disease in two different populations (12, 13).
  • the inventors have isolated new markers between D13S59 and D13S31 and have used them to construct a long range restriction map of the WND region (14, 15).
  • Three CA repeat markers, D13S314, D13S133 and D13S316, have been positioned in a 300 kb region within this map (16). These markers show high allelic association with WND and allowed the identification of specific Wilson disease haplotypes in the region (16).
  • D13S314 was used to define the proximal boundary of the Wilson disease region using a recombination event that is present in one of the Wilson disease families tested.
  • the present invention provides a DNA sequence containing the gene defective in Wilson disease.
  • the present invention provides a cDNA sequence containing the gene for a copper transporting ATPase (ATP7B) defective in Wilson disease.
  • the present invention provides a nucleotide sequence containing the copper transporting ATPase (ATP7B) which comprises the DNA sequence as illustrated in Figure 10, and the complementary strand or any modified derivative or fragment thereof.
  • the complete sequence of the metal binding ATPase defective in Wilson disease forms part of the present invention.
  • the DNA sequence includes six copper binding domains, a phosphate domain, a transduction, several potential transmembrane, phosphorylation and ATP binding domains. The sequence of each of these domains forms part of the invention. The 5' and 3' untranslated regions and stated intron sequences are included in the application.
  • the present application also includes nucleotide sequence adjacent to each exon, shown in Figure 11, which allows the amplification, by PCR, of each exon or a segment of it. Primers have been designed to amplify each exon or segment, however other similar primers can be selected from the sequenced regions, and the patent includes all such
  • the present invention also provides a use of the DNA sequence for Wilson disease or any modified derivative or fragment thereof (including the above-mentioned domains) to detect for Wilson disease.
  • the present invention further includes the use of the DNA sequence for Wilson disease or a modified derivative or any fragment of the given DNA sequence, derived in any way, including amplification by the polymerase chain reaction of RNA or genomic DNA, for the diagnosis of Wilson disease (hepatolenticular degeneration) or the heterozygous state, or for the identification of mutations.
  • This might include such methods as direct sequencing of PCR amplified frag ments, or the examination of differences in small amplified fragments using such methods as single strand confirmation polymorphism (SSCP), denaturing gradient gel electrophoresis, or heteroduplex analysis. Mutations can also be identified by sequencing of the complete gene, in segments, by an automated sequencer. Using the intron primers to amplify each exon, 29 different mutations have been identified. These examples, included in the present application, have been detected by single strand conformation polymorphism analysis (SSCP) and confirmed by sequencing.
  • SSCP single strand conformation polymorphism analysis
  • the present invention also includes the use of the DNA sequence for Wilson disease or a modified derivative or any fragment of the given DNA sequence, derived in any way, including amplification by the polymerase chain reaction, for use in plasmids or any other vector for therapy of Wilson disease or Menkes disease (another disorder of copper transport).
  • the application also includes use for enhancing heavy metal transport in humans or any other organism.
  • the present invention further includes the use of the DNA sequence for Wilson disease or a modified derivative or any fragment of the given DNA sequence, derived in any way, including amplification by the polymerase chain reaction, to obtain portions of the Wilson disease gene for deriving the homologous ATPase gene from other species.
  • these sequences have been used to obtain the homologous gene from the rat.
  • the rat sequence having been derived from the human sequence is included in the present application. This includes the coding region and the 5' and 3' untranslated regions, shown in Figure 17.
  • the cloning of the rat gene has allowed the finding of the basic defect in a mutant rat, the Long Evans Cinnamon (LEC) rat, which is now a model for Wilson disease and can be used to study methods of gene therapy.
  • LEC Long Evans Cinnamon
  • human DNA sequence that is presented here can be used for a variety of other species.
  • sequence is currently being used to sequence the mouse gene and to determine if a mutation in the toxic mouse lies in this same gene. The mouse can then be used to test gene therapies.
  • the present invention also includes the use of the DNA sequence for Wilson disease or any modified derivative or fragment thereof, derived by any means such as cloning or PCR amplification to obtain the corresponding gene from any other species.
  • the patent includes the use of the DNA sequence in any way to identify proteins which bind to the DNA sequence and may be involved in conrol of transcription and ultimately in the control of metal related processes.
  • the present invention further includes the use of the DNA sequence for Wilson disease or any modified derivative or fragment thereof in therapy to remove heavy metal from an organ.
  • the present invention yet also includes the use of the DNA sequence for Wilson disease, or any modified derivative or fragment thereof in animal breeding, for example to enhance the excretion of copper and other heavy metals.
  • the invention also includes all of the DNA markers associated with the Wilson disease that the inventors have developed.
  • the present invention includes DNA markers D13S314, D13S315, D13S316, D13S296 and D13S301 as well as any other markers that detect the same dinucleotide repeat polymorphisms as these five markers.
  • the invention further includes the use of the above- described DNA makers to detect Wilson disease.
  • the invention further provides a diagnostic kit for detecting Wilson disease comprising at least one marker selected from the group consisting of D13S314, D13S315, D13S316, D13S296 and D13S301.
  • Fig. 1 Schematic map of the WND candidate region. The positions of the markers relative to the YACs are indicated. D13S314 and D13S315 were derived from cosmids identified by endclones of 27D8 and 235H9 respectively. D13S316 was derived from a cosmid identified by D13S196. The established flanking markers D13S31 and D13S59 are also shown. A physical map of this region has been constructed (Bull and Cox, 1993). YAC isolation and characterization has been described elsewhere (Bull et al. submitted).
  • Fig. 2 Statistically significant allele distributions. The number of chromosomes carrying a specific allele is shown in white for normal and black for WND chromosomes.
  • Fig. 3 Isolation of Wc1.
  • X XbaI. MluI and NotI sites within YAC 27D8 are shown above and below the line respectively. Three other YACs mentioned in the text (95C3, 53C12 and 68F4) were isolated using PCR primers specific to the right (proximal) end of 27D8. b) Hybridization of probes Me1. a and cosL.d to YACs from the Wilson disease region. YACs 232H4 and 68F4 are overloaded.
  • Fig. 4 Chromosome mapping of cDNA fragments. a) Each cDNA was hybridized at high stringency to EcoRI and HindIII digests of genomic DNA from Human, hybrid ICD, YAC 95C3 and Hamster. The pattern obtained by hybridization of cDNA clone Wc1.i7 to EcoRI digests of these samples is shown. This clone cross hybridized to a single hamster fragment
  • Fig. 5 Partial DNA sequence of Wc1. Alignment of the amino acid sequences of Wc1 with MNK is indicated, amino acid identities in MNK are indicated with a period. Insertions and deletions in MNK compared to Wc1 are indicated as is the position of a splice site that occurred in one of the cDNA clones. The amino acid numbers of MNK are shown.
  • Fig. 6 Alignment of cDNA fragments with Me1.
  • the coding region of the Me1 cDNA is represented at the top of the figure by a thick stippled line. The location of functional elements are indicated.
  • the relative positions of probes Me1. a and Mc1.b are shown below with narrow stippled lines.
  • the relative positions of cDNA clones from Wc1 are indicated with thin black lines.
  • the Thick black line shows the part of the Wc1 transcript that has been sequenced.
  • Clone Wc1.f8 contains an unspliced intron.
  • Fig. 7 Alignment of nucleotide and derived amino acid sequence of the metal binding domains of Me1 and Wc1.
  • the nucleotide sequence of each domain of Wc1 is shown.
  • a translation of the domains and the corresponding domains of Me1 are shown directly below.
  • Nucleotides and amino acids that are conserved in Me1 are underlined. Residues that are also conserved in bacterial metal binding domains (see Fig. 6) are indicated at the bottom of the figure with an asterisk.
  • Nucleotides that form part of an unspliced intron within the cDNA clone containing the Cu5 domain are shown in italics.
  • Fig. 8 Alignment of derived amino acid sequence with other genes. Derived amino acid sequences of Wc1 were aligned with Me1 and other proteins from bacteria.
  • Residues found in both Me1 and Wc1 that are also conserved in one or more of the other proteins are underlined. Those that are invariant in the aligned sequences are shown in bold type. Abbreviations are E. hirae Cu ⁇ Enterococcus hirae copper transporting ATPase (26)) E. coli Hg (Escherichia coli mercuric transport protein merP (27)) S. aureus Cd ⁇ Staphylococcus aureus cadmium efflux ATPase (28))
  • Fig. 9 Northern blot hybridization.
  • Probe Wc1.c8 was hybridized to a Northern blot (Clontech) containing 2 mg of polyA+ RNA from a selection of tissues.
  • Fig. 10 Complete DNA sequence of the Wilson Disease gene.
  • Fig. 11 Sequences of exons and flanking intron regions. Exons 9/10/11 and 17/18 have been completely sequenced across intervening introns. Exonic sequence is shown in caps, intronic in lower case. Start ATG and termination TGA are shown in bold. Liver specific sequence is shown in italics. Numbers are for cDNA sequence only and start at the ATG codon. The number refers to the last cDNA nucleotide of that line. Exons 20 and 21 contain alternate 3' exons, expressed in different tissues.
  • Fig. 12 Exon structure of the ATP7B gene. Exons are shown as numbered, alternating black and white boxes and the location of the various functional regions are
  • Fig. 14 Diagram of the ATP7B region. The relative locations of the markers in this study are shown.
  • Positions of the markers have been determined by analysis of recombinant families (12) or overlapping YAC CLONES (46).
  • Fig. 15 Location of the mutations within the ATP7B gene. Only mutations listed in table 13 are shown. Exons shown as alternating black and white boxes.
  • Fig. 16 Splice site mutations in ATP7B. Sequences surrounding the alterations are shown and exon/intron boundaries are indicated. The bases affected are incidated with both letters. Translations are shown to indicate the downstream stop codons. Potential splice sites are shown in italics. A: 1615-1G ⁇ C, B: 2482+1G ⁇ C, C: 3463+1G ⁇ A.
  • Fig. 17 Nucleotide sequence (top) and amino acid sequence (bottom) of the homologous rat gene, including 5' and 3' regions, and coding region.
  • FIG. 18 Schematic of structure of the rat gene.
  • Fig. 19 Southern blot showing mapping of the ATP7B gene deletion in the LEC rat.
  • Peripheral blood was collected from 28 Canadian families, consisting of 22 of Northern European, three of Southern European, two of Oriental and one of Indian origin, and 23 families from the United Kingdom, consisting of nine of Northern European, four of Southern European, four of Indian, three of Sardinian and three of Middle Eastern origin. 21 of these families have been described elsewhere (12, 13). The remaining 30 kindreds consisted of 56 parents, 47 patients and 21 unaffected individuals. The ethnic origin of the parents of each patient was determined where possible. Diagnosis of Wilson disease was originally established by clinical symptomatology, slit lamp examination for Kaiser-Fleischer rings of the cornea, and biochemical tests (plasma copper, ceruloplasmin concentrations and urinary copper excretion, and in most cases, measurement of liver copper levels). In some cases, diagnosis was confirmed by radioactive copper studies (39).
  • DNA was extracted from whole blood collected in EDTA by a salt precipitation method (40).
  • D13S316 was derived from a cosmid identified by D13S196, an anonymous marker derived by Alu-PCR of a hybrid containing the upper half of chromosome 13 (14).
  • D13S314 and D13S315 were derived from cosmids identified by endclones of YACs 27D8 (identified by D13S196) and 235H9 (identified by D13S31) respectively (14). Endclones were obtained by an inverse PCR method (36). The probes were labelled using a T7
  • Allele sizes were determined by amplification of DNA from the original cosmid clone from which the marker was derived. Consistency in allele size determination was checked by including reactions performed on the DNA of 2 to 3 samples of known genotype on each gel. Gels were read independently by two individuals and ambiguous results were repeated.
  • D13S314 was derived from the endclone of the 27D8 YAC and anchors the proximal end of the candidate region through a recombination event we have described (12). All markers centromeric to this, including D13S315, are also recombinant and the event does not include D13S133 or D13S316. The location of D13S133 relative to the other three markers is based on the pattern of amplification of YACs in the region. Rare recombination events were observed for two markers. One obligate crossover occurred in 68 meioses informative for D13S314 and in 100 meioses informative for D13S315, no crossovers were found in 44 meioses for D13S133 and 124 meioses for D13S316.
  • haplotypes which differ by no more that two bp at a single marker were grouped.
  • Haplotype C is exclusively associated with allele 5 at D13S315 and allele 4 is present on all chromosomes carrying haplotypes D and E. A specific allele of this marker does not segregate exclusively with the other disease haplotypes.
  • haplotypes are in fact different mutations present on a similar haplotype, and the technique of grouping similar haplotypes should be used with caution.
  • haplotypes A and B only one variant is present on WND chromosomes and the several normal haplotypes have been grouped, therefore any error in classifying haplotypes results in a more conservative estimate of the relative frequency of these WND chromosomes.
  • haplotypes C, D, and E the very low frequency of alleles 5 and 6 on normal chromosomes (0 of 44 haplotypes) as well as the exclusive association with particular alleles of D13S315, make it likely that these groupings are justified. The validity of this technique will be determined once mutations have been identified in the WND gene.
  • haplotypes were characteristic of a particular area.
  • 10 chromosomes carrying haplotype A six are of British, one French, one Dutch, one German and one of unknown origin.
  • Haplotype B chromosomes include seven of British and one of Jewish origin.
  • the seven haplotype C disease chromosomes include two German, two Polish, one
  • Chromosomes with haplotype D consist of three British, three French and one German, and the haplotype 5-11-11, found on a chromosome of French origin is also likely related because it shares an extended haplotype, including D13S315, D13S228 and both RFLPs at D13S31, with other group D disease chromosomes.
  • Haplotype E is found on three chromosomes of German and two of British origin.
  • Chromosomes from other geographical/ethnic groups show no significant differences from those present on normal chromosomes due to small sample size.
  • D13S316 provides strong support for a candidate gene (Wc1) which we have identified on this YAC as discussed in
  • D13S315 failed to detect significant levels of association with Wilson disease despite its location between Wc1 and two markers (D13S31 and D13S228) which have previously been demonstrated to be in disequilibrium with the WND locus (13) and its specific association with three common Northern European haplotypes (C,D and E). This marker is furthest from the disease gene and is likely too mutable to detect association over this large distance. Another explanation is that the association previously seen is due to the chance association of alleles with WND chromosomes. However, other studies have found similar patterns of high and low disequilibrium across a disease region (42).
  • haplotypes found commonly on WND chromosomes also provides clues as to the number of
  • haplotypes A, B, C, and E are similar, the large number of chromosomes of German, Dutch and Polish origin carrying haplotypes C and E suggest that this is a separate mutation from that present on haplotypes A and B, which are found predominantly on chromosomes of British origin.
  • Haplotypes D and G are very different and almost certainly represent separate mutation events.
  • Evidence of common origins or admixture in the European population can be seen in the existence of the common German haplotypes on three chromosomes of British and one of French origin.
  • Haplotype D is present on three of six French haplotypes while three British and one German family also carry this haplotype. It is also possible that this represents different mutations that have occurred on the same haplotype in these populations.
  • the additional five haplotypes observed on single WND chromosomes may represent separate mutational events or rearrangements of more common haplotypes by an ancestral recombination event, as is likely the case with haplotype 5-11-11 which is present in a French family and is likely related to haplotype D (5-11-5), and haplotype 6-17-12 which is similar to haplotypes C and E (6-17-10/11).
  • haplotypes A and B there are likely to be at least three common Northern European mutations; one found on haplotypes A and B, another on haplotypes C and E, and a third on haplotype D as well as four or more rare mutations on haplotype G and three of the singly represented haplotypes.
  • haplotypes present in our two Sardinian families are interesting because they appear to define three distinctly different haplotypes (4-4-7,
  • the 7-17-11 haplotype appears on three of six disease chromosomes of Italian origin, in a Jewish family, and is the most common haplotype present on WND chromosomes in the British families. This haplotype may indicate the presence of a common wide-spread mutation instead of different mutations on the same haplotype.
  • haplotypes A or B would not provide information while the presence of haplotypes C through G could provide support for presence of Wilson disease, but would not be definitive.
  • specific mutations may be defined to provide a more definitive diagnosis.
  • New markers for Wilson Disease have been isolated in the region of the gene on chromosome 13q14.3 as described in Experiment 1. The markers were used to construct a long range restriction map and to obtain 19 YACs in the region. Using the copper-binding motif of the ATPase defective in Menkes disease, a homologous region was identified on three overlapping YACs and on cosmids from a chromosome 13 library. Cosmids were used to isolate cDNA clones by a direct PCR-based cDNA selection strategy.
  • the sequence of the isolated gene shows considerable homology with the Menkes ATPase throughout all its functional domains, including at least 6 copper-binding
  • the gene is expressed in the liver where there is no expression of the MNK gene. This is compatible with the defect in copper transport in the liver observed in
  • Markers are an established marker for Wilson disease with alleles that exhibit strong allelic association with WND (12). D13S196 and D13F71S1 were isolated by Alu-PCR and mapped to the region of WND as described in
  • EHR4 was rescued from the distal end of YAC 235H9 (see Table 6).
  • ICD is a human-hamster somatic cell hybrid containing the proximal half of chromosome 13 as the only human component (14).
  • YACs YACs. YACs were identified from pooled YAC DNA and then isolated from the CEPH YAC library by D. LePaslier using the primers shown in Table 6. All of them are located between the two established markers for Wilson disease D13S31 and D13S59 (11). Genomic DNA was isolated as described in (35) and sizes were determined by pulsed-field gel electrophoresis (PFGE). YAC 27D8 was characterized in detail.
  • probes (27L and 27R) from the left and right ends of the YAC were amplified by inverse PCR (36) and hybridized to Mlu I, Nru I and Not I digests of ICD as described in (15), enabling the YAC to be aligned within the long range restriction map. Further verification was achieved through restriction analysis of the YAC by complete and partial digests with Mlu I and Not I. Partial digestion was achieved using 1-5 min incubation in the presence of 1U restriction enzyme. DNA was separated using a CHEF DR II PFGE system (Biorad) (using a 4-40s pulse for 16h at 200V) and hybridized to probes specific to the right or left arms of the pYAC4 vector. YACs 53C12, 95C3 and 68F4, isolated using primers for probe 27R, have not been fully
  • YAC 232 H4 used as a negative control, does not hybridize to any of the probes in the Wilson disease region.
  • MNK Menkes
  • Probe Me1. a was amplified from the reverse transcribed template by PCR (14) using primers within the putative copper binding region of MNK: Mc967 (5' CAATGATTC AACAGCCACTT3') and Mc1965(5' TTAATATGTGCTTTGTTGGTTG 3'). Thirty five cycles were performed using an annealing temperature of 60°C. An additional probe Mc1.b was
  • Probe Me1. a was hybridized (14) at 50°C to HindIII digests of genomic DNA isolated from YACs in the Wilson disease region (Table 6). Filters were washed once in 2XSSC and once in 0.2XSSC at room temperature and exposed overnight. Similar hybridization conditions were used to screen 100,0000 cosmids from a chromosome 13 specific library (Los Alamos).
  • cDNA fragments for Wc1 were isolated by a direct selection strategy (22, 23) using purified insert from cosmids cosL and cosJ. The DNA was immobilized on filters and incubated with a combination of primary cDNA pools made from adult and fetal liver.
  • cDNAs were subcloned into Bluescript vector (Stratagene). Two hundred colonies were picked at random and arrayed. The colonies were screened at low stringency with probes Me1. a and Mc1.b. To check their localization, positively hybridizing clones were hybridized, under normal conditions of stringency, firstly to EcoRI digests of cosL and cosJ and secondly to HindiII and EcoRI digests of YAC 95C3, human and hamster genomic DNA and the human-hamster somatic cell hybrid ICD. Having confirmed that the clones mapped to the correct region of chromosome 13, they were sequenced and placed into contigs based on an overlap region of at least 50bp.
  • Sequencing data was used to align the clones with each another and with the known sequence of MNK.
  • Clone Wc1. f3 was found to be the most 3' fragment.
  • fragment Wcl.f3 was labelled to a specific activity of 1 ⁇ 10 8 cpm/ ⁇ g and used as a probe to screen four human cDNA libraries. 2 ⁇ 10 6 plaques were screened from an adult liver libraries (Stratagene), 1 ⁇ 106 from a second adult liver library (Clontech) and 1 ⁇ 10 6 from an human hepatoma library and 1.10 6 from an adult kidney library (Clontech).
  • a cDNA fragment (Wc1.bl-1) was amplified using an upper primer (350U: GTG GCT AGC ATT CAC CTT TCC) developed from the 3' end of close Wc1.f3 and a lower primer developed from an arm of the cloning vector.
  • An additional cDNA fragment (Wcl.87-90) was isolated by RT-PCR (described above). The first strand was extended on l ⁇ g of poly A+ fetal liver RNA (Clontech) from primer
  • F3L (5'ATGCGTATCCTTCGGACAGT3'). Forty cycles of PCR were performed using primers 1009U (5'GGCACATGCAGTACCACTCT3' ) and 1662L (5 ' TCTGTCTGGGAGATGTGCTT3 ' ) with an annealing temperature of 67°C.
  • Cosmid mapping Cosmids cosJ and cosL were digested to completion with Not I and then partially digested with XbaI , EcoRI or HindIII . Southern blots of the digested DNA were hybridized to probes derived from the arms of the cosmid vector. Fragments detected were used to construct restriction maps of the two cosmids.
  • Alignment of derived amino acid sequences Alignment of the cDNA clones to MNK and other proteins was carried out at NCBI using the BLAST network service.
  • cDNA Wc1.c8 was labelled to a specific activity of 1 ⁇ 10 8 cpm/ ⁇ l and hybridized for 20h to the filter in a solution containing 50% formamide, 6 ⁇ SSC, 0.1% SDS. The filter was washed once in 2 ⁇ SSC, and once in 0.2 ⁇ SSC, 0.1% SDS at 65°C. The filter was exposed to X-Ray film (Kodak) for 6 days. In addition, a Northern blot containing polyA+ RNA from heart, brain, placenta, lung, liver, muscle, kidney and pancreas (Clontech) was probed with Wc1.c8 using conditions recommended by the manufacturer.
  • Probe Me1 a from the proposed copper binding region of MNK (nucleotides 965-1965 (17) ) was hybridized at low stringency to the 19 YACs listed in Table 6. Above the background hybridization, shown by all YACs (represented in Fig. 3b by YACs 232H4 and 68F4) additional bands were observed in three overlapping YACs : 27D8, 95C3 and 53C12
  • Fig. 3b Two fragments (2.5 kb and 8.9 kb) were detected only in these YACs.
  • the location of YAC 27D8 with respect to the established marker D13S31 is shown in Fig. 3a.
  • probe Me1. a was used to screen a chromosome 13 specific cosmid library under the same conditions of low stringency. Two overlapping cosmids (cosJ and cosL) were isolated. From cosL, a non-repetitive probe (cosL.d) was isolated that was also found to cross hybridize to Me1. a.
  • cosL.d To check the localization of cosL.d, it was hybridized under normal conditions of stringency to YACs 27D8, 53C12 and 95C3 (Fig. 3b). The same 2.5 kb and 8.9 kb fragments were detected.
  • cosmids cosL and cosJ were used to isolate expressed sequences from liver using a direct PCR based cDNA selection strategy (22, 23).
  • a direct PCR based cDNA selection strategy 22, 23.
  • 200 selected cDNA clones were arrayed and screened at low stringency with probe Me1. a and a probe (Mc1.b) more towards the 3' end of the MNK cDNA in the ATP-binding region (nucleotides
  • each cDNA was hybridized to EcoRI and HindIII digests of cosJ and cosL, YAC 95C3, and a chromosome 13 hybrid, ICD.
  • a representative result is shown in Fig. 4a. All fragments detected by the clones used for further analysis mapped only within cosJ or cosL with no other homologous regions on hybrid ICD.
  • one of the cDNA fragments was hybridized to Mlu I and Not I digests of DNA from hybrid ICD that had been separated by pulsed field gel electrophoresis (PFGE). The probe detected a 2200 kb i ⁇ 7ru I fragment and 2100 kb and 1200 kb Mlu I fragments that are all common to the established marker D13S31 (15) (Fig. 4b).
  • the order of the cDNA clones was established by mapping each clone on cosmids cosL and cosJ digested with several restriction enzymes (Fig. 3a).
  • the gene covers a region of at least 20 kb.
  • Fig. 7 Alignment of these domains with the corresponding domains in MNK is shown in Fig. 7.
  • the six Wc1 copper domains in the figure show a mean amino acid identity of 65 percent with the corresponding copper domains one to six of MNK.
  • One of the clones Wc1.f ⁇ seems to be unspliced message since it contains a splice donor site. The site is also present in genomic DNA (not shown).
  • Both MNK and Wc1 also contain highly conserved domains characteristic of the P-type family of cation transporting ATPases. This family includes magnesium, calcium,
  • DKTGT Asp-Lys-Thr-Gly-Thr
  • MNK and Wc1 C-terminal to the transduction and phosphorylation domains is a highly conserved ATP-binding domain including a Gly-Asp-Gly (GDG) motif. Alignments of MNK and Wc1 around these three domains are shown in Fig. 8. The identity between MNK and Wc1 is 86 percent throughout the transduction/phosphorylation domains and 79 percent throughout the ATP-binding domain.
  • Fig. 7 Also shown in Fig. 7 is the alignment and homology of the functional domains of Wc1 with various heavy metal transporting ATPases from bacteria (for a review see (25)). As has previously been demonstrated for MNK (17, 18, 19), the functional domains of Wc1 are more closely related to these prokaryotic genes than to any characterized
  • MNK eukaryotic gene
  • copA from the gram positive bacteria Enterococcus hirae a gene involved in copper transport (26) Alignments are also shown with a mercuric transporting plasmid encoded protein merP from Escherichia coli (27), a cadmium
  • ATPases These are, two cysteine residues, flanking the invariant proline in the transduction domain and a proline situated 8 residues C-terminal to it. These residues may be involved in conferring metal specificity to the proteins (17).
  • DNA sequence is being submitted to genbank.
  • RNA was analyzed from brain, lung, spleen, heart, esophagus, muscle, liver and lymphoblasts. Transcript was detected only in the liver, and in relatively low abundance, only a small fraction based on the actin control (data not shown).
  • Poly A+ RNA was analyzed from a number of tissues (Fig. 9). Transcript of 7.5 kb was detected at an almost equal abundance in the liver and kidney. A slight trace of message of a similar size was also detected in heart, brain, lung, muscle, placenta and pancreas. The transcript appeared to be slightly smaller than the MNK transcript which is approximately 8.0-8.5 kb (17, 18, 19).
  • the placenta appeared to have an additional transcript of about 7 kb.
  • Wc1 gene encodes a copper transporting protein.
  • the gene shows high homology with MNK, which is proposed to be involved in transporting copper from intestinal and other cells. Sequence identity is observed in functionally important regions: the energy transduction, phosphorylation and ATP binding domains are 79% identical or greater.
  • Wc1 is predicted to be the Wilson disease gene because it lies within a region of chromosome 13 that is known to contain WND.
  • Wc1 is flanked proximally by D13S314 and distally by D13S133 and D13S316 (16).
  • Wc1 and MNK are very different. MNK is expressed in lung, skeletal muscle and heart, but is scarcely detectable in the liver or kidney. In contrast, Wc1 is expressed mainly in the liver and kidney. This tissue expression is appropriate for Wilson disease. A key feature in Wilson disease is accumulation of copper in the liver. The expression in kidney is consistent with the occurrence of kidney damage, believed to be due to copper toxicity, in many Wilson disease patients. Abnormalities of renal tubular function include aminoaciduria, proteinuria, uricosuria, hypercalciuria, defective urinary acidification, renal stones, and occasionally full blown Fanconi syndrome (29, 1).
  • Wilson disease The two main biochemical characteristics of Wilson disease are the disruption of incorporation of copper into ceruloplasmin in the liver and a severe reduction of copper excretion from the liver into the bile (5). Any candidate gene must have potential for involvement in both processes. Ceruloplasmin deficiency, almost always associated with Wilson disease (30) has been recognized as being very closely related to the basic defect. The localization of the ceruloplasmin locus to chromosome 3 (31) showed that a defect in the ceruloplasmin molecule could not be the basic defect in Wilson disease. However, the deficiency is present in patients in early life, before high levels of copper accumulate in the liver. Ceruloplasmin is a 132 kDa glycoprotein containing six atoms of tightly bound copper per molecule, synthesized in hepatocytes (32), and a possible donor of copper to tissues and enzymes (3).
  • Wc1 contains CXXC motifs in each of its metal binding domains, together with one CXC motif in the transduction domain. Similar motifs are
  • Wc1 and MNK are the only such metal transporters isolated to date from eukaryotes, but the high degree of homology preserved between the toxic metal binding ATPases of organisms as evolutionarily divergent as bacteria and humans indicates the fundamental importance of this type of molecule.
  • the ATP7B gene encodes a 7.5 kb transcript, of which 4.2 kb codes for the protein (44).
  • the gene is highly similar to the gene responsible for Menkes disease (ATP7A), which has recently been cloned, and spans 150 kb of genomic sequence on the X chromosome (17-19).
  • ATP7A Menkes disease
  • Exon/intron boundaries within the sequence of the ATP7B cDNA were identified by sequencing of genomic DNA in three cosmids which span the region of the gene. The isolation of two of the cosmids (J and L) has been described elsewhere and a third was isolated from the same chromosome 13 specific library using a cDNA clone from the 3' end of the transcript (Wc1.f3) as a probe (44). Primers were designed from cDNA sequence using the OLIGO program
  • Genomic Restriction Map A total of 10 ⁇ g of DNA from each cosmid was digested to completion with NotI to excise the insert and then digested with 5 units of BamHI, EcoRI, HindIII, KpnI or Sacl for 1,2,5,10,20 and 60 minutes in 10 ⁇ l volumes.
  • the partially digested cosmid DNA was electrophoresed through 0.6% agarose at 56 Vhr/cm to resolve large fragments (>6kb) and 1.2% agarose at 40 Vhr/cm to resolve small fragments and blotted on nylon membrane (Hybond N+, Amersham).
  • Each cosmid was probed with primers flanking the insert (45) to generate a restriction map. This map was confirmed by total digestion with the same five enzymes and their double digest combinations and by probing with exonic fragments as described below.
  • Primers were designed to amplify each exon and tested on cosmid and genomic DNA. Table 7 lists the primers used and the MgCl 2 concentrations that are optimal for amplification of 10 ng of genomic DNA. Also listed are restriction enzymes which cleave each amplimer into two fragments of appropriate size for SSCP analysis. The use of these primers from SSCP is described elsewhere (46). Individual exons were then used to probe cosmid DNA digested to completion with the enzymes described above.
  • Exons were labelled by amplification of 10 ng of cosmid DNA in 20 ⁇ l volumes containing 50 mM KCl, 10 mM Tris, pH 8.0, 10 mg/ml BSA, 1.5 mM or 3 mM MgCl 2 (table 1), 200 ⁇ M each of dATP, dGTP and dTTP, 25 ⁇ M dCTP, 2 ⁇ Ci [ ⁇ 32 P]-dCTP and 0.5 units of Amplitaq (Perkin Elmer). Amplification was performed in an MJ research PTC-100-96V Programmable Thermal Controller with 35 cycles of 30 seconds denaturation at 94°C, 30 seconds annealing at 55°C, and 30 seconds extension at 72°C. Unincorporated label was removed by sephadex G-50 spin column and hybridization was carried out by standard methods.
  • Exon 21 was mapped by probing cosmid DNA with a primer derived from the cDNA sequence in this region.
  • the primer is located immediately 5' of the stop codon in this exon and its sequence is GGACAGCGGCAGAGCCAGGAAAC.
  • Sequencing of the WND cosmids with cDNA primers identified a total of 20 exons in the liver transcript.
  • the sequence of each exon is shown in Figure 11.
  • the first and last nucleotide of each exon, its length, splice sites and domains are listed in table 8.
  • the locations of the functional regions of the gene within the exons are shown in figure 12.
  • the results obtained from sequencing primers in the 5' end of the cDNA sequence include sequence that is further upstream than that published.
  • the 5' untranslated portion of the cDNA is expected to be contained within one or more exons located upstream of this point.
  • the 3' end of the gene is different in kidney and liver cDNA clones (44, 47).
  • the final coding exon of the liver-derived sequence is contiguous with the genomic sequence and defines a single exon, designated as exon 20 in table 8.
  • the kidney-derived sequence in this region is identical to the liver cDNA for the first 73 base pairs of exon 20 and then diverges. There is a consensus splice donor site at this point, indicating that the difference between the two tissue transcripts is most likely due to the use of alternative splice sites. Therefore, at least some portion of the transcripts in the kidney have an additional exon added at this point.
  • the portion of the restriction map of the three WND cosmids containing the coding region of ATP7B and the locations of each of the exons are shown in figure 13.
  • the coding portion of the gene spans approximately 40 kb of genomic DNA.
  • the three overlapping cosmids from which the map was derived span a total region of about 80 kb, with 20 kb on each side of the map shown.
  • Introns 9, 10, and 17 have been completely sequenced and are each less than 200 bp in length (data not shown) thus exons 9-11, and 17-18 are shown as single blocks.
  • exons in the ATP7B gene is a necessary first step in order to make large scale mutation screening of Wilson disease possible.
  • the exons have also been placed on a genomic map of the region derived from cosmid DNA.
  • the coding region of the transcript is contained within 21 exons. Most of the exons are less than 300 base pairs in length, with the notable exception of exons 1 and 20. Exon 1 is 1.2 kb in length and includes copper binding domains (Cu) 1-4, while exon 20 includes the 271 bp of coding sequence before the termination codon and an undetermined number of bases beyond this point, and is greater than 400 bp in length. The size of exon 21 is unknown. This makes amplification of individual exons and subsequent SSCP relatively simple and sequencing of the PCR products can be accomplished with one reaction. We have divided exon 1 into six overlapping amplimers in order to cover the entire region and only the coding portion of exon 20 is amplified.
  • the restriction mapping of the WND region shows that the ATP7B coding region spans 40 kb of genomic DNA.
  • An additional 20 kb of DNA on either end of cosmid contig presumably contains the 5' and 3' noncoding regions and possibly the promoter and other regulatory portions of the gene. This is in contrast to the reported 150 kb for
  • Peripheral blood was collected from 34 Canadian families (consisting of 25 of Northern European, 4 of
  • Selected patient samples were screened for mutations by the use of single strand conformational polymorphism (SSCP) analysis on individual exons.
  • Patient samples were selected on the basis of the haplotypes derived from markers in the WND region (46), such that all haplotypes were represented at least once.
  • the primers used for each exon, their product length, and MgCl2 concentrations for optimal amplification are given in experiment 3 (51).
  • Exons were amplified under conditions identical to those used for the CA repeats (46), with an annealing temperature of 55°C, and digested for 2 hours with the appropriate restriction enzyme (51).
  • the samples were then diluted with one volume of SSCP buffer (0.2 M NaOH, 1% SDS) and three volumes of loading buffer (95% formamide, 15 mM EDTA, 0.03% each of xylene cyanol and bromphenol blue) and electrophoresed through 6% non-denaturing polyacrylamide gels at either room temperature with 10% glycerol for 18-24 hours at 10W or at 4°C with no glycerol for 5 hours at 35 W before drying and exposure to film.
  • SSCP buffer 0.2 M NaOH, 1% SDS
  • loading buffer 95% formamide, 15 mM EDTA, 0.03% each of xylene cyanol and bromphenol blue
  • Patient samples exhibiting shifts relative to normal samples on SSCP were subjected to direct sequencing to determine the nature of the mutation.
  • Patient and parents (where available) were amplified as above for 35 cycles with 200 ⁇ M cold dATP and no [ ⁇ 35 S]-dATP. Products were purified with a QiaQuick spin column (Qiagen), cycle sequenced (Circumvent, New England Biolabs ) using the PCR primers , and electrophoresed through 6% denaturing polyacrylamide gels.
  • experiment 1 (46) have been used to examine the haplotypes present on both normal and WND chromosomes. The locations of these markers relative to the disease gene are shown in figure 14. The locations of D13S133, D13S314 and D13S316 have been described in experiment 1. The position of D13S296 telomeric to ATP7B is based on its location near D13S133 (50). D13S301 has been placed centromeric to WND by a recombinant family that we have described previously (12).
  • 1652insT was identified on a single chromosome in an Sikh patient and also results in termination at the same point.
  • 2065delA was found on a single affected chromosome in a family of Italian origin, and also results in an immediate stop codon.
  • 2206insC was found on four chromosomes; 2 in British families, and one each in Scottish and Italian kindreds. The frameshift results in a termination codon 27 amino acids downstream in the next exon.
  • the 2881de1C mutation was found to be homozygous in a Sardinian and British patient and heterozygous in another British
  • L905X is caused by a T to A change at the second base of the codon and was found to be present on both chromosomes in a Saudi Arabian family and one chromosome in a Greek kindred.
  • R1288X was found on a single chromosome in a British family and is due to a C to T change at the first base of codon 1288.
  • the mutation 2482+1G ⁇ C alters the invariant G residue at the start of intron 9 which would result in the addition of the intronic DNA to the transcript (figure 16B) . This results in 36 amino acids after the end of exon 9 and then a premature termination codon. There is a near consensus splice donor site 52 bases into the intron which might be used in this transcript but the resulting product would have a frameshift. This mutation was found to be heterozygous in a British patient.
  • the third splice mutation, 3463+lG ⁇ A, was found on a single chromosome in a British family and also alters the invariant first base of the donor site, in intron 15
  • the other three base alterations that affect splice sites are not expected to alter splicing of the transcript.
  • the first change alters the splice site toward the consensus and the second does not change consensus.
  • the last base change alters the splice site away from the consensus but this change is found on approximately 40% of affected chromosomes and has been detected on two normal chromosomes by direct sequencing.
  • M738V and V964A were found on single chromosomes of British and Sikh origin, respectively, L735V was found on single chromosomes in two British patients and M1138V was found in a British and an Italian patient. Allele frequencies of these polymorphisms within the normal populations were not determined.
  • the identification of mutations in the ATP7B gene is a necessary first step in order to make direct diagnosis of Wilson disease possible.
  • the mutational data obtained has been compared to extended haplotypes of Wilson disease patients to assess the usefulness of CA repeat haplotypes in prediction of the disease state.
  • haplotype analysis has resulted in a more complicated picture of the number of mutations present in the populations studied.
  • the two haplotypes previously described (46) as being the most common in the Northern European populations (A and B) have been shown to be a collection of different haplotypes when more markers are added. This is likely due to the fact that the alleles of D13S314, D13S133, and D13S316 present on these original haplotypes are the alleles most commonly found on normal chromosomes in this population, and represent a common background on which a number of mutations may have
  • haplotypes C, D, and E are seen to remain tightly grouped with only slight variations (no more than 2 bp at the D13S301 locus, 1 bp at D13S316 or one haplotype with an 8 bp change at the D13S296 locus).
  • Groups C and E can be seen to share a common origin in that the majority of both haplotypes carry the H1038Q mutation.
  • Group D is also associated exclusively with a single mutation, G1235K.
  • the large number of subtypes within groups A and B indicate that there are a large number of mutations present in the Wilson disease population.
  • the mutation present on most group C and E haplotypes has been identified as H1038Q.
  • a few chromosomes carry the group C or E haplotype but do not have this mutation, and the changes present on these chromosomes have yet to be identified.
  • These two groups of haplotypes differ by no more that 4 bp at a given locus, most of the variation occurring at the D13S301 marker.
  • the correlation of haplotype group to mutation supports our previous system of grouping haplotypes that differ by no more than 2 bp at one locus (46).
  • the mutation on group D chromosomes has also been identified (G1235K).
  • Group C, D and E chromosomes represent approximately 37% of the Wilson disease chromosomes present in the Northern European population.
  • Careful examination of the haplotypes reveals that the D13S316 marker is diagnostic for these two mutations, in that most chromosomes carrying allele 6 at this marker have the H1038Q mutation and all chromosomes carrying allele 5 have the G1235K mutation.
  • These alleles are not present in the normal chromosomes in our families. This haplotype/ mutation association can be used to rapidly identify chromosomes likely to carry one of these mutations and this could be then confirmed by a single sequencing reaction.
  • Southern European population One Southern European haplotype (SE2) is identical to the group C/E haplotypes in the Northern Europeans and indeed carries the same mutation, indicating a common origin for these chromosomes. This may prove true for other haplotypes from all of the ethnic groups. Indeed, the existence of the same mutations in different ethnic groups (ie. 750delC) lends support to this prediction.
  • haplotypes which differ by more than 4 bp at several markers.
  • the 2206insC mutation is present on three very different haplotypes which cannot be explained by recombination.
  • this mutation is a insertion of a C into a series of six within the cDNA sequence and thus may represent a spot where DNA polymerase is more likely to make an error during replication. Therefore, these three haplotypes may represent independent origins of the same mutation.
  • R747L is present on three different haplotypes and is less likely than 2206insC to represent independent occurrences of the same mutation. This may represent a case of gene conversion that has resulted in the mutation being transferred to another haplotype.
  • haplotypes There are also cases of different mutations present on chromosomes with identical haplotypes. Several group C/E haplotypes do not carry the H1038Q mutation, presumably having another lesion. This haplotype is not found within the general population is not likely to be a common background on which two mutations have occurred
  • Table 14 lists a number of polymorphisms that have been identified within the ATP7B gene, only four of which are rare. These polymorphic bases, some of which alter restriction enzyme sites, could be readily typed in Wilson disease patients in order to extend the haplotype data. The marker loci would not be subject to the polymerase slippage that may cause large variation between related haplotypes consisting solely of CA repeat markers.
  • the Long-Evans Cinnamon (LEC) rat (54) shares many clinical and biochemical features with Wilson disease.
  • This inbred rat strain was originally established in 1975 from a closed colony of non-inbred Long-Evans (LE) parental rats through successive generations of sibmating. Spontaneous acute hepatitis with severe jaundice occurred in a male rat from the F 24 generation at five months of age (54, 55). The mutant allele causing the hepatitis was fixed in subsequent generations through inbreeding, and backcross experiments demonstrated an autosomal recessive pattern of inheritance for this condition (55, 56). LEC rats spontaneously develop acute hepatitis about four months after birth, with clinical features similar to those seen in human fulminant hepatitis, sometimes a feature of Wilson disease.
  • the cDNA library was made from the liver of an adult Sprague-Dawley male rat using oligo-dT/random hexamers as primers (Clonetech #RL1023a). Library screening was done according to the standard procedure, using human ATP7B cDNA segments Wc1.g1, Wc1.c8, Wc1.87-90 and Wc1.gb10 as probes (2). Inserts from positive
  • DNA probes were labelled with ⁇ - 32 P-dCTP using random priming method (25) and hybridization was carried out in 5x SSC, 0.1% SDS, 5x Denhardt, 100 ⁇ g/ml sheared and denatured salmon sperm DNA and 10% dextran sulphate at 65°C overnight and final washing conditions were at the same temperature and in 0.1x SSC and 0.1% SDS. Autoradiography was done with Kodak films at -70°C for 1-3 days. RT-PCR analysis. Poly(A+) RNA was extracted from liver tissues of a female LEC and a control female LE rat, using the Fasttrack mRNA isolation kit (Invitrogen).
  • RNA was reverse-transcribed in a volume of 33 ⁇ l, using random hexamers and murine reverse transcriptase and other reagents in the First-strand cDNA synthesis kit (Pharmacia) according to supplier's protocol.
  • PCR was carried out in 20 ⁇ l containing 0.5 ⁇ l of the reverse-transcribed cDNA templates, 20 pmole of each primer, 100 ⁇ M of each dNTPs, 50 mM KCl, 10 mM Tris/pH 8.3,1.5 mM MgCl 2 and 1.0 U Taq polymerase.
  • the step-cycle mode amplification started with one cycle at 95°C for 3 min, 60°C for 2 min and 72°C for 3 min, followed by 30 cycles each at 94°C for 1 min, 60°C for 0.5 min and 72°C for 1 min and terminated with a final extension at 72°C for 15 min. About 10 ⁇ l of the reaction products were analysed by electrophoresis on a 1.5% agarose gel.
  • Sequences of the primers are: 1) 0114F: GACATGGGATTCGAAGCTGC; 2) 0108R: CACTTCTGTGATGCTGTTCC; 3) DF1 : AATGCTCATGGCTCTGTGCTC; 4) DR1: CCACAGCCAGAACCTTCCTG; 5) DR2 : CCAGCATACTTTCCACGTTGC;
  • cDNA clones for ATP7B were isolated from a rat liver cDNA library, using as probes cDNA sequences for the human ATP7B gene (2).
  • a consensus sequence of about 4.7 kilobases (kb) was derived from these overlapping clones and its nucleotide sequence determined.
  • the sequence has a single large open reading frame (ORF) and includes 300 bp of 3' untranslated region.
  • the first in-frame methionine codon starts 17 nucleotides (nt) downstream of the 5' end of the sequence and there is a second in-frame methionine codon located 32 amino acids downstream.
  • the ATP7B cDNA sequence is highly homologous to its human counterpart and, as in humans, is predicted to encode a copper transporting P-type ATPase.
  • Overall structure as well as each of the individual functional domains are well conserved between the rat and human, with the exception of the lack of metal binding motif 4 in the rat (Figs. 17 & 18).
  • the sequence divergence between the two species in this region was confirmed by sequencing (two clones) and restriction mapping (five clones) independent cDNA clones.
  • the ATP7b cDNA sequences were used to probe Southern blots of genomic DNA digests from an LEC rat and a control LE rat.
  • a mixture of probes p7-5, p7-6 and p7-1 representing the entire 4.7 kb rat cDNA sequence
  • restriction enzyme digests of the genomic DNA from the two rats gave rise to different hybridization patterns (data not shown).
  • BamHI, HindIII, HincII, PvuII and TaqI the LEC DNA was found to have, in addition to some hybridization fragments shared with the control LE sample, at least one fragment difference, either missing altogether or altered in size, when compared with the LE pattern. This discrepancy was clearly not due to a polymorphism but could only be
  • probes p7-5, p 1-8 and p7-1 representing different parts of ATP7B, were used separately to localize the deleted region.
  • Probes p7-5 and pi-8 gave rise to identical hybridization patterns between the LEC and the control rats, whereas probe p7-1 revealed the same hybridization abnormalities noted before in the LEC DNA sample
  • Enzymes used for the digestion (Hd, Hindlll; Hc, Hincl) and probes used with each blot are indicated on the top.
  • the relative positions of these probes in the Atp7b cDNA sequence are indicated in Fig. 18. While data are shown only for the two enzymes, results using BamHl, Pstl and Taql gave the same conclusion.
  • 7RsF1 and 7RsF2 detected bands in both the LEC and the control rat DNA samples: 7RsF1 gave rise to identical hybridization patterns in the LEC and the control (an 8.5 kb fragment); 7RsF2 revealed an altered HindIII (5.8 1:b) fragment instead of the normal 11 kb fragment from the LEC sample (Fig. 19). Size alteration was also seen in the BamHI digest tested with 7RsF2 (data not shown). In contrast, 7RsF3 and 7RsF4 hybridized only to the control samples but not to the LEC DNA.
  • sequences represented by the probes 7RsF2 and 7RsF3 and the deletion extends to the most 3' end of the cloned region. Refining the deletion breakpoint
  • RT-PCR Reverse transcription-polymerase chain reaction
  • Lanes 1, 5, 7, 9 and 11 contain amplification products from the control LE sample; lanes 2, 6, 8, 10 and 12 contain those from the LEC rat. Primer pairs used are:
  • Lanes 3 and 4 show Rsal digests of the 844 bp fragments amplified with the primer pair 0114F/0108R from the LE and LEC cDNA samples, respectively. There were some weak nonspecific bands in lanes 5, 6, 10 and 12. As a control, amplifications with LE rat genomic DNA as template were done for all pairs of primers and no amplification products were detectable on the ethidium bromide-stained get for any primer pairs tested. DNA size markers are indicated on the left.
  • ATP7B cDNA-specific primer pair 0114F and 0108R amplified an 844 bp fragment from both the LEC and the control LE cDNA samples, a size expected from the normal cDNA sequence we determined.
  • the authenticity of these transcripts was further confirmed by digestion with the enzyme Rsal: four fragments of the expected sizes (374 bp, 290 bp, 112 bp and 68 bp) were produced from both the LEC and LE 844 bp fragments.
  • RT-PCR analysis was then applied to refine the proximal deletion breakpoint in the coding region.
  • a sense strand primer, DF1 was chosen in an area that is known to be present in the LEC ATP7B gene (based on the Southern hybridization results) and a series of three complementary strand primers, DR1, DR2 and DR3 were selected adjacent to the inferred deletion breakpoint region (Fig. 18).
  • rat homologue of the human Wilson disease gene ATP7B The coding sequences of the gene show a strong homology between rat and human. Overall structure and particularly individual functional domains are well conserved, confirming the functional importance of these regions.
  • the rat gene has each of the six deletions and insertions (from 3 to 78 amino acids in length) found in the human ATP7B gene in comparison with the Menkes gene, ATP7A (2). It is
  • LEC rats Another feature of the LEC rats is the extremely high incidence of hepatocellular carcinoma in those rats that survive the initial attack of hepatitis at age 12 months or older. This is in contrast with Wilson disease, in which patients rarely develop liver cancer. This difference might be due, at least partially, to many biological differences between the two species.
  • transgenic mice with excessive storage of abnormal Z ⁇ 1 -antitrypsin, as in human ⁇ 1 -antitrypsin deficiency also develop hepatocellular carcinoma (65), while this rarely occurs in the human patients.
  • patients with untreated Wilson disease may not survive long enough to develop cancers. The mechanisms leading to carcinogenesis in rats are still poorly understood, however, it is likely that the
  • abnormally high level of copper accumulation plays an important role in the process possible actions of the toxic level of copper include DNA-damaging effects from copper ions directly, or indirectly such as through the generation of free radicals (66, 67) and disturbance of expression of those genes controlled by certain zinc-finger transcription factors through replacement of zinc ion by copper.
  • This rat model can also be used to gain information on normal copper transport. Copper transport in the plasma has been well studied, but the mechanism of efflux from the liver is not well understood. The function of the Wilson disease gene must be closely associated with incorporation of copper into ceruloplasmin, as reflected by a very low ceruloplasmin concentration in most patients and in LEC rats. Questions of how the transport takes place and whether other proteins are involved can now be addressed. Also, because of the high degree of similarity between the cadmium and copper transporting ATPases in bacteria and the Wilson and Menkes disease genes (68), other heavy metals such as cadmium, may share or interact with the same transport system (69).
  • markers we have developed While other markers have been developed by others in this region, ours are particularly useful in that they are within about 200 kb of the Wilson disease gene, are very highly polymorphic, and the combination of these alleles, or haplotypes, have been studied both in our patients and in normal individuals (see Experiment 1).
  • the markers we have found particularly useful are as follows:
  • Wilson disease Diagnosis of Wilson disease is particularly difficult for those with liver disease, since copper accumulation, characteristic of Wilson disease, also occurs in other liver diseases which have a biliary obstructive component. Every abnormal biochemical test in Wilson disease can be found to be abnormal in some other type of liver disease. For example, in addition to high liver copper, ceruloplasmin typically decreased in Wilson disease, may be elevated into the normal range.
  • haplotypes we have developed with our DNA markers, along with D13S133, can be used to increase the certainty of a diagnosis of WND that a patient has Wilson disease. This is because some of the haplotypes which occur in patients are rare in the general population. If a patient has one of these haplotypes, the chances of having a Wilson disease mutation are high. In combination with biochemical data, positive support for a diagnosis of Wilson disease could be obtained and treatment initiated immediately. Examples of haplotypes which are considerably more common in Wilson disease, and have not been found in the normal population are as follows: (refer to Experiment 1 for further description of haplotypes). These haplotypes are comprised of the following markers:
  • Haplotype C 6 - 17 - 10 - 5 (particularly in German patients)
  • Haplotype D 5 - 11 - (5 or 4) - 5 (particularly in French patients)
  • Haplotype E 6 - 17 - 11 - 4 (particularly in German and
  • haplotypes represent 40% of a series of 47 random patients. This suggests that the haplotype approach could be useful in a relatively large proportion of cases.
  • the proposed sequence can be used for the analysis of specific mutations in patients with Wilson disease.
  • the direct analysis of such mutations has important implications for diagnosis.
  • All regions of the sequence can be analyzed by methods such as the polymerase chain reaction, with primer sequences from within the cDNA region as given, or from intron sequences not presented as part of the present sequence.
  • Any of the sequence which is amplified is included in the invention, whether amplified from sequences given or from sequences lying immediately
  • the amplified portions of the sequence also include similar sequences which may have one or a few nucleotides altered, with the end result being amplilfication of the sequence given. Regions of 250 to
  • 300 base pairs can be analyzed through mutation analysis by direct sequencing.
  • Another method for detecting mutations is through the examination of fragments of 200 to 300 base pairs, which are then analyzed by single strand polymorphism confirmation (SSCP) analysis or by heteroduplex analysis. Either of these meethods can detect differences from the normal sequence. The exact mutation can then be confirmed by sequencing. However, once mutations are established, such a survey will be useful for direct mutation detection.
  • SSCP single strand polymorphism confirmation
  • sequence we have obtained is useful for the direct detection of mutations. Based on this sequence, we have developed PCR primers to amplify the functional motifs of the protein: copper binding, energy transduction, phosphorylation, and ATP binding. From our sequence, we have developed sequencing primers to sequence PCR products to identify mutations.
  • the intron exon boundaries we have sequenced will provide a useful source for PCR primers to amplify exons of the gene for the further search for mutations.
  • Certain bacteria have adapted to survive high copper conditions by replicating a high copy number of a plasmid which contains a sequence to encode an ATPase with a copper binding domain, very similar to the Wilson disease gene.
  • Partial hepatectomy can improve the stability of targeted DNA (Wilson et al. J. Biochem.
  • Example 2 We have outlined in Example 2 that the Wilson disease gene is similar to genes on cadmium resistance and mercury resistance plasmids in bacteria. The similarity exists through all of the functional domains; metal binding, transduction phosphorylation and ATP binding. The Wilson disease gene could therefore be used, if incorporated into a plasmid construct, to remove excess cadmium or mercury from tissues. As expressed above, this is feasible for removal from the liver. Cadmium is particularly carcinogenic in the kidney, and it is of interest that the Wilson disease gene is expressed in kidney (Experiment 2).
  • a construct containing the Wilson disease gene could potentially be used to overcome the defect in Menkes disease, since the copper binding region is very similar.
  • a new process of targeting tissues with DNA-coated gold pellets suggest that the intestinal cells, the site of the defect in Menkes disease, could be induced to incorporate Wilson disease DNA to allow transport of copper out of that tissue.
  • Introduction of the plasmid into the intestinal epithelial cells seems also to be feasible.
  • Wilson and Menkes disease Another approach, for both Wilson and Menkes disease would be to induce overexpression of the defective gene, which may be possible if there is residual activity of the gene product.
  • Wilson disease gene could be targeted into the germ line of organisms for which the accumulation of toxic metals is a problem.
  • targeting of the Wilson disease or of similar sequence into a plasmid into the germ line of fish stocks could increase the ability of such stocks to eliminate heavy metals, in regions which have naturally-occurring or pollution induced metal contamination.
  • Copper toxicity has been noted as a problem in sheep, as may also be a problem in other domestic species. It is possible that this toxicity in sheep is due to particularly low levels of expression of the homologous gene to the P-type ATPase described in this application for WND.
  • the sequence presented may therefore have some application in therapy for toxicity in sheep, or in other animal species, or could be used in breeding to produce sheep, or other species which are more copper resistant.
  • the sheep is given as only one example of an animal sensitive to copper toxicity.
  • Other uses are also envisioned for the removal of copper or other toxic metals not only from sheep, but a variety of other organisms, including the removal of mercury from fish or any other species.
  • the DNA sequence of the present invention can be used to obtain the equivalent gene from the mouse, to study the homologous gene.
  • the human sequence in this application could be used to facilitate obtaining the sequence for the homologous gene in the toxic milk mouse, an inbred strain of mutant mouse, the defect in copper metabolism which may be identical to that of Wilson disease. Any use of the human sequence or a portion of it to be used for study of the toxic milk mouse and its normal counterpart are ineluded in this application.
  • D13S133 See Ref. (Petrukhin et al. 1993) a Reference genotypes from CEPH family 1332. Numbers are allele sizes in base pairs.
  • Intronic sequences are shown as lowercase letters, exonic sequences are uppercase. The invariant residues at start and end of each intron are shown in bold.
  • the given splice donor site for exon 20 is the site located 73 bp into die exon and appears to be used in kidney transcripts.
  • New haplotypes do not include D13S133.
  • IP Indian/Pakistani
  • S Sardinian
  • SE Southern European
  • Haplotype group (tables 3 and 4) is given in parentheses after the haplotype.
  • the numbering of bases begins at the ATG initiator codon.
  • Rhizobium melitoli fixGHI sequence predicts involvement of a specific cation pump in symbiotic nitrogen fixation. J bacteriol 171, 929-939 (1989).
  • Wilson disease gene is a putative copper
  • Thomas GR, Roberts EA, Walshe JM, Cox DW The Wilson disease gene: mutations and haplotypes. Am J Hum Genet 1994; 54: 71-78.
  • Mulligan R.C Correction of the genetic defect in hepatocytes from the Watanabe heritable hyperlipidemic rabbit. Proc. natn. Acad. Sci. U.S.A. 85, 4421-4425 (1988).

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Abstract

La maladie de Wilson (dégénérescence hépatolenticulaire) est un trouble récessif autosomique du transport du cuivre, entraînant une accumulation toxique de cuivre dans le foie et le cerveau. Le gène de cette maladie, (locus WND), a été cartographié sur la bande q14.3 du chromosome 13. Une séquence analogue aux motifs proposés de liaison de cuivre de l'ATPase (MNK) présumée défectueuse dans la maladie de Menkes a été identifée sur trois chromosomes de levure artifiels à chevauchement (YAC) de cette région. Il a été démontré que cette séquence fait partie d'un gène d'ATPase de type P (Wc1) qui est très semblabe à MNK, avec au moins six domaines de liason métallique présumés, homologues à ceux présents dans des transporteurs de métaux lourds procaryotiques. Ce gène se trouve dans une région de 300 kb qui a été identifiée comme un emplacement probable du locus WND. Ce gène est exprimé dans le foie et les reins. Des mutations ont été identifiées dans le gène chez des patients souffrant de la maladie de Wilson. Il a ainsi été confirmé que cette ATPase de transport de cuivre désignée comme ATP7B est le gène défectueux impliqué dans la maladie de Wilson.
PCT/CA1994/000519 1993-09-21 1994-09-21 Gene de la maladie de wilson WO1995008641A1 (fr)

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CA002108927A CA2108927C (fr) 1993-09-21 1993-10-21 Gene de la maladie de wilson

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003038125A1 (fr) * 2001-10-18 2003-05-08 Bio Gene Technologies, Inc. Procede modifie de criblage de mutation par pcr-sscp
KR101076612B1 (ko) 2009-01-14 2011-10-26 재단법인 아산사회복지재단 윌슨병 진단용 조성물
US9415067B2 (en) 2009-04-08 2016-08-16 Mars, Incorporated Genetic test for liver copper accumulation in dogs and low copper pet diet
US9827314B2 (en) 2003-12-08 2017-11-28 Mars, Incorporated Edible compositions which are adapted for use by a companion animal
US10150997B2 (en) 2011-12-06 2018-12-11 Mars, Incorporated Genetic test for liver copper accumulation in dogs
WO2019191270A1 (fr) * 2018-03-27 2019-10-03 The Board Of Trustees Of The University Of Illinois Restauration du transport du cuivre transmembranaire
US11077208B2 (en) 2015-12-18 2021-08-03 Ucl Business Ltd Wilson's disease gene therapy
US11578327B2 (en) 2018-02-14 2023-02-14 Deep Genomics Incorporated Oligonucleotide therapy for Wilson disease

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C. VULPE ET AL.: "Isolation of a candidate gene for Menkes disease and evidence that it encodes a copper-transporting ATPase", NATURE GENETICS, vol. 3, no. 1, January 1993 (1993-01-01), NEW YORK, US;, pages 7 - 13 *
E.A. STEWARD ET AL.: "Polymorphic microsatellites and Wilson Disease (WD)", AM. J. HUM. GENET., vol. 53, no. 4, October 1993 (1993-10-01), UNIV. CHICAGO PRESS, CHICAGO, US;, pages 864 - 873 *
G.R. THOMAS ET AL.: "Haplotype studies in Wilson disease", AM. J. HUM. GENET., vol. 54, no. 1, January 1994 (1994-01-01), UNIV. CHICAGO PRESS, CHICAGO, US;, pages 71 - 78 *
J. CHELLY ET AL.: "Isolation of a candidate gene for Menkes disease that encodes a potential hevy metal binding protein", NATURE GENETICS, vol. 3, no. 1, January 1993 (1993-01-01), NEW YORK, US;, pages 14 - 19 *
J.F.B. MERCER ET AL.: "Isolation of a partial candidate gene for Menkes disease by positional cloning", NATURE GENETICS, vol. 3, no. 1, January 1993 (1993-01-01), NEW YORK, US;, pages 20 - 25 *
K. PETRUKHIN ET AL.: "Mapping, cloning and genetic characterization of the region containing the Wilson disease gene", NATURE GENETICS, vol. 5, no. 4, December 1993 (1993-12-01), NEW YORK, US;, pages 338 - 343 *
P.C. BULL AND D.W.COX: "Long range restriction mapping of 13q14.3 focused on the Wilson disease region", GENOMICS, vol. 16, no. 3, June 1993 (1993-06-01), ACADEMIC PRESS, NEW YORK, US;, pages 593 - 598 *
P.C. BULL ET AL.: "The Wilson disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene", NATURE GENETICS, vol. 5, no. 4, December 1993 (1993-12-01), NEW YORK, US;, pages 327 - 337 *
R.E. TANZI ET AL.: "The wilson disease gene is a copper transporting ATPase with homolohy to the Menkes disease gene", NATURE GENETICS, vol. 5, no. 4, December 1993 (1993-12-01), NEW YORK, US;, pages 344 - 350 *
R.H.J. HOUWEN ET AL.: "DNA markers for the diagnosis of Wilson disease", J. HEPATOLOGY, vol. 17, no. 3, March 1993 (1993-03-01), ELSEVIER, AMSTERDAM, NL;, pages 269 - 276 *
Y. YAMAGUCHI ET AL.: "Expression of the Wilson disease gene is deficient in the Long-Evans Cinnamon rat", BIOCHEM. J., vol. 301, no. 1, 1 July 1994 (1994-07-01), BIOCHEMICAL SOC.,LONDON,UK;, pages 1 - 4 *
Y. YAMAGUCHI ET AL.: "Expression of the Wilson disease gene is deficient in the Long-Evans cinnamon rat", EMBL/GENBANK DATA LIBRARIES, 19 February 1994 (1994-02-19) *
Y. YAMAGUCHI ET AL.: "Isolation and characterization of a human liver cDNA as a candidate gene for Wilson disease", BIOCHEM. BIOPHYS. RES. COMMUN., vol. 197, no. 1, 30 November 1993 (1993-11-30), ACADEMIC PRESS, N.Y., US;, pages 271 - 277 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003038125A1 (fr) * 2001-10-18 2003-05-08 Bio Gene Technologies, Inc. Procede modifie de criblage de mutation par pcr-sscp
US9827314B2 (en) 2003-12-08 2017-11-28 Mars, Incorporated Edible compositions which are adapted for use by a companion animal
US12059465B2 (en) 2003-12-08 2024-08-13 Mars, Incorporated Edible compositions
KR101076612B1 (ko) 2009-01-14 2011-10-26 재단법인 아산사회복지재단 윌슨병 진단용 조성물
US9415067B2 (en) 2009-04-08 2016-08-16 Mars, Incorporated Genetic test for liver copper accumulation in dogs and low copper pet diet
US10150997B2 (en) 2011-12-06 2018-12-11 Mars, Incorporated Genetic test for liver copper accumulation in dogs
US12227805B2 (en) 2011-12-06 2025-02-18 Mars, Incorporated Genetic test for liver copper accumulation in dogs
US11077208B2 (en) 2015-12-18 2021-08-03 Ucl Business Ltd Wilson's disease gene therapy
US11578327B2 (en) 2018-02-14 2023-02-14 Deep Genomics Incorporated Oligonucleotide therapy for Wilson disease
WO2019191270A1 (fr) * 2018-03-27 2019-10-03 The Board Of Trustees Of The University Of Illinois Restauration du transport du cuivre transmembranaire

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