Note: Descriptions are shown in the official language in which they were submitted.
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BACKGROUND OF THE INVENTION
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 pro-
teins. 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 hydrox-
ide radicals, which can damage cell membranes, mitochondria, pro-
teins and DNA1.
Copper homeostasis requires appropriate mechanisms for
copper absorption, cellular transport, incorporation into protein,
storage, and excretion. In mammalian systems, various proteins or
peptides have been recognized for these functions2: albumin (and
copper histidine) for copper transport in the blood, ceruloplasmin
as a possible copper donor to tissues and enzymes3, and metallo-
thionein for intracellular copper storage4. 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 review5). However, these two
diseases affect different tissues. In X-linked Menkes disease,
copper export is defective in many tissues5 but is normal in the
liver6. 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 incor-
porate copperinto ceruloplasmin in the liver, and failure to
excrete copper from the liver into bile. This results in toxic
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accumulation of copper particul.arly 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 identi-
fy the gene responsible for Wilson disease in order t:o develop new
diagnostic and therapeutic strategies useful in the detection and
treatment of the disease.
SUMMARY OF THE INVENTION
The Wilson disease gene (WND) has been assigned to a
single locus by genetic linkage first to esterase D7, then to a
cluster of polymorphic markers on chromosome 13 band q14.38,9,10.
Multipoint linkage analysis indicates that WND is flanked proxi-
mally by marker D13S31 and distally by D13S59 at distances of 0.4
cM and 1.2 cM respectively11. Marker D13S31 is sufficiently close
to show allelic association with the disease in two different
populations12,13.
The inventors have isolated new markers between D13S59
and D13S31 and have used them to construct a long range restric-
tion map of the WND region14,15. Three CA repeat markers,
D13S314, D13S133 and D13S316, have been positioned in a 300 kb
region within this map16. These markers show high allelic
association with WND and allowed the identification of specific
Wilson disease haplotypes in the region16. In addition 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.
2
CA 02108927 2006-09-08
To try and isolate the Wilson disease gene, a probe
from the proposed copper binding region of Menkes (MNK)
was hybridized at low stringency to 19 YACs isolated from
a 1-1.5 Mb region immediately distal to marker D13S31.
This strategy has lead to the isolation of a gene with all
the characteristics required for a copper transporting
ATPase, which is predicted to be the gene for Wilson
disease.
Accordingly, the present invention provides a DNA
sequence containing the gene for Wilson disease.
More specifically, the present invention provides a
nucleic acid molecule comprising the DNA sequence as
illustrated in Figure 10, the complementary sequence
thereof or an allelic variant 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,
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 invention provides a nucleic acid molecule
comprising the underlined DNA sequence designated as Cul
in Figure 10.
The present invention provides a nucleic acid molecule
comprising the underlined DNA sequence designated as Cu2
in Figure 10.
The present invention provides a nucleic acid molecule
comprising the underlined DNA sequence designated as Cu3
in Figure 10.
- 3 -
CA 02108927 2008-05-12
The present invention provides a nucleic acid molecule
comprising the underlined DNA sequence designated as Cu4 in
Figure 10.
The present invention provides a nucleic acid molecule
comprising the underlined DNA sequence designated as Cu5 in
Figure 10.
The present invention provides a nucleic acid molecule
comprising the underlined DNA sequence designated as Cu6 in
Figure 10.
The present invention provides a nucleic acid molecule
comprising the underlined DNA sequence designated as Pt/T in
Figure 10.
The present invention provides a nucleic acid molecule
comprising the underlined DNA sequence designated as Tm in
Figure 10.
The present invention provides a nucleic acid molecule
comprising the dotted underlined DNA sequence designated as
Ph in Figure 10.
The present invention provides a nucleic acid molecule
comprising the underlined DNA sequence designated as ATP-
hinge in Figure 10.
The present invention also provides use of a nucleic acid
molecule comprising the DNA sequence for Wilson disease, the
complementary sequence thereof or an allelic variant thereof
(including the above-mentioned domains) to detect Wilson
disease.
The present invention further includes the use of nucleic
acid molecule comprising the DNA sequence for Wilson disease
or an allelic variant or any fragment of the given DNA
sequence, derived in any way, including amplification by the
polymerase chain reaction, 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
fragments, or the examination of differences in small
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CA 02108927 2008-05-12
amplified fragments using such methods as single strand
conformation polymorphism (SSCP) or heteroduplex analysis.
Thus, the present invention provides a use of a primer to
detect a mutation in the Wilson disease gene in a Wilson
disease patient, wherein said primer comprises 5' TGT AAT CCA
GGT GAC AAG CG 3' or 51 CAC AGC ATG GAA GGG AGA G 3'.
The present invention also includes the use of a nucleic
acid molecule comprising the DNA sequence for Wilson disease
or an allelic variant 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). This includes short
term use of plasmid containing any part of the sequence in
this application as initial therapy for rapid removal of
copper, in the early phases of treatment, when patients are
at risk from hemolysis and other complications from rapid
release of copper. The application also includes use for
enhancing heavy metal transport in humans or any other
organism.
Thus, the present invention provides use of a nucleic
acid molecule comprising the DNA sequence for Wilson disease,
the complementary sequence thereof, or an allelic variant
thereof to treat Wilson disease.
The present invention further includes the use of a
nucleic acid molecule comprising the DNA sequence for Wilson
disease or an allelic variant 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 gene from
other organisms. Specifically, this includes the use of the
described nucleic acid molecule to obtain the homologous gene
in the rat, for the study of the Long-Evans Cinnamon (LEC)
mutant, and the mouse gene, for the study of the toxic milk
- 5 -
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mouse, both of the above as potential models for Wilson
disease.
The present invention yet also includes the use of a
nucleic acid molecule comprising the DNA sequence for Wilson
disease or an allelic variant or fragment thereof, derived by
any means such as cloning or PCR amplification to obtain the
corresponding gene from any other species.
Thus, the present invention provides use of a nucleic
acid molecule comprising the DNA sequence for Wilson disease,
the complementary sequence thereof, or an allelic variant
thereof to isolate the Wilson disease gene, or fragment
thereof, from a mammal.
The present invention further includes the use of a
nucleic acid molecule comprising the DNA sequence for Wilson
disease or an allelic variant or fragment thereof in therapy
to remove heavy metal from an organ.
The present invention yet also includes the use of a
nucleic acid molecule comprising the DNA sequence for Wilson
disease, or an allelic variant or fragment thereof in animal
breeding, for example to enhance the excretion of copper and
other heavy metals.
The present invention provides a use of a nucleic acid
molecule comprising the DNA sequence of Figure 10, the
complementary sequence thereof or an allelic variant thereof
to reduce metal toxicity in an animal.
The invention also includes all of the DNA markers associated
with the Wilson disease that the inventors have developed.
In particular, the present invention includes
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CA 02108927 2006-09-08
DNA markers D13S314, D13S315 and D13S316 as well as any
other markers that detect the same dinucleotide repeat
polymorphisms as these three markers.
The present invention provides a DNA marker associated
with the gene for Wilson disease characterized in that it
detects the same dinucleotide repeat polymorphism as DNA
marker D13S314, wherein said marker can be amplified using
primers comprising sequences 5' GAG TGG AGG AGG AGA AAA GA
3' and 5' GTG TGA CTG GAT GGA TGT GA 3'; a DNA marker
associated with the gene for Wilson disease characterized
in that it detects the same dinucleotide repeat
polymorphism as DNA marker D13S315, wherein said marker
can be amplified using primers comprising the sequences
5' GCC ATC CAG AGT TAA ACC A 3' and 5' TTA TAG CTT TTC TCA
TGC ATT C 3'; and a DNA marker associated with the gene
for Wilson disease characterized in that it detects the
same dinucleotide repeat polymorphism as DNA marker
D13S316, wherein said marker can be amplified using
primers comprising the sequences 5' GCA GCA ATG CTT TGT
GCA TAA 3' and 5' TGT TTC CCA CCA ATC TTA CCG 3'.
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 pair of
primers selected from the group consisting of
a) 5' GAG TGG AGG AGG AGA AAA GA 3' and
5' GTG TGA CTG GAT GGA TGT GA 3';
b) 5' GCC ATC CAG AGT TAA ACC A 3' and
5' TTA TAG CTT TTC TCA TGC ATT C 3'; and
c) 5' GCA GCA ATG CTT TGT GCA TAA 3' and
5' TGT TTC CCA CCA ATC TTA CCG 3'; and instructions for
detecting Wilson disease.
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BRIEF DESCRIPTION OF THE DRAWINGS
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 end-
clones 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. sub-
mitted).
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 Wcl.a) Restriction maps. At the
top is a long range restriction map around the proximal flanking
marker for WND, D13S31. Wcl and markers D13F71S1, D13S196 and
D13S31 map to the intervals shown with thick black bars. The
location of three microsatellite markers are shown with asterisks.
At the bottom are restriction maps of cosmids cosL and cosJ. cDNA
clones were mapped to the intervals shown with thick bars.
Restriction sites are : M=Mlu I; R=Nru I; N=Not I; H=HindIII;
E=EcoRI; 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 Mcl.a and cosL.d to YACs from the Wilson disease region.
YACs 232H4 and 68F4 are overloaded.
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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 Ham-
ster. The pattern obtained by hybridization of cDNA clone Wcl.i7
to EcoRI digests of these samples is shown. This clone cross
hybridized to a single hamster fragment (indicated by an
asterisk). b) Linkup of clone Wcl.i7 with marker D13S31. M1uI
and Nru I digests of the chromosome 13 hybrid ICD were separated
with a CHEF PFGE system (Biorad) through 1% agarose with a
300-2000s ramped pulse at 50V for 150h. Sizes (in kb) of detected
fragments are indicated on the left. Cross hybridization of
Wc(i7) to the hamster component of ICD is indicated with an
asterisk.
Fig. 5 Partial DNA sequence of Wcl. Alignment of the
amino acid sequences of Wcl with MNK is indicated, amino acid
identities in MNK are indicated with a period. insertions and
deletions in MNK compared to Wcl 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 Mc1. The
coding region of the Mc1 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 Mcl.a
and Mcl.b are shown below with narrow stippled lines. The rela-
tive positions of cDNA clones from Wcl are indicated with thin
black lines. The Thick black line shows the part of the Wcl
7
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transcript that has been sequenced. Clone Wcl.f8 contains an
unspliced intron. This is indicated with a dashed line.
Fig. 7 Alignment of nucleotide and derived amino acid
sequence of the metal binding domains of Mc1 and Wcl. The nucleo-
tide sequence of each domain of Wcl is shown. A translation of
the domains and the corresponding domains of Mcl are shown direct-
ly below. Nucleotides and amino acids that are conserved in Mc1
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 Mc1 and other proteins from bacteria. Residues found in both
Mc1 and Wcl 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 ATPase26) E. 'coli Hg
(Escherichia coli mercuric transport protein merP27) S. aureus
Cd(Staphylococcus aureus cadmium efflux ATPase28) R. meliloti
Fix(Rhizobium meliloti nitrogen fixation protein Fix138).
Fig. 9 Northern blot hybridization. Probe Wcl.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.
8
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DETAILED DESCRIPTION OF THE INVENTION
EXPERIMENT 1 - HAPLOTYPE STUDIES IN WILSON DISEASE
Summary
In 51 families with Wilson disease, we have studied DNA
haplotypes of dinucleotide repeat polymorphisms (CA repeats) in
the 13q14.3 region to examine these markers for association with
the Wilson disease gene (WND). In addition to a previously
described marker (D13S133), we have developed three new highly
polymorphic markers (D13S314, D13S315, D13S316) close to the WND
locus. We have examined the distribution of marker alleles at the
loci studied and have found that D13S314, D13S133, and D13S316
each show non-random distribution on chromosomes carrying the WND
mutation. We have studied haplotypes of these three markers and
have found that there are highly significant differences between
WND and normal haplotypes in Northern European families. These
findings have important implications for mutation detection and
molecular diagnosis in Wilson disease families.
Materials and Methods
Families Studied
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 elsewhere12,13. The remaining 30
kindreds consisted of 56 parents, 47 patients and 21 unaffected
9
CA 02108927 2004-06-21
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 studies39
DNA Analysis
DNA was extracted from whole blood collected in EDTA by a salt
precipitation method40.
The markers used in this study are listed in Table 1. All three
new markers were derived from cosmid clones isolated from a flow-
sorted chromosome 13 library. 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 1314, D13S314 and
D13S315 were derived from cosmids identified by endclones of YACs 27D8
(identified by D13S196) and 235H9 (identified by D13S31)
respectively14. Endclones were obtained by an inverse PCR method36
The probes were labelled using a T7 Quickprime' random labelling kit
(Pharmacia), hybridized to filter lifts of the cosmid library as
described previously1zand exposed to film. Primary positive signals
were picked and replated for secondary and if necessary, tertiary
screening. DNA was isolated from the clones, digested with three to
five of enzymes, electrophoresed in 1% agarose and transferred to
nylon membrane (Hybond N+, Amersham). The blot was probed with poly-
CA 02108927 2004-06-21
dCdA (Pharmacia) to identify bands containing potentially poly-morphic
repeat units. Positive bands were subcloned and sequenced to
determine the length of the repeat. Primers were designed using the
OLIGOTM software package (Research Genetics).
All markers were typed by amplification of the poly-dCdA tract by
the polymerase chain reaction. The reactions were carried out in 10
l volumes containing 50 mM KC1, 10 mM Tris, pH 8.0, 10 mg/ml BSA, 1.5
mM MgC12, 200 M each of dCTP, dGTP and dTTP, 25 M dATP, 0.2 Ci [a
35S] -dATP and 0.5 units of AmplitaqTM (Perkin Elmer). Amplification was
performed in an MJ research PTC-100-96V Programmable Thermal
ControllerTM with 30 cycles of 30 seconds denaturation at 94 C, 30
seconds annealing at the appropriate temperature (Table 1), and 30
seconds extension at 72 C. The samples were then electrophoresed
through 6% denaturing polyacrylamide gels, which were dried and
exposed to film at room temperature for 1 to 5 days.
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.
Results
All four markers were typed in the 51 Wilson disease families. The
data are shown in Tables 3 and 4. The relative positions of the
markers used in this study are summarized in Figure 1. Also shown is
the location of a candidate for WND gene
11
0 c~
~+1089c r~
~ ~ 75415-2
(Wcl) which we have recently identified as discussed in Experi-
ment 2 and in reference 43. 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 described12. All
markers centromeric to this, including D13S315, are also recombin-
ant and the event does not include D13S133 or D13S316. The loca-
tion of D13S133 relative to the other three markers is based on
the pattern of amplification of YACs in the region. Rare recom-
bination 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.
Alleles associated with the normal and disease chromo-
somes were determined. Statistically significant deviations
between normal and WND chromosomes were seen in the Northern
European families for D13S133, D13S314 and D13S316. These
distributions are shown graphically in Figure 2. Tests of signi-
ficance were done by the x2 method for large contingency tables
with the following results: D13S133 gave a value of 1,9.94 (11
d.f., p < 0.05), D13S314 yielded a value of 22.58 (9 d.f.,
p< 0.01), and D13S316 resulted in values of 26.62 (8 d.f.,
p 0.001). Haplotypes of the three markers which gave signi-
ficant evidence of association were constructed for each family.
Table 5 summarizes the haplotypes present on Northern European WND
chromosomes and their corresponding frequencies on normal chromo-
somes. It has been shown that allele differences of single repeat
units in microsatellite markers arise more frequently than larger
12
CA 02108927 2004-06-21
deviations41. Therefore, because of the variability of CA repeat
markers due to single allele slippage and the possibility of mis-
assignment of alleles, haplotypes which differ by no more than two bp
at a single marker were grouped. The data are significant at
p 0.0001 (X2 = 51.44, 8 d.f.). While D13S315 does not show
significant amounts of association, several of the haplotypes can be
seen to extend to this marker. Haplotype C is exclusively associated
with allele 5 at D135315 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. There is
the possibility that the variant haplotypes are in fact different
mutations present on a similar haplotype, and the technique of
grouping similar haplotypes should be used with caution. However, in
the case of 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. In the case of 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.
Northern European families were further subdivided into more
specific ethnic groups in order to determine if any haplotypes were
characteristic of a particular area. Of the 10
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0 2108,927 75415-2
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 Dutch, one French and one of British origin. Chromo-
somes 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 chromo-
somes due to small sample size.
Discussion
We have studied four highly polymorphic dinucleotide repeat
polymorphisms near the WND locus and have found that three of them
exhibit significant levels of allelic association with the dis-
ease. The high degree of association seen between the disease and
the markers D13S314 and D13S316 provides strong support for a
candidate gene (Wcl) which we have identified on this YAC as dis-
cussed in Experiment 2 and in reference 43. These three markers
also form haplotypes around Wcl which can be shown to be present
on disease chromosomes but not on the normal chromosomes in the
same population.
One marker (D13S315) failed to detect significant levels
of association with Wilson disease despite its location between
14
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Wcl and two markers (D13S31 and D13S228) which have previously
been demonstrated to be in disequilibrium with the WND locus13 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 region42.
The existence of haplotypes found commonly on WND
chromosomes also provides clues as to the number of possible muta-
tions present in the Northern European population. While haplo-
tvnP.q A_ R_ C. and E are similar, the larae number of chromosomes
-1r----= -= -= ---- , ~
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 chromo-
somes 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 muta-
tional events or rearrangements of more common haplotypes by an
0 2108927 75415-2
ancestral recombination event, as is likely the case with haplo-
type 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). We conclude that
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.
The number of patients in other geographical/ethnic
groups are fewer in number it is not possible to get statistically
significant data regarding association and haplotypes in most
cases. The haplotypes present in our two Sardinian families are
interesting because they appear to define three distinctly
different haplotypes (4-4-7, 4-7-10, and 7-17-11), unexpected in
this island population. This suggests the possibility of at least
three different mutations in the Sardinian population. The
7-17-11 haplotype appears on three of six disease chromosomes of
Italian origin, in a Jewish family, and is the most common haplo-
type present on WND chromosomes in the British families. This
haplotype may indicate the presence of a common widespread muta-
tion instead of different mutations on the same haplotype.
The rarity of observed recombination events make these
markers ideal for DNA diagnosis of families in which an affected
child is available for testing. The presence of two highly poly-
morphic loci on either side of the candidate gene ensures that any
family will most likely be informative for presymptomatic
16
0 2108927 75415-2
diagnosis of sibs of patients. Occasionally, diagnosis of Wilson
disease is difficult to establish and haplotype analysis at the
present time could provide information in some cases. The
presence of 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. With the
identification of a candidate gene, specific mutations may be
defined to provide a more definitive diagnosis.
EXPERIMENT 2 - ISOLATION OF THE WILSON DISEASE GENE
Summary
New markers for Wilson Disease have been isolated in the
region of the gene on chromosome 13q14.3 as described in Experi-
ment 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 iso-
late 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 domains, trans-
duction, phosphorylation and ATP-binding domains. 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 patients with Wilson disease.
17
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2108927 75415-2
Methodology
General methods. Southern blotting, and PCR were performed as
described in14. Pulsed-field gel electrophoresis (PFGE) is
described previously in15. DNA Sequencing was done using a T7
sequencing kit (USB).
Markers. Marker D13S31 is an established marker for Wilson
disease with alleles that exhibit strong allelic association with
WND12. D13S196 and D13F71S1 were isolated by Alu-PCR and mapped
to the region of WND as described in14. The three markers were
used in the construction of a long range restriction map of the
WND region15. Probe EHR4 was rescued from the distal end of
YAC 235H9 (see Table 6).
Cell lines. ICD is a human-hamster somatic cell hybrid containing
the proximal half of chromosome 13 as the only human componentl4.
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 D13S5911. Geno~nic DNA was
isolated as described in35 and sizes were determined by pulsed-
field gel electrophoresis (PFGE). YAC 27D8 was characterized in
detail. To confirm that it was non-chimeric, probes (27L and 27R)
from the left and right ends of the YAC were amplified by inverse
PCR36 and hybridized to Mlu I, Nru I and Not I digests of ICD as
described in15, 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
18
CA 02108927 2004-06-21
1-5 mm incubation in the presence lU restriction enzyme. DNA was
separated using a CHEF DR II PFGETM 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 characterized. YAC
232 H4, used as a negative control, does not hybridize to any of the
probes in the Wilson disease region. Isolation of Menkes (MNK) cDNA
probes. To isolate probes (Mcl.a and Mcl.b) for the MNK gene, reverse
transcription was performed with a M-MTV reverse transcription kit
(BRL) using 0.5 mg total RNA from cultured human myotubes as template
and primer Mc4062 (5' GC(A/G)TCATTGAT(T/G)CC(A/G)TC(C/T)CC 3')
corresponding to position 4062 of the MNK cDNA17) in the presence of
40U RNase inhibitor (Pharmacia). Probe Mcl.a was amplified from the
reverse transcribed template by PCR14using primers within the putative
copper binding region of MNK: Mc967 (5' CAA TGA TTC AAC AGC CAC TT3 ')
and Mcl965(5' TTA ATA TGT GCT TTG TTG GTT G 3'). Thirty five cycles
were performed using an annealing temperature of 60 C. An additional
probe Mci.b was amplified using primers Mc2942
(5' TTT GCA GAC AAA CTC AGT GG 3') and Mc3835
(5' GTC TGC AAT GGC TAT CAA GC 3'). PCR products were directly
subcloned into a T-tailed vector (Promega). The relative location of
the probes within the MNK cDNA is shown in Fig. 5.
Low stringency hybridization.
Probe Mcl.a was hybridized14at 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 over-
19
CA 02108927 2004-06-21
night. Similar hybridization conditions were used to screen 100,000
cosmids from a chromosome 13 specific library (Los Alamos)
Isolation of cDNA fragments.
cDNA fragments for Wcl were isolated by a direct selection
strategy22,23 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. Following two rounds of
hybridization, cDNAs were subcloned into BluescriptTM vector
(Stratagene). Two hundred colonies were picked at random and arrayed.
The colonies were screened at low stringency with probes Mcl.a and
Mcl.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 Wcl.f3 was found to be the most
3' fragment. To isolate the 3' end of the cDNA, fragment Wcl.f3 was
labelled to a specific activity of 1x108 cpm/ g and used as a probe to
screen four human cDNA libraries.
2x106 plaques were screened from an adult liver libraries
(Stratagene), 1x106 from a second adult liver library (Clontech) and
1x106 from an human hepatoma library and 1x106 from an adult kidney
library (Clontech). From a positively hybridizing clone
0 2108927
75415-2
isolated from the Kidney library, a cDNA fragment (Wcl.bl-1) was
amplified using an upper primer (350U: GTG GCT AGC ATT CAC CTT
TCC) developed from the 31 end of close Wcl.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 670C.
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.
Northern blot hybridization. 10 g of total RNA isolated from
each of the following tissues: brain, lung, spleen, heart,
stomach, esophagus, muscle, liver and lymphoblasts, was separated
using a 1% agarose gel containing formamide37. The RNA was trans-
ferred onto nylon filters(HybondN+, Amersham). cDNA Wcl.c8 was
labelled to a specific activity of 1x108 cpm/ l and hybridized for
20h to the filter in a solution containing 50% formamide, 6xSSC,
0.1% SDS. The filter was washed once in 2xSSC, and once in
21
= 2108927
. 75415-2
0.2xSSC, 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 Wcl.c8 using
conditions recommended by the manufacturer.
Isolation of Wc1
Probe Mcl.a from the proposed copper binding region of MNK
(nucleotides 965-196517) 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. To
isolate the cross hybridizing sequence, probe Mcl.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 tb Mcl.a. 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.
The cosmids cosL and cosJ were used to isolate expressed
sequences from liver using a direct PCR based cDNA selection
strategy22,23. To isolate clones from regions of Wcl that were
similar to MNK, 200 selected cDNA clones were arrayed and screened
at low stringency with probe Mcl.a and a probe (Mcl.b) more
22
= (~ ~ ~ ~ ~ ~ '
75415-2
towards the 3' end of the MNK cDNA in the ATP-binding region
(nucleotides 2940-383017). Thirteen individual cDNA clones of
500-1000 bp in length were isolated, of which eight were char-
acterized in detail. To check that they were all located within
the correct region of chromosome 13, 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. In addition, one of the cDNA fragments
(Wcli7) was hybridized to Mlu I and Not I digests of DNA from
hybrid ICD that had been separated by pulsed field gel electro-
phoresis (PFGE). The probe detected a 2200 kb Nru I fragment and
2100 kb and 1200 kb Mlu I fragments that are all common to the
established marker D13S3115 (Fig. 4b).
In an attempt to isolate larger cDNA fragments, a total
of 4x106 colonies from three liver cDNA libraries were screened
with clone Wcl.f3. No further cDNAs were obtained.
The order of the cDNA clones was established by mapping
each clone on cosmids cosL and cosJ digested with several restric-
tion enzymes (Fig. 3a). The gene covers a region of at least
20 kb.
DNA sequence analysis
DNA sequence was obtained for all the clones shown in
Fig.3a. Sequence analysis of the cDNA clones revealed that Wcl
is very similar to MNK. This enabled the isolated clones to be
aligned with the MNK cDNA as shown in Figs. 5, 6 and 10. This
23
0 2108927 75415-2
alignment agreed with the position of the clones on the cosmid
map.
Translation of the nucleotide sequence revealed six
putative heavy metal binding domains very similar to the six do-
mains found at the 5' end of MNK17,18,19. Alignment of these
domains with the corresponding domains in MNK is shown in Fig. 7.
The six Wcl 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 Wcl.f8 seems to be unspliced
message since it contains a splice donor site. The site is also
present in genomic DNA (not shown). The cDNA selection method we
used occasionally selects such products22.
Both MNK and Wcl also contain highly conserved domains
characteristic of the P-type family of cation transporting
ATPases. This family includes magnesium, calcium, potassium,
sodium and proton pumps from various organisms. Members of the
family contain a highly conserved region containing the motif
Asp-Lys-Thr-Gly-Thr (DKTGT), that includes an aspartate residue
which forms a phosphorylated intermediate during the cation trans-
port cycle. Forty three residues N-terminal to this aspartate is
a proline residue thought to be involved in transduction of the
energy from ATP hydrolysis to cation transport24. C-terminal to
the transduction and phosphorylation domains is a highly conserved
ATP-binding domain including a Gly-Asp-Gly (GDG) motif. Align-
ments of MNK and Wcl around these three domains are shown in
Fig. 8. The identity between MNK and Wcl is 86 perceht throughout
24
2108927
75415-2
the transduction/phosphorylation domains and 79 percent throughout
the ATP-binding domain.
Also shown in Fig. 7 is the alignment and homology of
the functional domains of Wcl with various heavy metal transport-
ing ATPases from bacteria (for a review see25). As has previously
been demonstrated for MNK17,18,19, the functional domains of Wcl
are more closely related to these prokaryotic genes than to any
characterized eukaryotic gene, except MNK. The most closely
related gene is copA from the gram positive bacteria Enterococcus
hirae a gene involved in copper transport26. Alignments are also
shown with a mercuric transporting plasmid encoded protein merP
from Escherichia coli27, a cadmium exporting ATPase from Staphyl-
ococcus aureus28 and a protein involved in nitrogen fixation
(FixI) from the symbiotic bacterium Rhizobium meli.loti26. In
addition to the N-terminal metal binding domains characteristic of
this sub-group of ATPases, three other conserved residues are
present that are not a general feature of P-type 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 speci-
ficity to the proteins17. DNA sequence is being submitted to
genbank.
Expression
To determine the tissue distribution of the Wcl message,
clone Wcl.C8 was hybridized to Northern blots containing RNA from
a variety of tissues. Total RNA was analyzed from brain, lung,
spleen, heart, esophagus, muscle, liver and lymphoblasts. Tran-
~ 2108927 75415-2
script 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 kb17,18,19.
The placenta appeared to have an additional transcript
of about 7 kb.
Discussion
There is strong evidence that the Wcl 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, phos-
phorylation and ATP binding domains are 79% identical or greater.
In comparing the metal-binding and transduction domains of Wcl,
MNK, and the copper-resistant bacteria E. hirae, there are certain
conserved residues that may be specific for copper transport
(Fig. 8).
Wcl is predicted to be the Wilson disease gene because
it lies within a region of chromosome 13 that is known to contain
WND. A cluster of three highly polymorphic markers D13S133,
D13S314 and D13S316, all located within YAC 27D8 and spanning a
region of about 300 kb, show strong allelic association with WND
26
2108927
75415-2
and together define a good candidate region for the gene. Wcl is
flanked proximally by D13S314 and distally by D13S133 and
D13S31616. No other homologous copper-binding doma.in,s, transduc-
tion, phosphorylation, or ATP-binding domains were found within
the Wilson disease region.
The expression patterns of Wcl and MNK are very differ-
ent. MNK is expressed in lung, skeletal muscle and heart, but is
scarcely detectable in the liver or kidney. In contrast, Wcl is
expressed mainly in the liver and kidney. This tissue expression
is appropriate for Wilson disease. A key feature in Wilson dis-
ease 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 amino-
aciduria, proteinuria, uricosuria, hypercalciuria, defective
urinary acidification, renal stones, and occasionally full blown
Fanconi syndrome29,1.
The two main biochemical characteristics of Wilson
disease are the disruption of incorporation of copper into cerulo-
plasmin in the liver and a severe reduction of copper excretion
from the liver into the bile5. Any candidate gene must have
potential for involvement in both processes. Ceruloplasmin defi-
ciency, almost always associated with Wilson disease30 has been
recognized as being very closely related to the basic defect. The
localization of the ceruloplasmin locus to chromosome 331 showed
that a defect in the ceruloplasmin molecule could not be the basic
defect in Wilson disease. However, the deficiency is present in
27
= 2108927 75415-2
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 hepato-
cytes32, and a possible donor of copper to tissues and enzymes3.
Copper is incorporated during the biosynthesis of ceruloplasmin
which is then secreted from the hepatocytes into the plasma32.
The two processes, copper incorporation and ceruloplasmin secre-
tion, appear to be independent of one another32,33. Incorporation
of copper into apoceruloplasmin in vitro can only be achieved
under reducing conditions32. It is therefore interesting to note
that Wcl contains CXXC motifs in each of its metal binding
domains, together with one CXC motif in the transduction domain.
Similar motifs are characteristic of many transition metal binding
proteins34. The motifs are abundant in metallothionein and bind
copper in the reduced (CuI) state4. Incorporation of copper into
ceruloplasmin might require close proximity of the two molecules,
and some affinity of ceruloplasmin to the membrane ATPase might be
predicted. The pathway involved in copper excretion into bile may
be similarly sensitive to the redox state of copper. Wcl there-
fore has the potential to play a direct role in copper incorpora-
tion into ceruloplasmin, and in copper excretion, by maintaining
the metal ion in the correct redox state (Cu I).
Although much is known about the role of copper in many
essential enzymes, and about its transport in the blood, the mech-
anism of copper transport between tissues has remained unclear.
The isolation of a second human gene for a putative copper trans-
porting ATPase, with contrasting tissue distribution, helps to
28 1
~ 2108927
75415-2
reveal exciting new directions in the study of copper transport in
health and disease. Wcl and MNK are the only such metal trans-
porters 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.
EXAMPLES OF APPLICATIONS OF THE INVENTION
The identification of the gene responsible for Wilson
disease as well as markers associated with the disease has
important implications for the development of new diagnostic and
therapeutic strategies for the disease. Below are some examples
of the use of the present invention in the diagnosis and treatment
of Wilson disease.
A. Diagnosis of presymptomatic sibs
After the first individual in a family is diagnosed with
Wilson disease, there is frequently difficulty in determining
whether other sibs, who have a one in four change of being affect-
ed, are actually patients. We have demonstrated in some families
(Cox, D.W. and Billingsley, G.D., The application of DNA markers
to the diagnosis of presymptomatic Wilson disease. Proceedings
of: Genetics of Psychiatric Diseases Wenner-Gren International
Symposium, et. L. Wetterberg, Stockholm, pp. 167,988; Houwen et
al., H. Hepatol. 17:269, 1993) that an incorrect diagnosis can be
made, even when all possible biochemical and radioactive studies
are carried out. Reliable diagnosis can be made with the markers
we have developed. While other markers have been developed by
others in this region, ours are particularly useful in that they
29
~ 2108927 75415-2
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:
D13S314 - 12 alleles
D13S315 - 9 alleles
D13S316 - 9 alleles
In addition, we have used, in our haplotypes, D13S133, a marker
which was developed by others, which we have identified as being
very close to the Wilson disease locus.
Our own markers can be used to reliably diagnose Wilson
disease in sibs, and because of the high variability are most
likely to be informative in all families. We have already
successfully carried out presymptomatic diagnosis in at least six
families.
B. Diagnosis of patients
Diagnosis of Wilson disease is particularly difficult
for those with liver disease, since copper accumulation, charac-
teristic of Wilson disease, also occurs in other liver diseases
which have a biliary obstructive component. Every abnormal bio-
chemical 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.
2108927 75415-2
1) Determination of haplotypes
In some cases, the 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 consid-
erably 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:
D13S316 - D16S133 - D13S314 - D13S315
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 British
patients)
Among our patients of Northern European origin, these
haplotypes represent 40% of a series of 47 random patients. This
suggests that the haplotype approach could be useful in a rela-
tively large proportion of cases.
This approach is useful even when the mutation is not
known. However, direct analysis of the mutation will of course be
more reliable. Typically, detection of all mutations for disease
31
CA 02108927 2004-06-21
takes a considerable length of time, and may not be complete for
years.
2) Mutation analysis
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 adjacent (in introns). The amplified portions of
the sequence also include similar sequences which may have one or a
few nucleotides altered, with the end result being amplification 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 methods 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.
We have used specific primers, as shown below, to amplify a 275
base pair portion of the WND gene, followed by single strand
conformation polymorphism (SSCP) and heteroduplex
32
= 2108927
75415-2
analysis: Two patients have been identified to date with this
specific mutation.
B8.3a, 21-mer,
5' TGT AAT CCA GGT GAC AAG CAG 3'
B8.3b, 19-mer,
5' CAC AGC ATG GAA GGG AGA G 3'
The same approach can be used to identify other mutations
throughout the 4120 base pair sequence of the gene.
a) Detection of point mutations
The 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.
b) Detection of deletions or duplications
We have also developed primers which will be useful for
the detection of deletions. We expect that a large proportion of
the mutations in the Wilson disease gene will involve deletion (or
duplications), particularly of the copper binding regions.
Because there are six very similar motifs in the copper binding
region, as we know from studies of the immunoglobulin heavy chain
region carried out in our laboratory, deletions and duplications
tend to occur frequently in the present of repeated sequences. In
fact this has been demonstrated for Menkes disease17. There are
about 16% of patients with Menkes disease who have deletions in
the copper binding region. The PCR primers we have developed can
be used directly to identify such deletions. All of these primer
33
CA 02108927 2004-06-21
sequences lie within the region we have submitted in this application.
For additional 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.
Therapy
Therapy for Wilson disease at the present time involves chelation
of excess copper through the use of a chelating agent such as
penicillamine, a potent agent which binds copper through its cysteine
residues. But there are problems with the use of this agent, and the
neurological symptoms can be worsened on initial treatment as copper
is released from to the liver and transfers to other tissues, for
example the brain. In addition, about 15% of the patients experience
side effects from the therapy, including depression of the immune
system, and reduction in the number of white and red blood cells.
Zinc therapy is being used in some cases, but tends to cause
intestinal irritation and is not tolerated well by some patients.
The new basis of therapy would involve introduction of the Wilson
disease gene in a plasmid. We have discussed the copper and mercury
containing plasmids in our publications (Bull et al. Nature Genetics
(1993) 5(4): 327-37; Bull and Cox Trends in Genetics (1994)10:246-
252). Copper is used extensively in agriculture as a fungicide and
bactericide. 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.
34
2108927 75415-2
Creation of a construct similar to that found in the copper
resistant bacteria therefore appears to be a possible approach.
Such a construct would then have to enter into the
liver. This experiment has already been shown to be successful in
the Watanabe rabbit, which has heritable hyperlipidemia, and
demonstrated that allogenic hepatocytes can be transplanted into
affected rabbits to ameliorate hypercholesterolemia (Wilson et al.
PNAS 85:4421, 1988). In this rabbit, the gene was introduced in a
plasmid construct attached to an asialoglycoprotein r'eceptor,
which targets to the liver cell. A similar approach is therefor
feasible for a plasmid containing the Wilson disease gene. Human
hepatocytes, cultured in vitro, can be transfected with an adeno-
virus containing the Wilson disease gene, and returned to the
affected donor into the peripheral circulation. This model has
already been tested in rats with the alphal-antitrypsin gene
(Jaffe et al. Nature Genetics 1, 1992). Partial hepatectomy can
improve the stability of targeted DNA (Wilson et al. J. Biochem.
267:963, 1992). It is of interest that in these studies, DNA in
the hepatocytes was present in stabilized plasmids, which do not
self-replicate. The Wilson disease therefore could be used
directly as a plasmid to be added to cultured hepatocytes, or
perhaps to be administered directly through the portal system.
Episomes which contained alphal-antitrypsin were found to remain
relatively stable and produced the product (alphal-antitrypsin)
for at least four months. Since even a low production of alphal-
antitrypsin product should avoid the copper accumulation which
takes place, this approach is technically feasible.
2108927 75415-2
Other potential therapies
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 (Experi-
ment 2). Targeting of the gene to the kidney could alleviate
cadmium toxicity in those who have been inadvertently exposed.
The metal binding regions are very similar for the Wilson disease
gene, and for the mercury and cadmium resistance plasmids. It is
very likely that this sequence will be found to bind the other
heavy metals. The differences outlined in Experiment 2, Figure 8,
may suggest that slight alteration in the copper binding region
could increase the specific binding for mercury and cadmium.
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 (Yang et al. PNAS
87:9568, 1993) 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.
36
! 210$927 75415-2
Introduction of the plasmid into the intestinal epithelial cells
seems also to be feasible.
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. We
have found from our haplotype studies that most patients with
Wilson disease appear to be genetic compounds, that is they prob-
ably carry two different mutations. At least one of these may
have residual activity.
Non-human applications
The Wilson disease gene could be targeted into the germ
line of organisms for which the accumulation of toxic metals is a
problem. For example, the 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
37
~ 2108927 75415-2
of copper or other toxic metals not only from sheep, but a variety
of other organisms, including the removal of inercury from fish or
any other species.
The DNA sequence of the present invention can be used to
obtain the equivalent gene from the rat, 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
Long-Evans Cinnamon (LEC) rat, an inbred strain of mutant rat, in
which the defect in copper metabolism may be similar to that of
Wilson disease. Any use of the human sequence or a portion of it
to be used for study of the LEC rat and its normal counterpart are
included in this application.
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 dis-
ease. 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
included in this application.
While the above refer to specific applications of the
present invention, it is to be appreciated that other uses, that
are conceivable by one skilled in the art, are also within the
scope of the present invention.
38
~ 2108927 75415-2
References
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39
~ 2108927
75415-2
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= 2108927
75415-2
18. Mercer, J. F. B., Livingstone, J., Hall, B., et al. Isolation
of a partial candidate gene for Menkes disease by positional
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19. Chelly, J., Tumer, Z., Tonnesen, T., et al. Isolation of a
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(1965).
21. Owen, C. A. Jr. Metabolism of radiocopper (64Cu) in the rat.
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24. Vilsen, B., Andersen, J. P., Clarke, D. M. & MacLennan, D. H.
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26. Odermatt, A., Suter, H., Krapf, R. & Solioz, M. Primary
structure of two P-type ATPases involved in copper
41
~ 2108927
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homeostasis in Enterococcus hirae. J. Biol. Chem. 268,
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27. Griffin, H. G., Foster, T. J., Silver, S. & Misra,
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42
~ 2108927
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35. Scherer, S. & Tsui, L-T. in Advanced techniques in chromosome
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39. Cox DW, Fraser FC and Sass-Kortsak A (1972) A genetic study
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40. Miller SA, Dykes DD and Polesky HF (1988) A simple salting
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41. Oudet C, Mornet E, Serre JL, Thomas F, Lentes-Zengerling S,
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52:297-304
43
2108927 75415-2
42. MacDonald ME, Lin C, Srinidhi L, Bates G, Altherr M, Whaley
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49:723-734
43. Bull PC, Thomas GR, Rommens JM, Forbes JR and Cox DW (1993)
Identification of a putative copper binding P--type ATPase
similar to the Menkes (MNK) gene: a candidate for the Wilson
disease gene. (submitted)
44
WtT'
~hlel 2108927
Markers tisecl in this sttidy
Annealing No.
Lcx us Primers Temr Alleles PIC 133201 133202
D13S314 GAGTOGAGGAGGAGAAAAGA 62' 12 0.76 141 / 141 145/ 141
GTGTGACTGGATGGATGTGA
D13S315 GCCATCCAGAGTTAAACCA 58' 8 0.45 164 / 1G2 1G4 / 162
TTATAGCTTi I'CTCATGCATTC
D 1 1S 116 GCAGCAATGCTTTGTGCATAA 02' 9 0.73 1401140 1401140
TGTTTCCCACCAATCTTACCG
D 11S 133 See Ref. (Petrukhin et al. 1993)
Rcfercnce genotylies froin CEPH f<unily 1332. Numhcrs arc allele sizes in hasc
pairs.
,_._.
2108927
Table 2
Ailele size detinitiuns
Aliete I)135316 D13S133 D13S314 D13S315
1 154 187 163 170
2 152 185 161 168
3 150 183 159 166
4 144 181 157 164
146 179 155 162
6 144 177 153 160
7 142 175 151 154
8 140 173 149 152
9 139 171 147
11) 169 145
11 167 141
12 163 137
13 157
14 153
138
16 136
17 134
Values rerresenl allele sizes in base pairs (bp)
46
~.~.~
ApU1e3 2108927
Marker distrihutivns on 'ilson disease family chrvmosomes
NE" SE' Sarc)" mE" Or" IP
Mlrker A11e)e N W N W N W N W N W N W
D13S316 1 2 l) t) O O O O O 0 0 0 0
2 1 1 2 0
t) O 0 Q 0 0 0 0
3 2 3 2 1 1 0 1 0 0 0 1 0
4 19 3 2 1 3 3 2 2 0 0 2 10
3 9 2 3 0 0 1 1 1 1 2 0
6 4 16 I 0 I 0 0 I U 0 0 0
7 19 26 2 R O 3 1 2 3 3 2 1
R 4 2 1 ( 0 0 0 0 0 0 3 0
.......... 1...... ct.......... a...... o........c~...... ~......... n.....
j)......... Q ...... o........ ....... o
Total 56 60 14 14 5 6 5 6 4 4 10 11
D13S114 1 0 0 0 0 0 0 1 0 0 0 0 0
2 t) 0 0 U l 0 I t) 0 0 0 0
3 1 0 0 O 0 0 0 0 0 0 0 0
4 3 3 0 1 0 0 0 0 0 0 0 0
5 5 7 1 0 O 0 1 0 0 0 I 3
6 1 0 0 0 0 0 0 0 0 0 2 2
7 5 5 0 2 1 1 0 1 0 2 0 2
R 5 0 4 0
0 O 0 0 0 0 0 0
9 3 0 1 0 0 0 t) 0 0 0 0 0
7 22 2 5 0 1 2 0 0 0 2 2
11 15 15 5 6 2 2 0 5 1 0 4 2
R ........ 7 ...... I........... 0...... !1......... 0...... 5?.........
0....... 1......... 0...... 1.... ....(1....... 5)
Tcital 52 53 13 14 4 4 14 17 1 2 10 11
" Families were grouped according to geographical origin. NE = Nortliern
European, SE = Soutliern Euro)xau-, Sard
=
Surdinian, ME = Middle Eastern, Or = Oriental, IP = Indian/Pakistani
47
2108927
#I)le 4
Marker (listr-ilmutiems oti ZVilsuti (lisease familv chrumesomes
NO SE'i S.vc!'t ME11 Or'r iP'r
Marker nllele N W N W N W N W N W N W
1)1 3S 1 33 I U U 0 U 0 O I 0 0 0 0
0
2 0 I l 0 0 U 1 0 0 0 0 0
3 2 2 0 0 1 0 0 0 0 0 0 0
4 3 0 0 2 0 1 0 0 0 0 0 0
0 l 2 0 1 0 0 0 0 0 2 5
6 3 3 2 I 0 0 0 0 0 0 1 2
7 6 O U 0 0 1 0 l 1 2 0 0
8 3 3 0 0 0 0 1 0 0 0 0 0
9 2 0 0 I 0 0 0 t U 0 1 1
t I 0 0 0 0 1 0 0 0 0 0
1( 5 7 0 0 0 0 0 0 0 0 1 0
12 0 0 0 0 0 0 0 1 0 0 0 0
13 U 0 t 0 0 0 0 0 0 0 0 0
14 0 0 1 0 0 0 0 0 U 0 0 0
I 0 0 0 0 O
0 0 0 0 0 0
16 2 0 0 0 l 0 l) 0 U U 1 O
.17...... _.22....40...........5...... . 8 .........
0......2........Ø.....:}.........3......2........2.......1
Total 49 58 12 12 4 4 5 6 4 4 10 11
D13S315 I 3 0 0 0 1 1 1 0 0 0 0 0
2 5 4 3 0 1 0 1 0 0 1 2 1
3 4 9 1 1 1 0 1 2 U 2 3 2
4 35 27 8 9 3 4 2 1 3 1 2 3
5 9 17 1 1 0 0 0 0 0 0 3 2
6 0 0 1 2 0 0 0 1 0 0 0 0
7 1 l 0 0 0 ( 0 2 1 0 0 ~
K......... ~)...... a........... (?...... I......... (t...... 0.........
(t...... 0......... (?...... U........ 51....... 0
Total 57 58 14 14 6 6 5 6 4 4 10 11
n See notes for table 2
48
2108927
We 5
Iiahlutype distrihutiun un cl)rumusumes of Nurthern I?urupean oril;in
WND Normal
GrcniF Flsihlotp}~cs No. Freq No. Frcq
A 7-17-1() 9 3
R-17-1O 1 1
0.21 4 0.09
B 7-17-I1 8 8
7-16-11 0 1
R-17-11 U 3
8 0.17 12 0.27
C 6-17-10 7 0.15 U 0.00
D 5-11-5 4 0
5-11-4 2 0
5-1O-5 1 O
7 0.15 0 0.00
E 6-17-1l 5 0.11 u (LW
F 7-17-5 1 0
7-17-4 1 0
R-17-5 1 0
3 0.06 0 0.00
0 2-6-7 1 0
3-6-7 1 0
2 0.04 u 0.00
5 others 5 0.11 4 0.09
IS otlrers 0 0.00 24 0.55
....................................................=--
..........................
"Tutsil 47 44
F{<qplutypes are given in the order: D 13S316 - D 13S 133 - D 13S314
49
2108927
75415-2
Table 6 YACs in the Wilson disease region
D Number Probe Primers YACs
D13F71S1/2 pB32.3 CCGGGTATCTTAATTGGTGT 11G2;102F4;296G5
CTGGGGCCAACAATGTATTA 355C2
D13S196 pB40.3 GCAAAGTTCATAGGAAACCAGG 27D8;86A3;90H11;
ACATTTTGGTCAGACACTGGC 220A9;298H2
27R ATTGGGCATCTCTTGCTGTT 9B2;53C12;68F3
TGCAGGAATTCACTGTGTGA 95C3;407F11;117E9
EHR4 GGCCAGAATGACAAAATTCA 378B12;215B5;407F3
GGCTTCATGAGTGTGGTCCT
D13S31 235H9
