Note: Descriptions are shown in the official language in which they were submitted.
CA 02312472 2000-05-30
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. =
õ
-1 =
SHORT GCG EXPANSIONS IN THE PAB II GENE FOR OCULO-
PHARYNGEAL MUSCULAR DYSTROPHY AND DIAGNOSTIC THEREOF
BACKGROUND OF THE INVENTION
(a) Field of the Invention
The invention relates to PAB II gene, and its
uses thereof for the diagnosis, prognosis and treatment
of a disease related with protein accumulation in
nucleus, such as oculopharyngeal muscular dystrophy.
(b) Description of Prior Art
Autosomal dominant oculopharyngeal muscular dys-
rophy(OPMD) is an adult-onset disease with a world-
wide distribution. It usually presents itself in the
sixth decade with progressive swallowing difficulties
(dys-phagia), eye lid drooping (ptosis) and proximal
limb weakness. Unique nuclear filament inclusions in
skeletal muscle fibers are its pathological hallmark
(Tome, F.M.S. & Fardeau, Acta Neuropath. 49, 85-87
(1980)). Using the full power of linkage analysis in
eleven French Canadian families, the oculopharyngeal
muscular dystrophy gene was fine mapped on human
chromosome 14 (Brais et al., 1997, Neuromuscular
Disorders 7 (Supp1.1):S70-74). A region of .75 cM was
thereby identified as a region containing the potential
and unknown OMPD gene (Brais et al., 1997, supra).
Unfortunately, the OMPD gene has yet to be isolated and
its nucleic acid or protein sequence have yet to be
cribbed.
It would be highly desirable to be provided with
a tool for the diagnosis, prognosis and treatment of a
disease related with polyalanine accumulation in the
nucleus, such as observed in oculopharyngeal muscular
dystrophy.
SUMMARY OF THE INVENTION
One aim of the present invention is to provide
a tool for the diagnosis, prognosis and treatment of a
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disease related with polyalanine accumulation in
nucleus, such as oculopharyngeal muscular dystrophy.
Herein, the poly(A) binding protein II (PAB II)
gene was isolated from a 217 kb candidate interval in
chromosome 14q11.A (GCG)6 repeat encoding a polyalanine
tract located at the N-terminus of the protein was
expanded to (GCG)8-13 in the 144 OPMD families
screened. More severe pheno-types were observed in
compound heterozygotes for the (GCG)9 mutation and a
(GCG)7 allele found in 2% of the population, whereas
homozygosity for the (GCG)7 allele leads to autosomal
recessive OPMD. Thus the (GCG)7 allele is an example of
a polymorphism which can act as either a modifier of a
dominant phenotype or as a recessive mutation.
Pathological expansions of the polyalanine tract may
cause mutated PAB II oligomers to accumulate as
filament inclusions in nuclei.
In accordance with the present invention there
is provided a' human PAB II gene containing a
transcribed polymorphic GCG repeat, which comprises a
sequence as set forth in Fig. 4, which includes introns
and flank-ing genomic sequence.
The allelic variants of GCG repeat of the human
PAB II gene are associated with a disease related with
protein accumulation in the nucleus, such as
polyalanine accumulation, or with a disease related
with swallowing difficulties, such as oculopharyngeal
muscular dystrophy.
In accordance with the present invention there
is also provided a method for the diagnosis of a dis-
ease associated with protein accumulation in the
nucleus, which comprises the steps of:
a) obtaining a nucleic acid sample of said patient;
and
b) determining allelic variants of a GCG repeat of
the human PAB II gene; thereby long allelic
variants are indicative of a disease related
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14. .14 ix
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with protein accumulation in the nucleus, such
as polyalanine accumulation and oculopharyngeal
muscular dystrophy.
The long allelic variants have from about 245 to
about 263 bp in length.
In accordance with the present invention there
is also provided a non-human mammal model for the human
PAB II gene, whose germ cells and somatic cells are
modified to express at least one allelic variant of
the RAE II gene and wherein said allelic variant of the
PAB II is being introduced into the mammal, or an
ancestor of the mammal, at an embryonic stage.
In accordance with the present invention there
is also provided a method for the screening of thera-
peutic agents for the prevention and/or treatment of
oculopharyngeal muscular dystrophy, which comprises the
steps of:
a) administering the therapeutic agents to the non-
human animal of the present invention or
oculopharyngeal muscular dystrophy patients; and
b) evaluating the prevention and/or treatment of
development of oculopharyngeal muscular dystro-
phy in this animal (such as a mammal) or in
patients.
In accordance with the present invention there
is also provided a method to identify genes-products
thereof, or part thereof, which interact with a
biochemical pathway affected by the PAB II gene, which
comprises the steps of:
a) designing probes and/or primers using the PAB II
gene and screening oculopharyngeal muscular
dystrophy patients samples with said probes
and/or primers; and
b) evaluating the role of the identified gene in
oculopharyngeal muscular dystrophy patients.
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In accordance with the present invention, there
is provided an isolated human PAB II nucleic acid
molecule comprising an allelic variant of a polymorphic
GCG repeat in exon I thereof, wherein the allelic
variant of the polymorphic GCG repeat has the sequence
ATG (GCG)6+,1 GCA, with n being selected from 1 to 7, and
wherein an allelic variant of the polymorphic GCG
repeat is associated with oculopharyngeal muscular
dystrophy (OPMD) disease in a human patient.
In accordance with the present invention, there
is also provided an isolated nucleic acid molecule
comprising an allelic variant of a polymorphic GCG
repeat of exon I of a human PAB II gene, wherein the
allelic variant of the polymorphic GCG repeat gene is
associated with oculopharyngeal muscular dystrophy
(OPMD) disease and wherein the allelic variant of the
GCG repeat has the sequence ATG(GCG)6_, GCA, wherein n
is selected from 1 to 7, as set forth in SEQ ID NOs: 3-
9, respectively.
In accordance with the present invention, there
is also provided a method for the diagnosis or
prognosis of oculopharyngeal muscular dystrophy (OPMD)
disease, said method comprising:
a) obtaining a nucleic acid sample of said
patient; and
b) determining allelic variants of a GCG repeat
in exon I of the PAB II gene in said sample,
said GCG repeat having the sequence ATG
(GCG)6+n GCA,wherein n is selected from 0 to
7, and
wherein when at least one allele of said GCG repeat has
an n equal to 1 to 7, said allele is associated with
OPMD disease, thereby diagnosing or prognosing OPMD
disease in said human patient.
In accordance with the present invention, there
is provided an isolated embryonic cell expressing at
least one allelic variant of a polymorphic GCG repeat
in exon I of the PAB II gene, wherein the allelic
variant of the polymorphic GCG repeat has the sequence
ATG (GCG)6.,õ GCA, with n being selected from 1 to 7, and
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wherein the polymorphic repeat in a human PAB II gene
is indicative of oculopharyngeal muscular dystrophy
(OPMD) disease.
In accordance with the present invention, there
is also provided a method for screening and identifying
an agent for the prevention or treatment of
oculopharyngeal muscular dystrophy (OPMD) disease
associated with a phenotype which is at least one of
protein accumulation in a cell nucleus, a swallowing
difficulty, or ptosis, the method comprising:
a) exposing a non-human transgenic animal to the
agent, wherein the non-human transgenic
animal is obtained from a fertilized embryo
of a non-human animal modified so as to
express at least one allelic variant of a
polymorphic GCG repeat in exon I of the PAB
II gene, wherein the allelic variant of the
polymorphic GCG repeat has the sequence ATG
(GCG)6,õ GCA, with n being selected from 1 to
7, and wherein the allelic variant of the
polymorphic repeat in a human PABII gene is
indicative of OPMD disease; and
b) evaluating the prevention or treatment of
development of the phenotype in the animal
exposed to the agent as compared to a control
animal not having been exposed to the agent.
In accordance with the present invention, there
is also provided a cell transformed with an expression
vector comprising a promoter operably linked to at
least one allelic variant of a human polymorphic GCG
repeat of exon I of the PAB II gene, wherein the
allelic variant of the polymorphic GCG repeat has the
sequence ATG (GCG)6_,õ GCA, with n being selected from 1
to 7, and wherein the allelic variant is associated
with protein accumulation in the nucleus of the cell.
In accordance with the present invention, there
is also provided a method for screening and identifying
an agent which modulates protein accumulation in the
nucleus of a cell, said method comprising:
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a) exposing the cell mentioned above to said
agent; and
b) evaluating said protein accumulation in said
nucleus of said exposed cell as compared to a
control cell not having been exposed to said
agent,
wherein an agent which modulates protein accumulation
in the nucleus of a cell is identified when said
protein accumulation in said nucleus of said exposed
cell is different than that of said control cell not
having been exposed to said agent.
In accordance with the present invention, there
is also provided an isolated human PAB II gene
comprising a polymorphic GCG repeat in exon I thereof,
wherein the repeat has the sequence ATG (GCG)6+n GCA,
wherein n is 0, and wherein the sequence is indicative
of absence of oculopharyngeal muscular dystrophy (OPMD)
disease, the OPMD disease being associated with at
least one of protein accumulation in a cell nucleus, a
swallowing difficulty, or ptosis in a human patient.
In accordance with the present invention, there
is also provided a method for diagnosing in a human
patient an oculopharyngeal muscular dystrophy (OPMD)
disease associated with a meiotically stable
trinucleotide expansion in a coding sequence of a gene,
the method comprising:
a) obtaining a nucleic acid sample from the
patient;
b) determining in the sample whether the gene
comprises at least one trinucleotide expansion
having the sequence ATG(GCG)6+nGCA,
with n being 1 to 7,
wherein the determination of one trinucleotide
expansion in the coding sequence of the gene
is indicative of OPMD disease in the patient.
In accordance with the present invention, there
is also provided a method for determining the presence
or absence of an allelic variant of a polymorphic GCG
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trinucleotide repeat in a human, the method comprising
determining the presence of the GCG
trinucleotide
repeat in exon I of a human PAB II gene in a nucleic
acid sample from the human, the GCG repeat having the
sequence ATG(GCG)6+nGCA, wherein when n is selected from
0 to 7, and wherein the presence of at least one of two
alleles of the GCG repeat having an n equal to 1 to 7
identifies the presence of an allelic variant of the
PAB II gene.
In accordance with the present invention, there
is also provided a method for determining whether a
human is at risk of developing oculopharyngeal muscular
dystrophy (OPMD) disease, the method comprising
conducting an assay on a nucleic acid sample from the
human to determine the presence or absence of a GCG
trinucleotide repeat in a human PAB II gene, the GCG
trinucleotide repeat having the sequence ATG(GCG)6+nGCA,
with n being 1 to 7, wherein the presence of the GCG
trinucleotide repeat is indicative that the human is at
risk for development of OPMD disease.
In accordance with the present invention, there
is also provided a method for determining whether a
human is negative for oculopharyngeal muscular
dystrophy (OPMD) disease, wherein the method comprises:
a) amplifying a nucleic acid sample from the
human using a pair of oligonucleotide primers
which specifically amplify a region of nucleic
acid containing GCG trinucleotide repeats in
exon I of a human PAB II gene to obtain an
amplification product, with the GCG
trinucleotide repeat having the sequence
ATG(GCG)6+nGCA, with n being 1 to 7;
b) determining the presence or absence of the GCG
trinucleotide repeat in the amplification
product,
wherein the absence the GCG trinucleotide repeat
is indicative that the human is negative for OPMD
disease.
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In accordance with the present invention, there
is also provided a method for identifying a human
having a normal GCG trinucleotide repeat in a human PAB
II gene, the method comprising analyzing a GCG
trinucleotide repeat region in exon I of a human PAB II
gene in a nucleic acid sample from the human, wherein
the normal GCG trinucleotide repeat in the human PAB II
gene has the sequence ATG(GCG)6GCA.
In accordance with the present invention, there
is also provided a method for identifying whether a
human patient has a predisposition to develop
oculopharyngeal muscular dystrophy (OPMD) disease, the
method comprising determining the presence or absence
of a GCG trinucleotide repeat in exon I of a human PAB
II gene in a nucleic acid sample from the patient,
wherein the presence of the GCG trinucleotide repeat
having the sequence ATG(GCG)6õGCA, with n being 1 to 7,
is indicative that the patient has a predisposition to
develop OPMD disease.
In accordance with the present invention, there
is also provided a method for diagnosing
oculopharyngeal muscular dystrophy (OPMD) disease in a
human patient, the method comprising determining the
presence or absence of a GCG trinucleotide repeat in
exon I of a human PAB II gene in a nucleic acid sample
from the patient, wherein the presence of the GCG
trinucleotide repeat having the sequence (GCG)6_, GCA,
wherein n is 1 to 7, is indicative of OPMD disease in
the patient.
In accordance with the present invention, there
is also provided a method for diagnosing
oculopharyngeal muscular dystrophy (OPMD) disease in a
human, the method comprising:
a) contacting nucleic acid from the human with a
pair of primers that amplify a region of
nucleic acid containing GCG trinucleotide
repeats in exon I of a human PAB II gene, the
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GCG trinucleotide repeat having the sequence
(GCG)6,n GCA, wherein n is 0 to 7; and
b) detecting an amplification product comprising
the GCG trinucleotide repeat region,
wherein when the GCG repeat detected has an n equal to
1 to 7, the detection is indicative of OPMD disease in
the human.
In accordance with the present invention, there
is also provided an isolated PAB II nucleic acid
molecule comprising a polymorphic GCG repeat having the
sequence ATG (GCG)6+GCA, wherein when:
a) n=0, the nucleic acid sequence is not
associated with oculopharyngeal muscular
dystrophy (OPMD) disease; or
b) n is selected from 1 to 7, the nucleic acid
sequence is associated with OPMD disease.
In accordance with the present invention, there
is also provided a method of determining the presence
or absence of an allelic variant of a polymorphic GCG
trinucleotide repeat in a human, the method comprising
determining the presence of the GCG trinucleotide
repeat in exon I of a human PAB II gene in a sample
from the human, the GCG repeat encoding the sequence
Met(Ala)6,Ala, wherein n is selected from 0 to 7, and
wherein the presence of at least one of two alleles of
the GCG repeat having n equal to 1 to 7 identifies the
presence of an allelic variant of the PAB II gene
associated with oculopharyngeal muscular dystrophy
(OPMD) disease.
In accordance with the present invention, there
is also provided an isolated human PAB II nucleic acid
molecule comprising a polymorphic GCG repeat in exon I
thereof, wherein the polymorphic GCG repeat encodes the
sequence Met (Ala)6,nAla, with n being selected from 1
to 7, and wherein the polymorphic Ala repeat is
associated with oculopharyngeal muscular dystrophy
(OPMD) disease in a human patient.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figs. 1A-B illustrate the positional cloning of
the PAB II gene;
Figs. 2A-G illustrate the OPMD (GCG)n expansion
sizes and sequence of the mutation site (SEQ ID NOS:4-
9);
Fig. 3 illustrates the age distribution of
swallowing time (st) for French Canadian OPMD carriers
of the (GCG)9 mutation; and
Fig. 4 illustrates the nucleotide sequence of
human poly(A) binding protein II (hPAB II)(SEQ ID
NO:3).
DETAILED DESCRIPTION OF THE INVENTION
In order to identify the gene mutated in OPMD,
a 350 kb cosmid contig was constructed between flanking
markers D145990 and D1451457 (Fig. 1A). Positions of
the PAB II-selected cDNA clones were determined in
relation to the EcoRI restriction map and the
Genealogy-based Estimate of Historical Meiosis (GEHM)-
derived candidate interval (Rommens, J.M. et al., in
Proceedings of the third international workshop on the
identification of tran-scribed sequences (eds.
Hochgeschwender, U. & Gardiner, K.) 65-79 (Plenum, New
York, 1994)).
The human poly(A) binding protein II gene (PAB
II) is encoded by the nucleotide-sequence as set forth
in Fig. 4.
Twenty-five cDNAs were isolated by cDNA selec-
tion from the candidate interval (Rommens, J.M. et al.,
in Proceedings of the third international workshop on
the identification of transcribed sequences (eds.
Hochgeschwender, U. & Gardiner, K.; 65-79; Plenum, New
York, 1994). Three of these hybridized to a common 20
kb EcoRI restriction fragment and showed high sequence
homology to the bovine poly(A) binding protein II
gene(bPAB II) (Fig. 1A). The PAB II gene appeared to be
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a good candidate for OPMD because it mapped to the
genetically defined 0.26 cM candidate interval in 14q11
(Fig. 1A), its mRNA showed a high level of expression
in skeletal muscle, and the PAB II protein is exclu-
sively localized to the nucleus (Krause, S. et al.,
Exp. Cell Res. 214, 75-82 (1994)) where it acts as a
factor in mRNA polyadenylation (Whale, E., Cell 66,
759-768 (1991); Whale, E. et al., J. Biol. Chem. 268,
2937-2945 (1993); Bienroth, S. et al., EMBO J. 12, 585-
594 (1993)).
A 8 kb HindIII genomic fragment containing the
PAB II gene was sub-cloned and sequenced (6002 bp;
GenBankTM: AF026029)(Nemeth, A. et al., Nucleic Acids
Res. 23, 4034-4041 (1995)) (Fig. 1B). Genomic structure
of the PAB II gene, and position of the OPMD (GCG)n
expansions. Exons are numbered. Introns 1 and 6 are
variably present in 60% of cDNA clones. ORF, open read-
ing frame; cen, centromere and tel, telomere.
The coding sequence was based on the previously
published bovine sequence (GenBankTM: X89969) and the
sequence of 31 human cDNAs and ESTs. The gene is
comuposed of 7 exons and is transcribed in the cen-qter
orientation (Fig. 1B). Multiple splice variants are
found in ESTs and on Northern blots (Nemeth, A. et al.,
Nucleic Acids Res. 23, 4034-4041 (1995)). In particu-
lar, introns 1 and 6 are present in more than 60% of
clones (Fig. 1B) (Nemeth, A. et al., Nucleic Acids Res.
23, 4034-4041 (1995)). The coding and protein sequences
are highly conserved between human, bovine and mouse
(GenBankTM: U93050). 93% of the PAB II sequence was
read-ily amenable to RT-PCR- or genomic-SSCP screening.
No mutations were uncovered using both techniques. How-
ever, a 400 bp region of exon 1 containing the start
codon could not be readily amplified. This region is
80% GC rich. It includes a (GCG)6 repeat which codes
for the first six alanines of a homopolymeric stretch
of 10 (Fig. 2G). Nucleotide sequence of the mutated
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region of PAB II as well as the amino acid sequences of
the N-terminus polyalanine stretch and position of the
OPMD alanine insertions is also shown in Fig. 2.
Special conditions were designed to amplify by
PCR a 242 bp genomic fragment including this GCG-
repeat. The (GCG)6 allele was found in 98% of French
Canadian non-OPMD control chromosomes, whereas 2% of
chromosomes carried a (GCG)7 polymorphism (n=86)
(Brais, B. et al., Hum. Mol. Genet. 4, 429-434 (1995)).
Screening OPMD cases belonging to 144 families
showed in all cases a PCR product larger by 6 to 21 bp
than that found in controls (Fig. 2A). (GCG)6 normal
allele (N) and the six different (GCG)n expansions
observed in 144 families.
Sequencing of these fragments revealed that the
increased sizes were due to expansions of the GCG
repeat (Fig. 2G). Fig. 2F shows the sequence of the
(GCG)9 French Canadian expansion in a heterozygous par-
ent and his homozygous child. Partial sequence of exon
1 in a normal (GCG)6 control (N), a heterozygote (ht.)
and a homozygote (hm.) for the (GCG)9-repeat mutation.
The number of families sharing the different (GCG)n-
repeats expansions is shown in Table 1.
Table 1
Number of families sharing the different dominant
(GCG)n OPMD mutations
Mutations Polyalaninet Families
(GCG)8 12 4
(GCG)9 13 99
(GCG)10 14 19
(GCG)11 15 16
(GCG)12 16 5
(GCG)13 17 1
Total 144
t, 10 alanine residues in normal PAB II.
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1 C C
_ 7
The (GCG)9 expansion shared by 70 French Cana-
dian families is the most frequent mutation we observed
(Table 1).The (GCG)9 expansion is quite stable, with a
single doubling observed in family F151 in an estimated
598 French Canadian meioses (Fig. 2C). The doubling of
the French Canadian (GCG)9 expansion is demonstrated in
Family F151.
This contrasts with the unstable nature of
preuviously described disease-causing triplet-repeats
(Rosenberg, R.N., New Eng. J. Med. 335, 1222-1224
(1996)).
Genotyping of all the participants in the clini-
cal study of French Canadian OPMD provided molecular
insights into the clinical variability observed in this
condition. The genotypes for both copies of the PAB II
mutated region were added to an anonymous version of
this clinical database of 176 (GCG)9 mutation carriers
(Brais, B. et al., Hum. Mol. Genet. 4, 429-434 (1995)).
Severity of the phenotype can be assessed by the swal-
lowing time (st) in seconds taken to drink 80 cc of
ice-cold water (Brais, B. et al., Hum. Mol. Genet. 4,
429-434 (1995); Bouchard, J.-P. et al., Can. J. Neurol.
Sci. 19, 296-297 (1992)).The late onset and progres-
sive nature of the muscular dystrophy is clearly illus-
trated in heterozygous carriers of the (GCG)9 mutation
(bold curve in Fig. 3) when compared to the average St
of control (GCG)6 homozygous participants(n=76, thinner
line in Fig. 3). The bold curve represents the average
OPMD St for carriers of only one copy of the (GCG)9
mutation (n=169), while the thinner line corresponds to
the average St for (GCG)6 homozygous normal con-
trols(n=76). The black dot corresponds to the St value
for individual VIII. Roman numerals refer to individual
cases shown in Figs. 2B, 2D and discussed in the text.
The genotype of a homozygous (GCG)9 patient and her
parents is shown in Fig. 2B. Independent segregation of
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CA 02312472 2000-05-30
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_ 8 s ¨
the (GCG)7 allele is also shown. Of note, case V has a
more severe OPMD phenotype (Fig. 2D).
Two groups of genotypically distinct OPMD cases
have more severe swallowing difficulties. Individuals
I, II, and III have an early-onset disease and are
homozygous for the (GCG)9 expansion (P < 10-5)
(Figs. 2B, F). Cases IV, V, VI and VII have more severe
phenotypes and are compound heterozygotes for the
(GCG)9 mutation and the (GCG)7 polymorphism (P < 10-5).
In Fig. 2D the independent segregation of the two
alleles is shown. Case V, who inherited the French
Canadian (GCG)9 mutation and the (GCG)7 polymorphism,
is more symptomatic than his brother VIII who carries
the (GCG)9 mutation and a normal (GCG)6 allele
(Figs. 2D and 3). The (GCG)7 polymorphism thus appears
to be a modifier of severity of dominant OPMD. Further-
more, the (GCG)7 allele can act as a recessive
mutaution. This was documented in the French patient IX
who inherited two copies of the (GCG)7 polymorphism and
has a late-onset autosomal recessive form of OPMD
(Fig. 2E). Case IX, who has a recessive form of OPMD,
is shown to have inherited two copies of the (GCG)7
polymorphism.
This is the first description of short trinu-
cleotide repeat expansions causing a human disease. The
addition of only two GCG repeats is sufficient to cause
dominant OPMD. OPMD expansions do not share the cardi-
nal features of "dynamic mutations". The GCG expansions
are not only short they are also meiotically quite sta-
ble. Furthermore, there is a clear cut-off between the
normal and abnormal alleles, a single GCG expansion
causing a recessive phenotype. The PAB II (GCG)7 allele
is the first example of a relatively frequent allele
which can act as either a modifier of a dominant pheno-
type or as a recessive mutation. This dosage effect is
reminiscent of the one observed in a homozygote for two
dominant synpolydactyly mutations. In this case, the
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patient had more severe deformities because she inher-
ited two duplications causing an expansion in the
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I I .
i = .
fVt"41'
polyalanine tract of the HOXD13 protein (Akarsu, A.N.
et al., Hum. Mol. Genet. 5, 945-952 (1996)). A duplica-
tion causing a similar polyalanine expansion in the a
subunit 1 gene of the core-binding transcription factor
5 (CBF(1)
has also been found to cause dominant cleido-
cranial dysplasia (Mundlos, S. et al., Cell 89, 773-779
(1997)). The mutations in these two rare diseases are
not triplet-repeats. The are duplications of "cryptic
repeats" composed of mixed synonymous codons and are
10 thought to
result from unequal crossing over (Warren,
S.T., Science 275, 408-409 (1997)). In the case of
OPMD, slippage during replication causing a reiteration
of the GCG codon is a more likely mechanism (Wells,
D.R., J. Biol. Chem. 271, 2875-2878 (1996)).
Different observations converge to suggest that
a gain of function of PAB II may cause the accumulation
of nuclear filaments observed in OPMD (Tome, F.M.S. &
Fardeau, Acta Neuropath. 49, 85-87 (1980)). PAB II is
found mostly in dimeric and oligomeric forms (Nemeth,
A. et al., Nucleic Acids Res. 23, 4034-4041 (1995)). It
is possible that the polyalanine tract plays a role in
polymerization. Polyalanine stretches have been found
in many other nuclear proteins such as the HOX pro-
teins, but their function is still unknown (Davies,
S.W. et al., Cell 90, 537-548 (1997)). Alanine is a
highly hydrophobic amino acid present in the cores of
proteins. In dragline spider silk, polyalanine
stretches are thought to form B-sheet structures impor-
tant in ensuring the fibers' strength (Simmons, A.H. et
al., Science 271, 84-87 (1996)). Polyalanine oligomers
have also been shown to be extremely resistant to
chemical denaturation and enzymatic degradation
(Forood, B. et al., Bioch. and Biophy. Res. Corn. 211,
7-13 (1995)). One can speculate that PAB II oligomers
comprised of a sufficient number of mutated molecules
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might accumulate in the nuclei by forming undegradable
polyalanine rich macromolecules. The rate of the
accuilmulation would then depend on the ratio of mutated
to non-mutated protein. The more severe phenotypes
observed in homozygotes for the (GCG)9 mutations and
compound heterozygotes for the (GCG)9 mutation and
(GCG)7 allele may correspond to the fact that in these
cases PAB II oligomers are composed only of mutated
proteins. The ensuing faster filament accumulation
could cause accelerated cell death. The recent descrip-
tion of nuclear filament inclusions in Huntington's
disease, raises the possibility that "nuclear toxicity"
caused by the accumulation of mutated homopolymeric
domains is involved in the molecular pathophysiology of
other triplet-repeat diseases (Davies, S.W. et al.,
Cell 90, 537-548 (1997); Scherzinger, E. et al., Cell
90, 549-558 (1997); DiFiglia, M. et al., Science 277,
1990-1993 (1997)). Future immunocytochemical and
expression studies will be able to test this patho-
physiological hypothesis and provide some insight into
why certain muscle groups are more affected while all
tissues express PAB II.
Methods
Contig and cDNA selection
The cosmid contig was constructed by standard
cosmid walking techniques using a gridded chromosome
14-specific cosmid library (Evans, G.A. et al., Gene
79, 9-20 (1989)). The cDNA clones were isolated by cDNA
selection as previously described (Rommens, J.M. et
al., in Proceedings of the third international workshop
on the identification of transcribed sequences (eds.
Hochgeschwender, U. & Gardiner, K.) 65-79 (Plenum, New
York, 1994)).
Cloning of the PAB II gene
Three cDNA clones corresponding to PAB II were
sequenced (SequenaseTM,
- - CA 02312472 2007-08-22 --
- 12 -
USB). Clones were verified to map to cosmids by South-
ern hybridization. The 8 kb HindIII restriction frag-
ment was sub-cloned from cosmid 166G8 into pBluescriptII"
(SK) (Stratagene). The clone was sequenced using prim-
ers derived from the bPABII gene and human EST
sequences. Sequencing of the PAB II introns was done by
primer walking.
PAB II mutation screening and sequencing
All cases were diagnosed as having OPMD on
clinical grounds (Brais, B. et al., Hum. Mol. Genet. 4,
429-434 (1995)). RT-PCR- and genomic SSCP analyses were
done using stan-dard protocols (Lafreniere, R.G. et
al., Nat. Genet. 15, 298-302 (1997)). The primers used
to amplify the PAB II mutated region were: 5'-
CGCAGTGCCCCGCCTTAGA-3' (SEQ ID NO:19) 1 and 5'-
ACAAGATGGCGCCGCCGCCCCGGC-3' (SEQ ID NO:20) =
PCR
reactions were performed in a total volume of 15 pl
containing: 40 ng of genomic DNA; 1.5 pg of BSA; 1 pM
of each primer; 250 pM dCTP and dTTP; 25 pM dATP; 125
pM of dGTP and 125 pM of 7-deaza-dGTP (Pharmacia); 7.5%
DMSO; 3.75 pCi135S]dATP, 1.5 unit of Taq DNA polymerase
and 1.5 mM MgC12 (Perkin Elmer). For non-radioactive
PCR reactions the [35S]dATP was replaced by 225 pM of
dATP. The amplification procedure consisted of an
initial denaturation step at 95 C for five minutes,
followed by 35 cycles of denaturation at 95 C for 15 s,
annealing at 70 C for 30 s, elongation at 74 C for
s and a final elongation at 74 C for 7 min. Samples
were loaded on 5% polyacrylamide denatur-ing gels.
30 Following electrophoresis, gels were dried and
autoradiographs were obtained. Sizes of the inserts
were determined by comparing to a standard M13 sequence
(Sequenase", USE). Fragments used for sequencing were
gel-purified. Sequencing of the mutated fragment using
the Amplicycle kit" (Perkin Elmer) was done with the
5'-CGCAGTGCCCCGCCTTAGAGGTG-3' (SEQ ID NO:21) primer at
an elongation temperature of 68 C.
CA 02312472 2000-05-30
t I I II ( 3
( I
- 13 = ' (r
Stability of (GCG)-repeat expansions
The meiotic stability of the (GCG)9-repeat was
estimated based on a large French Canadian OPMD cohort.
It had been previously established that a single
ancestral OPMD carrier chro-mosome was introduced in
the French Canadian population by three sisters in
1648. Seventy of the seventy one French Canadian OPMD
families tested to date segregate a (GCG)9 expansion.
However, in family F151, the affected brother and
sister, despite sharing the French Canadian ancestral
haplotype, carry a (GCG)12 expansion, twice the size of
the ancestral (GCG)9 mutation (Fig. 2C). In this
founder effect study, it is estimated that 450 (304-
594) historical meioses shaped the 123 OPMD cases
belonging to 42 of the 71 enrolled families. The
screening of the full set of participants allowed an
identification of another 148 (GCG)9 carrier
Chromosomes. Therefore, it is estimated that a single
mutation of the (GCG)9 expansion has occurred in 598
(452-742) meioses.
Genotype-phenotype correlations
176 carriers of at least one copy of the (GCG)9
mutation were examined during the early stage of the
linkage study. All were asked to swallow 80 cc of ice-
cold water as rapidly as possible. Testing was stopped
after 60 seconds. The swallowing time (st) was
validated as a sensitive test to identify OPMD cases
(Brais, B. et al., Hum. Mol. Genet. 4, 429-434 (1995);
Bouchard, J.-P. et al., Can. J. Neurol. Sci. 19, 296-
297 (1992)). The st values for 76 (GCG)6 homozygotes
normal controls is illustrated in Fig. 3. Analyses of
variance were computed by two-way ANOVA (SYSTAT
package). For the (GCG)9 homozygotes their mean st
value was compared to the mean value for all (GCG)9
heterozygotes aged 35-40 (P < 10). For the (GCG)9 and
(GCG)7 compound heterozygotes their mean st value was
,4ET
CA 02312472 2000-05-30
t
!C
ti 411( .1 II
/I II
- 14
compared to the mean value for all (GCG)9 heterozygotes
aged 45-65 (P < 10).
While the invention has been described in con-
nection with specific embodiments thereof, it will be
understood that it is capable of further modifications
and this application is intended to cover any vari-
ations, uses, or adaptations of the invention follow-
ing, in general, the principles of the invention and
including such departures from the present disclosure
as come within known or customary practice within the
art to which the invention pertains and as may be
applied to the essential features hereinbefore set
forth, and as follows in the scope of the appended
claims.
A474nMf,:NEFT:
CA 02312472 2011-08-23
. .
SEQUENCE LISTING
<110> McGill University
Rouleau, Guy A.
Brais, Bernard
<120> Short GCG Expansions in the PAB II Gene for Oculopharyngeal
Muscular Dystrophy and Diagnostic Thereof
<130> 13180.5
<140> 2,312,472
<141> 1998-12-07
<150> CA 2,218,199
<151> 1997-12-09
<160> 21
<170> PatentIn version 3.2
<210> 1
<211> 30
<212> DNA
<213> Homo sapiens
<400> 1
atggcggcgg cggcggcggc ggcagcagca
30
<210> 2
<211> 24
<212> DNA
<213> Homo sapiens
<400> 2
atggcggcgg cggcggcggc ggca
24
<210> 3
<211> 27
<212> DNA
<213> Homo sapiens
<400> 3
atggcggcgg cggcggcggc ggcggca
27
<210> 4
<211> 30
<212> DNA
<213> Homo sapiens
<400> 4
atggcggcgg cggcggcggc ggcggcggca
30
CA 02312472 2011-08-23
16
<210> 5
<211> 33
<212> DNA
<213> Homo sapiens
<400> 5
atggcggcgg cggcggcggc ggcggcggcg gca 33
<210> 6
<211> 36
<212> DNA
<213> Homo sapiens
<400> 6
atggcggcgg cggcggcggc ggcggcggcg gcggca 36
<210> 7
<211> 39
<212> DNA
<213> Homo sapiens
<400> 7
atggcggcgg cggcggcggc ggcggcggcg gcggcggca 39
<210> 8
<211> 42
<212> DNA
<213> Homo sapiens
<400> 8
atggcggcgg cggcggcggc ggcggcggcg gcggcggcgg ca 42
<210> 9
<211> 45
<212> DNA
<213> Homo sapiens
<400> 9
atggcggcgg cggcggcggc ggcggcggcg gcggcggcgg cggca 45
<210> 10
<211> 19
<212> PRT
<213> Homo sapiens
<400> 10
Met Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Ala Ala Gly Gly
1 5 10 15
Arg Gly Ser
CA 02312472 2011-08-23
17
<210> 11
<211> 16
<212> PRT
<213> Homo sapiens
<400> 11
Met Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Ala Ala Gly
1 5 10 15
<210> 12
<211> 17
<212> PRT
<213> Homo sapiens
<400> 12
Met Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Ala Ala
1 5 10 15
Gly
<210> 13
<211> 18
<212> PRT
<213> Homo sapiens
<400> 13
Met Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Ala
1 5 10 15
Ala Gly
<210> 14
<211> 19
<212> PRT
<213> Homo sapiens
<400> 14
Met Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly
1 5 10 15
Ala Ala Gly
CA 02312472 2011-08-23
=
. . . . _...... _ .
.
=
18 .
=
<210> 15
<211> 20
<212>- PRT
<213> Homo sapiens
<400> 15
Met Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
1 5 10 15
Gly Ala Ala Gly
<210> 16
<211> 21
<212> PRT
<213> Homo sapiens
<400> 16
Met Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
1 5 10 15
Ala Gly Ala Ala Gly
<210> 17
<211> 22
<212> PRT
<213> Homo sapiens
<400> 17
Met Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
1 5 10 15
Ala Ala Gly Ala Ala Gly
<210> 18
<211> 6002
<212> DNA
<213> Homo sapiens
<220>
<221> misc feature
<222> 46l6) (4616)
<223> n A, T, C or G
=
0891 bqopoqbobo boqp6bobbo obBeo5e5o5 elpobb6s6be e66e6q56oo oe66e544e3
0Z91 obobboebbb bb000v6466 6e6oq6b4oe 56BooBeb5? 56pbbs.65vb peopereobbo
0951 poop6E656o 44.5b400b615 q000bbBxl. ofteibBoopo 3oobo6po3p b6poop6oD6
00ST pbbebyeboo obybopobeE, 6DobP5opo6 ebbqobqobq obebbebqop bRbElqopebb
0t,f71 pbqoqbebbq oDbboeubbb oe43eb666-e 063E6E666o 3036)5666e6 6bboo6bebb
09ET ftqb5oobb6 boo gyopEo66o5 bob.66boo.66 6opqobbbbo 18505E6354
0zET o6666.6obeo Beobsoboob B35635E:off) D6bob64ebo 5bobv5404b ep000bbbo6
09Z1 bobbboobqe opoolowbe obobboobbq boolool6P5 qeewbp.61.1 oftobfreobb
puT 6opfq.664vo qobp4ov4be b5qepopooq qeq.E.vqbveo obqiebiqqv qqp6D6-466p
01711 bv4q306033 fq6po5o5.64 q4egbe61.-4-4 eftifooeqoo oqql.e.62D65
epe5qqp644
0801 546ob3e56,5 qbbebbeqp6 o4ov66ve4o op4Teqqe45 5o6646o6eo pollo4e4pe
mil b6eobveft6 65boo66-e46 3=53=656 363-44pe4e3 opq6o6e6bo po4ve3o6.66
096 v4obvqopbo 4.5eop4qqoo qbbqqebpeb boqpbobbpb qbEobqopop 8554Debubq
006 ogeoboo4v? pobvpabogo goebol4poo peoqfiqbebo eeeboeo464 oppoqqbfte
Of/8 3666660q1.1 ooq6vo65oo obeebqqq6u eoopqqq565 3qooe5q6qe 3q6e4o66q3
OBL ubbeqoppoo b6obbgebee oez beeobbqobe qoRoqobepe epobboopeD
0zL qopbbblqqq bqoqobqabq oeeetyaoolq qbeeopqebu 5oq6bqo3q4 3.645b4op4o
099 4e33Dquoeu obt,D4qqgpo 34Doeeoeoq 4yolqoblPE, 55.qopq300q obpeqemps.6
009 6hfiqs56qop ueqevoofq5 bqu6Tebblb u4obebv5b5 3546640q4q obqopoqopq
OP5 ve65?5qoe5 6golo5pi5y 43y.64644pe ftyouBeDu6 bbobb5p6pe oboeqop66p
0817 vfieobub666 qpeebb4eue vfmeeeebbp op464o6PP5 54.4eoe6q1,6 6.6p6b4qvbe
OZV ob4b5o6s4q bqoqfpepobb 6686uveobv pobowoeqq qoe6e6444q fthoelq4ebq
09E b3ob.62e6o4 egbobblobq eoeopse-46p eeopoogeo2 upeee6bpp6 bqbebeebeb
00E peeffyeeyno oeoeqbbeob qb6bgpe5eo epfibt,ftlqb TeopqoE,656 6bue.64.4e6q
OPZ 4.46Eritua64 qq.66654052 E5bb5eb4e2 eb6bqopq61. 4oepoo56s4 eov56-4boob
081 o5obep44ee oePvP4beop eeDbeq4Elpq ooppovoqob D66E.Q6qoge oq000pbooe
OZT sb6egeeegy geebobqopo ebgboo6beo p64;ygeobp qbbqfigego4 upuobeofte
09 qp;Poeevoo epoqeeoqqb qoo64vueoe qee33p0be4 PVPOOOPOP6 6466eehqee
81 <00f7>
61,
EZ-80-TTOZ ZLVZTEZO VD
CA 02312472 2011-08-23
gaggcccaga gctcgggcga gcggtggcag gcggggggtg gggttgggcg gggaataacg 1740
tggctggggc gggtcgggcc ggggatgggt cagcgatcac tacaaggggc ccgactggct 1800
tgattcgggc gtcacgggtg cctagtgttg ttctagagag ggtagctttt cttttatcac 1860
gaccctcgca tggggcgagg gaaatggccg agcatggctg aggcgcgctc tggccgagag 1920
cagggcacag cccctgcgtt ggttcctctt aagctgtcct ccataccctc cccacttata 1980
ttaggagctg gaagctatca aagetcgagt cagggagatg gaggaagaag ctgagaagct 2040
aaaggagcta cagaacgagg tagagaagca gatgaatatg agtccacctc caggcaatgc 2100
tgagtaactg gcggttgcac gcggagcccg ggttctcggg ttggaagggt tgtggggagg 2160
atggggaatg tggggttaga tactcggcac cctggagctg cttgtctgag ctattatgac 2220
tgtgccgcgg tcatagtccg ttgtgtgttc ctctgacctt tgtgaggcag aactgatatt 2280
ttggtggtgg tagccttgtg cctccctttg tcctgttata attgtgttgc tctttattct 2340
tagtctacgt ctatctttct ttggtagagg ttgcgtgctc gcatttgacc ttcaaatcta 2400
atagtttttc ctccaattgg agacgcttta ggattctaag agaaagcaag ctggaagggg 2460
tttccocttt aaattctaga aatgtggagt ctcagcccac ttaattttgc tcactcttaa 2520
aagcatttca accaaagcca ttcattaggg atttgatttg gagggcagga gggattccta 2580
tactgtttta agtgtgtatt aattctttca atttatcgaa ttatttagtg agtaacctgc 2640
tatgcactag gcactattct cggcttgtgg gtacagcagg gaacagcaca gaccaaaatc 2700
tttgccttca ctgagcttat gggatagtgc tggtggtgga agtgcaacat attggtcaag 2760
tagaaaacaa gtgtgtggtt tttgtaaaaa attatttttt cctgatagct ggcccggtga 2820
tcatgtccat tgaggagaag atggaggctg atgcccgttc catctatgtt ggcaatgtga 2880
cgtactgggg ctctgactgg ggttgggggc aagttcttct tttggggaat tatttaatag 2940
tcctgaaaga acatctccgg gatagatgtg gttttgggtg tggagggagt gtgggaagga 3000
ggttaaaggt aatggaatga tcagtaatca gcaaaggctc tgggtttgga aggaaaagag 3060
attaattcct caaattacca gatttcatgt gctttggtgt atgatggccc agaccaaagg 3120
ctcgggaggg ttcttttgag acaggaattt gcctggtgcc tgtgaaattt ttctcctctc 3180
atcaggtgga ctatggtgca acagcagaag agctggaagc tcactttcat ggctgtggtt 3240
cagtcaaccg tgttaccata ctgtgtgaca aatttagtgg ccatcccaaa ggtaaagtaa 3300
aggggagtaa gttgagataa tttaaattac agtgtacaaa tagataaatt atgttttata 3360
CA 02312472 2011-08-23
=
21
ttgagcagta agttatttgg tgttaacaca ggtgatctgt gtcatttaag atcatggcat 3420
taatgttgat atatcaggag ttgcacctaa atgtcttcag aggccagata acaaaaatga 3480
aggctagatg tgggtgggat tacgaactag aaggggaggg gcagcttcta cttggcctat 3540
tatggcatat ggaaattcag gccctgtgtg tcttattttt acaaatttca aagagtagct 3600
ggaaatttta aaatttaaat gatttcgaat gattgaaatt ttccatttag aagaattttg 3660
acaaataaaa aatataactg cattgtagcc caaaacgaag catgcctgca ggttgaattt 3720
gacctgtgag gtatttgtaa cctcagagag atacaatgac aattcttttc aggtttgcgt 3780
atatagagtt ctcagacaaa gagtcagtga ggacttcctt ggccttagat gagtccctat 3840
ttagaggaag gcaaatcaag gtaagcctat gtccattgct gttctagttg tgtataaact 3900
ctccaggttg cctttaaggc tatcatttgt tcatctctga ctcaggtgat cccaaaacga 3960
accaacagac caggcatcag cacaacagac cggggttttc cacgagcccg ctaccgcgcc 4020
cggaccacca actacaacag ctcccgctct cgattctaca gtggttttaa cagcaggccc 4080
cggggtcgcg tctacaggtc aggatagatg ggctgctcct ctttcccccg cctcccgtga 4140
gccccgtatg cttcctcctc tctggtctga ggaacotccc tccccccacc cctccccgtg 4200
gtcttcagga actttgtctc ctgcctgtgc aggttgagga aggtagttgc aggccaggcc 4260
agaaggcagc ctcatcatct tttctgcagt agaaattggt gataagggct gcatccctcc 4320
cttggttcaa agaggcttcc acccccagcc ttttttttct tgggagttgg tggcatttga 4380
aggtgtttgc ggacaaaact gggaggaaca gggcctccag gaagttgaaa gcactgcttg 4440
gacatttgtt acttttttcg gagttaggga gggattgaag actgaacctc ccttggaaga 4500
ataccagagg ctagctagtt gatcctccca acagccttgt gggaggattt tgagatactt 4560
attctttatt tgagccagtc ttgcaaggtt aacttctcac tgggcctagt gtggtnccca 4620
ggtttttgcc ttgcttcact tctgtctcta catttaaata gacgggttag gcatataaac 4680
cttggctttt cataagctct acctgcctat ccccaggagt tagggaggat ctatttgtga 4740
aggccctagg gtttaaaaac tgtggaggac tgaaaaactg gataaaaagg gggtcctttt 4800
ccttgcccct gtctctcact cagatgcgct tctttttcgc cactgtttgg caaagttttc 4860
tgttaagccc ccctccccct gccccagttc tcccaggtgc gttactattt ctgggatcat 4920
ggggtcggtt ttaggacact tgaacacttc ttttccocce ttcccttcac agtaactggg 4980
gcaggggcct acggggaggg gcttgtactg aactatctag tgatcacgtt aacacctaac 5040
tctccttctt tcttccaggg gccgggctag agcgacatca tggtattccc cttactaaaa 5100
CA 02312472 2011-08-23
22
aaagtgtgta ttaggaggag agagaggaaa aaaagaggaa agaaggaaaa aaaaaagaat 5160
taaaaaaaaa aaaaagaaaa acagaagatg accttgatgg aaaaaaaata ttttttaaaa 5220
aaaagatata ctgtggaagg ggggagaatc ccataactaa ctgctgagga gggacctgct 5280
ttggggagta ggggaaggcc cagggagtgg ggcagggggc tgcttattca ctctggggat 5340
tcgccatgga cacgtctcaa ctgcgcaagc tgcttgccca tgtttccctg cccccttcac 5400
ccccttgggc ctgctcaagg gtaggtgggc gtgggtggta ggagggtttt ttttacccag 5460
ggctctggaa ggacaccaaa ctgttctgct tgttaccttc cctcccgtct tctcctcgcc 5520
tttcacagtc ccctcctgcc tgctcctgtc cagccaggtc taccacccac cccacccctc 5580
tttctccggc tccctgcccc tccagattgc ctggtgatct attttgtttc cttttgtgtt 5640
tctttttctg ttttgagtgt ctttctttgc aggtttctgt agccggaaga tctccgttcc 5700
gctcccagcg gctccagtgt aaattcccct tccccctggg gaaatgcact accttgtttt 5760
ggggggttta ggggtgtttt tgtttttcag ttgttttgtt tttttgtttt ttttttttcc 5820
tttgcctttt ttccctttta tttggaggga atgggaggaa gtgggaacag ggaggtggga 5880
ggtggatttt gtttattttt ttagctcatt tccaggggtg ggaatttttt tttaatatgt 5940
gtcatgaata aagttgtttt tgaaaataaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 6000
aa 6002
<210> 19
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide
<400> 19
cgcagtgccc cgccttaga 19
<210> 20
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide
<400> 20
acaagatggc gccgccgccc cggc 24
CA 02312472 2011-08-23
23
<210> 21
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide
<400> 21
cgcagtgccc cgccttagag gtg 23
