CA2312472C - Short gcg expansions in the pab ii gene for oculopharyngeal muscular dystrophy and diagnostic thereof - Google Patents

Abstract

The present invention relates to a human PAB II gene containing transcribed polymorphic GCG repeat, which comprises a sequence as set forth in SEQ ID NO:3, which includes introns and flanking genomic sequence. The allelic variants of GCG repeat of the human PAB II gene are associated with a disease related with protein accumulation in nucleus, such as polyalanine accumulation, a disease related with swallowing difficulties, such as oculopharyngeal muscular dystrophy. The present invention also relates to a method for the diagnosis of a disease with protein accumulation in nucleus, which comprises the steps of: a) obtaining a nucleic acid sample of said patient; and b) determining allelic variants of GCG repeat of the gene of claim 1, and wherein long allelic variants are indicative of a disease related with protein accumulation in nucleus.

Description

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-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 AMENDED SHEET

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 AMENDED SHEET

14. .14 ix I

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.
AMENDED SHEET

- 3a -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 - 3b -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:

- 3c -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

- 3d -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.

- 3e -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 - 3f -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.

-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 WENDED SHEET

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 i 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.
AMENDED SHEET

_ 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 timENDED SliEET
=

, .
_ 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 AMENDED SHEET

_ 9 , -patient had more severe deformities because she inher-ited two duplications causing an expansion in the AMENDED SHEET

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 AMENDED SHEET

. - 11 -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, 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.

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

t !C
ti 411( .1 II
/I II

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:

. .
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 <210> 2 <211> 24 <212> DNA
<213> Homo sapiens <400> 2 atggcggcgg cggcggcggc ggca <210> 3 <211> 27 <212> DNA
<213> Homo sapiens <400> 3 atggcggcgg cggcggcggc ggcggca <210> 4 <211> 30 <212> DNA
<213> Homo sapiens <400> 4 atggcggcgg cggcggcggc ggcggcggca <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 Arg Gly Ser <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 <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 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 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 Ala Ala Gly =
. . . . _...... _ .
.
=
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 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 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 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, 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 =

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 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 <210> 21 <211> 23 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide <400> 21 cgcagtgccc cgccttagag gtg 23

Claims (86)

WHAT IS CLAIMED IS:

1. An isolated human PAB II nucleic acid molecule comprising an allelic variant of a polymorphic GCG repeat in exon I thereof, wherein said allelic variant of said polymorphic GCG repeat has the sequence ATG (GCG)6+n GCA, with n being selected from 1 to 7, and wherein said allelic variant of said polymorphic GCG repeat is associated with oculopharyngeal muscular dystrophy (OPMD) disease in a human patient.

2. The nucleic acid molecule of claim 1, wherein said OPMD disease is associated with a phenotype which is at least one of:
(a) protein accumulation in a cell nucleus;
(b) a swallowing difficulty; or (c) ptosis.

3. The nucleic acid molecule of claim 1 or 2, wherein n is selected from 2 to 7, and wherein said allelic variant is associated with an increased severity of the disease.

4. The nucleic acid molecule of claim 3, wherein the phenotype associated with said allelic variant is dominant.

5. The nucleic acid molecule of claim 1 or 2, wherein a first allele of said GCG repeat has an n which is equal to 1.

6. The nucleic acid molecule of claim 5, wherein a second allele of said GCG repeat has an n selected from 2 to 7, and wherein said first allele is a modulator of the severity of the phenotype associated with said second allele.

7. The nucleic acid molecule of any one of claims 1-5, wherein said human patient is homozygous for said polymorphic GCG repeat.

8. The nucleic acid molecule of any one of claims 1-6, wherein said human patient is heterozygous for said polymorphic GCG repeat.

9. An isolated nucleic acid molecule comprising an allelic variant of a polymorphic GCG repeat of exon I of a human PAB II gene, wherein said allelic variant of said polymorphic GCG repeat is associated with oculopharyngeal muscular dystrophy (OPMD) disease and wherein said allelic variant of said GCG repeat has the sequence ATG (GCG)6+n GCA, wherein n is selected from 1 to 7, as set forth in SEQ ID NOs: 3-9, respectively.

10. The nucleic acid molecule of claim 9, wherein said OPMD disease is associated with a phenotype which is at least one of:
(a) protein accumulation in a cell nucleus;
(b) a swallowing difficulty; or (c) ptosis.

11. The nucleic acid molecule of claim 9 or 10, wherein n is selected from 2 to 7, as set forth in SEQ ID NOs: 4-9, respectively, and wherein said allelic variant is associated with an increased severity of said disease.

12. The nucleic acid molecule of claim 10, wherein the phenotype associated with said allelic variant is dominant.

13. The nucleic acid molecule of claim 12, wherein a first allele of said GCG repeat in said patient has an n which is equal to 1, as set forth in SEQ
ID NO: 3, and a second allele of said GCG repeat has an n selected from 2 to 7, as set forth in SEQ ID NOs: 4-9, respectively, and wherein said first allele is a modulator of the severity of the phenotype associated with said second allele.

14. A method for the diagnosis or prognosis of oculopharyngeal muscular dystrophy (OPMD) disease, said method comprising:
a) obtaining a nucleic acid sample from a human patient; and b) determining allelic variants of a GCG repeat in exon I of the PAB ll 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.

15. The method of claim 14, wherein said OPMD disease is associated with a phenotype which is at least one of:
(a) protein accumulation in a cell nucleus;
(b) a swallowing difficulty; or (c) ptosis.

16. The method of claim 14 or 15, wherein n is selected from 2 to 7, and wherein said allelic variant is associated with an increased severity of said disease.

17. The method of claim 16, wherein said GCG repeat has a sequence which is:
a) SEQ ID NO:4, when n = 2;
b) SEQ ID NO:5, when n = 3;
c) SEQ ID NO:6, when n = 4;
d) SEQ ID NO:7, when n = 5;
e) SEQ ID NO:8, when n = 6; or f) SEQ ID NO:9, when n = 7.

18. The method of claim 16, wherein said phenotype associated with said allelic variant is dominant.

19. The method of claim 14 or 15, wherein one allele has an n equal to 0 and a GCG repeat having the sequence set forth in SEQ ID NO:2.

20. The method of claim 14 or 15, wherein a first allele of said GCG
repeat has an n which is equal to 1.

21. The method of claim 20, wherein said GCG repeat having an n which equals 1 has the sequence set forth in SEQ ID NO:3.

22. The method of claim 20 or 21, wherein a second allele of said GCG
repeat has an n selected from 2 to 7, and wherein said first allele is a modulator of the severity of the phenotype associated with said second allele.

23. An isolated embryonic cell expressing at least one allelic variant of a polymorphic GCG repeat in exon I of the PAB II gene, wherein said allelic variant of said polymorphic GCG repeat has the sequence ATG (GCG)6+n GCA, with n being selected from 1 to 7, and wherein said polymorphic repeat in a human PAB II gene is indicative of oculopharyngeal muscular dystrophy (OPMD) disease.

24. The isolated embryonic cell of claim 23, wherein said OPMD disease is associated with a phenotype which is at least one of:
(a) protein accumulation in a cell nucleus;
(b) a swallowing difficulty; or (c) ptosis.

25. The isolated embryonic cell of claim 23 or 24, wherein said allelic variant of the PAB II gene is a human allelic variant.

26. 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, said method comprising:
a) exposing a non-human transgenic animal to said agent, wherein said 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 said allelic variant of said polymorphic GCG
repeat has the sequence ATG (GCG)6+n GCA, with n being selected from 1 to 7, and wherein said allelic variant of said polymorphic repeat in a human PABII gene is indicative of OPMD disease; and b) evaluating the prevention or treatment of development of said phenotype in said animal exposed to said agent as compared to a control animal not having been exposed to said agent.

27. 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 said allelic variant of said polymorphic GCG repeat has the sequence ATG (GCG)6+n GCA, with n being selected from 1 to 7, and wherein said allelic variant is associated with protein accumulation in the nucleus of said cell.

28. The cell of claim 27, wherein said cell is a mammalian cell.

29. A method for screening and identifying an agent which modulates protein accumulation in the nucleus of a cell, said method comprising:
a) exposing the cell of claim 27 or 28 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.

30. An isolated human PAB II gene comprising a polymorphic GCG
repeat in exon I thereof, wherein said repeat has the sequence ATG (GCG)6+n GCA, wherein n is 0, and wherein said sequence is indicative of absence of oculopharyngeal muscular dystrophy (OPMD) disease, said OPMD disease being associated with at least one of protein accumulation in a cell nucleus, a swallowing difficulty, or ptosis in a human patient.

31. The human PAB II gene of claim 30, wherein said gene is as set forth in SEQ ID NO: 2.

32. 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, said method comprising:
a) obtaining a nucleic acid sample from said patient;
b) determining in said sample whether said gene comprises at least one trinucleotide expansion having the sequence ATG(GCG)6+n GCA, with n being 1 to 7, wherein the determination of one trinucleotide expansion in said coding sequence of said gene is indicative of OPMD disease in said patient.

33. The method of claim 32, wherein said OPMD disease is associated with a phenotype which is at least one of:
(a) protein accumulation in a cell nucleus;
(b) a swallowing difficulty; or (c) ptosis.

34. The method of claim 33, wherein said disease is associated with protein accumulation in a cell nucleus.

35. A method for determining the presence or absence of an allelic variant of a polymorphic GCG trinucleotide repeat in a human, said 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 said human, said GCG
repeat having the sequence ATG(GCG)6+n GCA, wherein when n is selected from 0 to 7, and wherein the presence of at least one of two alleles of said GCG repeat having an n equal to 1 to 7 identifies the presence of an allelic variant of the PAB
II
gene.

36. The method of claim 35, wherein the presence of said allelic variant of said polymorphic GCG trinucleotide repeat is determined by amplification of nucleic acid containing the GCG trinucleotide repeat using a pair of oligonucleotide primers which specifically amplify the region of nucleic acid containing said GCG trinucleotide repeat and detecting amplified products containing said GCG trinucleotide repeat region.

37. The method of claim 35, wherein the presence of said allelic variant of said polymorphic GCG trinucleotide repeat is determined by performing a polymerase chain reaction with a pair of oligonucleotide primers which specifically amplify the region of nucleic acid containing said GCG
trinucleotide repeat and detecting amplified products containing said GCG
trinucleotide repeat region.

38. The method of claim 36 or 37, comprising the additional step of sequencing the amplified products.

39. The method of claim 37 or 38, wherein the pair of oligonucleotide primers comprise the oligonucleotides of SEQ ID NO:19 and SEQ ID NO:20.

40. The method of any one of claims 35-39, wherein the presence of said allelic variant of said GCG trinucleotide repeat is indicative of the presence of oculopharyngeal muscular dystrophy (OPMD) disease in said patient.

41. The method of claim 40, wherein said OPMD disease is associated with a phenotype which is at least one of:
(a) protein accumulation in a cell nucleus;
(b) a swallowing difficulty; or (c) ptosis.

42. The method of claim 35, wherein the presence of two alleles of said GCG repeat having an n = 0 identifies the absence of an allelic variant of the PAB II gene that is associated with OPMD disease.

43. A method for determining whether a human is at risk of developing oculopharyngeal muscular dystrophy (OPMD) disease, said method comprising conducting an assay on a nucleic acid sample from said human to determine the presence or absence of a GCG trinucleotide repeat in a human PAB II gene, said GCG trinucleotide repeat having the sequence ATG(GCG)6+n GCA, with n being 1 to 7, wherein the presence of said GCG trinucleotide repeat is indicative that said human is at risk for development of OPMD disease.

44. The method of claim 43, wherein said assay is probe hybridization, direct sequencing, restriction enzyme fragment analysis or fragment electrophoretic motility.

45. The method of claim 43, wherein said assay comprises:
a) amplifying a target portion of the nucleic acid using a pair of oligonucleotide primers which specifically amplify the region of nucleic acid containing GCG trinucleotide repeats to obtain an amplified product; and b) determining the presence or absence of said GCG trinucleotide repeat in said nucleotide sequence of said amplified product.

46. The method of claim 45, wherein said target portion is amplified by polymerase chain reaction with suitable oligonucleotide primers.

47. The method of claim 46, wherein said oligonucleotide primers comprise the oligonucleotides of SEQ ID NO:19 and SEQ ID NO:20.

48. A method for determining whether a human is negative for oculopharyngeal muscular dystrophy (OPMD) disease, wherein said method comprises:
a) amplifying a nucleic acid sample from said 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 11 gene to obtain an amplification product, with said GCG
trinucleotide repeat having the sequence ATG(GCG)6+n GCA, with n being 1 to 7;
b) determining the presence or absence of said GCG trinucleotide repeat in said amplification product, wherein the absence said GCG trinucleotide repeat is indicative that said human is negative for OPMD disease.

49. A method for identifying a human having a normal GCG trinucleotide repeat in a human PAB II gene, said method comprising analyzing a GCG
trinucleotide repeat region in exon I of a human PAB II gene in a nucleic acid sample from said human, wherein said normal GCG trinucleotide repeat in said human PAB II gene has the sequence ATG (GCG)6GCA.

50. A method for identifying whether a human patient has a predisposition to develop oculopharyngeal muscular dystrophy (OPMD) disease, said 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 said patient, wherein the presence of said GCG trinucleotide repeat having the sequence ATG(GCG)6+n GCA, with n being 1 to 7, is indicative that said patient has a predisposition to develop OPMD disease.

51. The method of claim 50, wherein the presence of said GCG
trinucleotide repeat is determined by amplification of nucleic acid containing the GCG trinucleotide repeat using a pair of oligonucleotide primers which specifically amplify the region of nucleic acid containing the GCG
trinucleotide repeat and detecting amplified products containing said GCG trinucleotide repeat region.

52. The method of claim 50, wherein the presence of said GCG
trinucleotide repeat is determined by performing a polymerase chain reaction with a pair of oligonucleotide primers which specifically amplify the region of nucleic acid containing the GCG trinucleotide repeat and detecting amplified products containing said GCG trinucleotide repeat region.

53. The method of claim 51 or 52, comprising the additional step of sequencing the amplified products.

54. The method of claim 51 or 52, wherein the pair of oligonucleotide primers comprise the oligonucleotides of SEQ ID NO:19 and SEQ ID NO:20.

55. A method for diagnosing oculopharyngeal muscular dystrophy (OPMD) disease in a human patient, said 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 said patient, wherein the presence of said GCG trinucleotide repeat having the sequence (GCG)6+n GCA, wherein n is 1 to 7, is indicative of OPMD disease in said patient.

56. The method of claim 55, wherein the presence of said GCG
trinucleotide repeat is determined by amplification of nucleic acid containing the GCG trinucleotide repeat using a pair of oligonucleotide primers which specifically amplify the region of nucleic acid containing the GCG
trinucleotide repeat and detecting amplified products containing said GCG trinucleotide repeat region.

57. The method of claim 55, wherein the presence of said GCG
trinucleotide repeat is determined by performing a polymerase chain reaction with a pair of oligonucleotide primers which specifically amplify the region of nucleic acid containing the GCG trinucleotide repeat and detecting amplified products containing said GCG trinucleotide repeat region.

58. The method of claim 56 or 57, comprising the additional step of sequencing the amplified products.

59. The method of claim 56 or 57, wherein the pair of oligonucleotide primers comprise the oligonucleotides of SEQ ID NO:19 and SEQ ID NO:20.

60. A method for diagnosing oculopharyngeal muscular dystrophy (OPMD) disease in a human, said method comprising:
a) contacting nucleic acid from said 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, said GCG trinucleotide repeat having the sequence (GCG)6+n GCA, wherein n is 0 to 7; and b) detecting an amplification product comprising said GCG
trinucleotide repeat region, wherein when said GCG repeat detected has an n equal to 1 to 7, said detection is indicative of OPMD disease in said human.

61. The method of claim 60, wherein the pair of oligonucleotide primers comprise the oligonucleotides of SEQ ID NO:19 and SEQ ID NO:20.

62. An isolated PAB II nucleic acid molecule comprising a polymorphic GCG repeat having the sequence ATG (GCG)6+n GCA, wherein when:
a) n=0, said nucleic acid molecule is not associated with oculopharyngeal muscular dystrophy (OPMD) disease; or b) n is selected from 1 to 7, said nucleic acid molecule is associated with OPMD disease.

63. The isolated PAB II nucleic acid molecule of claim 62, wherein said OPMD disease is associated with a phenotype which is at least one of:
(a) protein accumulation in a cell nucleus;
(b) a swallowing difficulty; or (c) ptosis.

64. The isolated nucleic acid molecule of claim 62 or 63, wherein n=0, and wherein said sequence comprises the sequence as set forth in SEQ ID NO:
18.

65. The isolated nucleic acid molecule of claim 62 or 63, wherein n=0, and wherein said molecule comprises the sequence as set forth in SEQ ID NO: 1.

66. The isolated nucleic acid molecule of claim 62 or 63, wherein n=0, and wherein said GCG repeat has the sequence set forth in SEQ ID NO: 2.

67. The isolated nucleic acid molecule of claim 62 or 63, wherein n=1, and wherein said GCG repeat has the sequence set forth in SEQ ID NO: 3.

68. The isolated nucleic acid molecule of claim 62 or 63, wherein n=2, and wherein said GCG repeat has the sequence set forth in SEQ ID NO: 4.

69. The isolated nucleic acid molecule of claim 62 or 63, wherein n=3, and wherein said GCG repeat has the sequence set forth in SEQ ID NO: 5.

70. The isolated nucleic acid molecule of claim 62 or 63, wherein n=4, and wherein said GCG repeat has the sequence set forth in SEQ ID NO: 6.

71. The isolated nucleic acid molecule of claim 62 or 63, wherein n=5, and wherein said GCG repeat has the sequence set forth in SEQ ID NO: 7.

72. The isolated nucleic acid molecule of claim 62 or 63, wherein n=6, and wherein said GCG repeat has the sequence set forth in SEQ ID NO: 8.

73. The isolated nucleic acid molecule of claim 62 or 63, wherein n=7, and wherein said GCG repeat has the sequence set forth in SEQ ID NO: 9.

74. A method of determining the presence or absence of an allelic variant of a polymorphic GCG trinucleotide repeat in a human, said method comprising determining the presence of the GCG trinucleotide repeat in exon I
of a human PAB II gene in a sample from said human, said GCG repeat encoding the sequence Met(Ala)6+n Ala, wherein n is selected from 0 to 7, and wherein the presence of at least one of two alleles of said 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.

75. The method of claim 74, wherein the presence of said allelic variant of said polymorphic GCG trinucleotide repeat is determined by amplification of nucleic acid containing the GCG trinucleotide repeat using a pair of oligonucleotide primers which specifically amplify the region of nucleic acid containing said GCG trinucleotide repeat and detecting amplified products containing said GCG trinucleotide repeat region.

76. The method of claim 74, wherein the presence of said allelic variant of said polymorphic GCG trinucleotide repeat is determined by performing a polymerase chain reaction with a pair of oligonucleotide primers which specifically amplify the region of nucleic acid containing said GCG
trinucleotide repeat and detecting amplified products containing said GCG
trinucleotide repeat region.

77. The method of claim 75 or 76, comprising the additional step of sequencing the amplified products.

78. The method of claim 77, wherein the pair of oligonucleotide primers comprise the nucleotide sequences set forth in SEQ ID NO:19 and SEQ ID
NO:20.

79. The method of any one of claims 74-78, wherein the presence of said allelic variant of said GCG trinucleotide repeat is indicative of the presence of oculopharyngeal muscular dystrophy (OPMD) disease in said patient.

80. The method of claim 79, wherein said OPMD disease is associated with a phenotype which is at least one of:
(a) protein accumulation in a cell nucleus;
(b) a swallowing difficulty; or (c) ptosis.

81. The method of claim 74, wherein the presence of two alleles of said GCG repeat having n = 0 identifies the absence of an allelic variant of the PAB II gene that is associated with OPMD disease.

82. The method of claim 79 or 80, wherein n is selected from 2 to 7, and wherein said allelic variant is associated with an increased severity of the disease.

83. The method of claim 79 or 80, wherein a first allele of said GCG repeat has an n which is equal to 1.

84. The method of claim 79 or 80, wherein a second allele of said GCG
repeat has an n selected from 2 to 7, and wherein said first allele is a modulator of the severity of the phenotype associated with said second allele.

85. An isolated human PAB II nucleic acid molecule comprising a polymorphic GCG repeat in exon I thereof, wherein said polymorphic GCG repeat encodes the sequence Met (Ala)6+n Ala, with n being selected from 1 to 7, and wherein said polymorphic Ala repeat is associated with oculopharyngeal muscular dystrophy (OPMD) disease in a human patient.

86. The nucleic acid molecule of claim 85, wherein said OPMD disease is associated with a phenotype which is at least one of:

(a) protein accumulation in a cell nucleus;
(b) a swallowing difficulty; or (c) ptosis.