Patent EvaluationShort GCG expansions in the PAB II gene for oculopharyngeal musculardystrophy Short GCG expansions in thePAB II gene

for oculopharyngeal muscular dystrophyand diagnostic thereof

McGill University: WO9929896

Mutations within the poly(A) binding protein II (PAB II) gene deterministicfor the condition of oculopharyngeal muscular dystrophy (OPMD) aredescribed. These mutations are short, stable, expansions of a trinucleotiderepeat (GCG) within exon 1 of the gene. These findings will be ofdiagnostic value to clinicians and will permit genotype phenotype correla-tions in OPMD. They are the prelude to the generation of animal models ofOPMD, which will facilitate increased understanding of OPMDpathogenesis and the design and testing of potential therapeutic agents.Such agents may also be applicable to other trinucleotide expansiondisorders characterised by intranuclear inclusions, and possibly to other‘conformational diseases’ with intra- or extracellular protein aggregations.

Keywords:aggregation, diagnosis, oculopharyngeal muscular dystrophy(OPMD), poly(A) binding protein II (PAB II) gene, treatment, trinucleotideexpansion

Exp. Opin. Ther. Patents (1999)9(11):1579-1581

1. Introduction

The syndrome of progressive bilateral ptosis, dysphagia, ophthalmoplegiaand proximal limb weakness, occurring as a familial trait, was firstdescribed in the early years of this century by Taylor [1]. Later studies of thesame pedigree [2] traced all the cases to a common ancestor who settled inQuebec in the 17th century. The condition, usually has its onset in the 5th or6th decade of life, but may occur earlier. Besides the original pedigree andother French-Canadian families, OPMD has also been described in BukharaJews [3] and in many other countries. The pattern of inheritance is usuallyautosomal dominant with complete penetrance, but occasional autosomalrecessive families have been reported. In the original French-Canadianfamily, transmission was exclusively through the female line [2,4] which hasraised the possibility of this being a mitochondrial disorder.

The clinical diagnosis of OPMD is generally not difficult to make, especiallyin older patients with a clear family history. The differential diagnosisincludes myasthenia gravis (although there is no muscle fatigability inOPMD), other ocular myopathies, and, particularly in younger patients orthose with prominent ophthalmoplegia, mitochondrial disorders(‘oculocraniosomatic syndrome’) [4]. Muscle biopsy in OPMD revealsrimmed vacuoles and pathognomonic filamentous intranuclear inclusions[5], although these are found in only 3 - 6.5% of muscle nuclei and so mayneed to be sought very diligently to establish the diagnosis. Morphologicalmitochondrial abnormalities have also been reported in muscle biopsies onoccasion [6] but their significance remains uncertain. There is no specific or

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Patent Evaluation

1. Introduction

2. Biology and action

3. Expert opinion

Bibliography

Patent details

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Expert Opinion on Therapeutic Patents

curative treatment for OPMD, but symptomatictherapies (surgical correction of ptosis, percutaneousgastrostomy feeding) may be used.

Recently, linkage of the OPMD disease locus tochromosome 14q11.2-q13 has been established [7-9].This observation was the prelude to identifying thegene responsible for OPMD, the subject of the currentpatent; some of the details have already beenpublished [10].

2. Biology and action

Using markers flanking the disease locus, standardpositional cloning techniques were used to identifythe genetic abnormality in OPMD; of the candidatecDNAs isolated, three showed high sequencehomology to the bovine PAB II gene [11]. PAB IIappeared to be a good candidate gene because itmapped to the candidate interval in 14q11, its mRNAshowed high expression in skeletal muscle, and thePAB II protein is located exclusively in the nucleuswhere it acts as a factor in mRNA polyadenylation [12].

Sequencing the PAB II gene in French-Canadiancontrol chromosomes showed a (GCG)6 allele in exon1 in 98% of individuals, and a (GCG)7 polymorphismin the remaining 2%. In all cases of OPMD, belongingto 144 families, a polymerase chain reaction (PCR)product 6 to 21 base pairs larger than that in controlswas found, constituting short expansions of the GCGtrinucleotide ranging from (GCG)8 to (GCG)13. The(GCG)9 mutation, shared by 70 French-Canadianfamilies, was the most frequent mutation observed.Hence, unlike previously described disease-causingtriplet repeats [13], this (GCG)9 expansion is meioti-cally quite stable.

OPMD patients with more severe swallowing difficul-ties, as assessed by the swallowing time (time to drink80 ml of ice cold water [7]), were found to havedifferent genotypes: either compound heterozygosityfor the (GCG)9 mutation and the (GCG)7 polymor-phism [10]; or homozygosity for (GCG)9 [14]. Thus,(GCG)7 appears to act as both a modifier of theseverity of autosomal dominant OPMD, and as arecessive mutation, since patients with autosomalrecessive OPMD have been shown to carry two copiesof the (GCG)7 polymorphism.

The availability of the deterministic mutations forOPMD in the PAB II gene may allow production ofmammalian models of OPMD, through modification

of germ cells or somatic cells, using establishedtechniques and allelic variants of the PAB II gene.Such models are claimed in the patent but no experi-mental details are provided.

3. Expert opinion

The delineation of the OPMD disease-causingmutation will permit a definitive diagnosis to be madein those individuals clinically suspected to have thedisease, without the need for muscle biopsy and thelaborious search for the pathognomonic intranuclearinclusions (INIs) [5]. Indeed, the test is already in usein neurogenetic laboratories around the world. Withgenetic diagnosis available, this will allow definitionof phenotype genotype correlations. The role of the(GCG)7 polymorphism as a modifier of diseaseseverity has already been mentioned [10]. It has alsobeen shown that individuals homozygous for PAB IIgene expansions have an earlier onset and moresevere form of OPMD, with a larger proportion ofmuscle nuclei showing the pathognomonic INIs [14].In this context, assessment of the phenotypic signifi-cance (i f any) of concurrent delet ions inmitochondrial DNA in OPMD [15,16] in a populationhomogeneous for PAB II mutations may be possible.

In the future, cell biological techniques, and thedevelopment of transgenic mice carrying disease-causing PAB II alleles, may allow elucidation of themechanism by which this particular trinucleotideexpansion causes disease. It is hypothesised [10] thathighly hydrophobic polyalanine stretches, asencoded by GCG expansions, may play a role inpolymerisation. Wild type PAB II is found mostly indimeric and oligomeric forms [11], and it may be thatin the presence of sufficient mutated PAB II,undegradeable polyalanine oligomers mightaggregate into filamentous INIs. The availability ofOPMD animal models will be the prelude to thetesting of potential therapies to block filamentformation, which may prevent or treat the disease.

Since several trinucleotide repeat disorders are nowdescribed [14,17], a number of which are character-ised by the accumulation of filamentous intranuclearmaterial (e.g., huntingtin in Huntington’s disease [18]and ataxins in spinocerebellar ataxias 1, 2, 3 and 7[19]), these future studies may provide generictherapies of use in all these disorders. Such therapiesmight also be of value in other ‘conformational

© Ashley Publications Ltd. All rights reserved. Exp. Opin. Ther. Patents(1999)9(11)

1580 Short GCG expansions in the PAB II gene for oculopharyngeal muscular dystrophy

diseases’ with intra- or extracellular protein aggrega-tions, such as Alzheimer’s disease [20].

Bibliography

Papers of special note have been highlighted as:• of interest•• of considerable interest

1. TAYLOR EW: Progressive vagus-glossopharyngealparalysis with ptosis: a contribution to the group offamily diseases. J. Nerv. Ment. Dis. (1915) 42:129-139.

2. VICTOR M, HAYES R, ADAMS RD: Oculopharyngealmuscular dystrophy: a familial disease of late lifecharacterized by dysphagia and progressive ptosis ofthe eyelids. New Engl. J. Med. (1962) 207:1267-1272.

3. BLUMEN SC, NISIPEANU P, SADEH M et al.: Clinicalfeatures of oculopharyngeal muscular dystrophyamong Bukhara Jews. Neuromusc. Disord. (1993)3:575-577.

4. ROWLAND LP, HIRANO M, DIMAURO S, SCHON EA:Oculopharyngeal muscular dystrophy, other ocularmyopathies, and progressive external ophthalmo-plegia. Neuromusc. Disord. (1997) 7(Suppl. 1):S15-S21.

5. TOMÉ FMS, FARDEAU M: Nuclear inclusions inoculopharyngeal muscular dystrophy. Acta Neuropa-thol. (Berlin) (1980) 49:85-87.

6. DE SEZE J, PASQUIER F, RUCHOUX M-M, HURTEVENT J-F,PETIT H: Les anomalies mitochondriales au cours de ladystrophie musculaire oculo-pharyngée. Rev. Neurol.(Paris) (1997) 153:335-338.

7. BRAIS B, XIE Y-G, SANSON M et al.: The oculopharyngealmuscular dystrophy locus maps to the region of thecardiac and mysoin heavy chain genes on chromo-some 14q11.2-q13. Hum. Mol. Genet. (1995) 4:429-434.

8. STAJICH JM, GILCHRIST JM, LENNON F et al.: Confirma-tion of linkage of oculopharyngeal musculardystrophy to chromosome 14q11.2-q13. Ann. Neurol.(1996) 40:801-804.

9. KRESS W, HALLIGER-KELLER B, GRIMM T et al.: Noevidence for heterogeneity in oculopharyngealmuscular dystrophy. J. Med. Genet. (1998) 35:613-614.

10. BRAIS B, BOUCHARD J-P, XIE Y-G et al.: Short GCGexpansions in the PAB2 gene cause oculopharyngealmuscular dystrophy. Nature Genet. (1998) 18:164-167.

•• Demonstration that expansions of the (GCG) trinucleotidein exon 1 of the PAB II gene are deterministic for OPMD.

11. NEMETH A, KRAUSE S, BLANK D et al.: Isolation ofgenomic and cDNA clones encoding bovine poly(A)

binding protein II. Nucleic Acids Res. (1995)23:4034-4041.

12. WAHLE E: A novel poly(A)-binding protein acts as aspecificity factor in the second phase of messengerRNA polyadenylation. Cell (1991) 66:759-768.

13. ROSENBERG RN: DNA-triplet repeats and neurologicdisease. New Engl. J. Med. (1996) 335:1222-1224.

14. BLUMEN SC, BRAIS B, KORCZYN AD et al.: Homozygotesfor oculopharyngeal muscular dystrophy have asevere form of the disease. Ann. Neurol. (1999)46:115-118.

• Study showing that homozygotes for (GCG)9 expansionshad a more severe OPMD phenotype than heterozygotes.

15. LEZZA AMS, CORMIO A, GERARDI P et al.: MitochondrialDNA deletions in oculopharyngeal musculardystrophy. FEBS Lett. (1997) 418:167-170.

16. MUQIT MMK, WOOD NW, LARNER AJ, LANE RJM:Morphological mitochondrial abnormalities andmitochondrial DNA deletions in monozygotic twinswith genetically-confirmed oculopharyngealmuscular dystrophy. (1999). (Submitted).

17. PERUTZ MF: Glutamine repeats and neurodegenerativediseases: molecular aspects. Trends Biochem. Sci. (1999)24:58-63.

18. DIFIGLIA M, SAPP E, CHASE KO et al.: Aggregation ofhuntingtin in neuronal intranuclear inclusions anddystrophic neurites in brain. Science (1997)277:1990-1993.

19. PAULSON HL, PEREZ MK, TROTTIER Y et al.: Intranuclearinclusions of expanded polyglutamine protein inspinocerebellar ataxia Type 3. Neuron (1997)19:333-344.

20. LARNER AJ: Tau protein as a therapeutic target inAlzheimer’s disease and other neurodegenerativedisorders. Exp. Opin. Ther. Patents (1999)9(10):1359-1370.

Patent details

Title: Short GCG expansions in the PAB II gene foroculopharyngeal muscular dystrophy and diagnosticthereof

Assignee: McGill Univ.

Inventors: Rouleau GA, Brais B

Priority data: 09/12/97 97CA-218199

Filing date: 07/12/98

Publication date: 17/06/99

Publication no.: WO9929896

© Ashley Publications Ltd. All rights reserved. Exp. Opin. Ther. Patents(1999)9(11)

Patent Evaluation 1581