nuclear accumulation of expanded pabp2 gene product in oculopharyngeal muscular dystrophy

ABSTRACT: Autosomal dominant oculopharyngeal muscular dystrophy(OPMD) is an adult-onset disease caused by (GCG) repeat expansions inexon 1 of the poly(A) binding protein 2 gene (PABP2). To elucidate themolecular mechanism underlying the disease, we raised an antiserumagainst a synthetic peptide fragment predicted from PABP2 cDNA. Thepeptide corresponded to amino acids 271–291 where a cluster of posttrans-lational arginine methylation occurs. We examined the subcellular localiza-tion of PABP2 in muscle specimens from five patients with OPMD, 14 pa-tients with various neuromuscular disorders, and three normal controls.All Japanese patients with OPMD have been shown to have expanded(GCG)8, 9, or 11 mutations in PABP2, as well as intranuclear tubulofilamen-tous inclusions (ITFI) of 8.5 nm. None of 50 separate Japanese controlindividuals were shown to have expanded (GCG) repeat in PABP2. Positiveimmunoreaction for polyclonal PABP2 was confined to the intranuclear ag-gregates of muscle fibers exclusively in patients with OPMD. Frequency ofthe nuclei positive for PABP2 (2%) was similar to that of ITFI detected byelectron microscopy (2.5%). There was no apparent relationship betweenthe frequency of PABP2-positive intranuclear aggregates and the severity ofmuscle fiber damage. In contrast, nuclear immunoreaction was not detectedin any samples from normal controls or from other neuromuscular diseases.These results suggest the presence of molecular modification of the productof expanded (GCG) repeat in PABP2, since the synthetic antigen peptidemay not recognize a highly dimethylated cluster of arginine residues of thenative PABP2, but may recognize the mutated form. Nuclear accumulationof expanded PABP2 product implies a causative role for ITFI.

© 2000 John Wiley & Sons, Inc. Muscle Nerve 23: 1549–1554, 2000

NUCLEAR ACCUMULATION OF EXPANDEDPABP2 GENE PRODUCT INOCULOPHARYNGEAL MUSCULAR DYSTROPHY

EIICHIRO UYAMA, MD,1 TOSHIFUMI TSUKAHARA, PhD, 2 KANAKO GOTO, BS, 2

YOSHIHIRO KURANO, PhD, 3 MEGUMU OGAWA, BS, 2 YEON-JEONG KIM, BS, 2

MAKOTO UCHINO, MD,1 and KIICHI ARAHATA, MD 2

1 Department of Neurology, Kumamoto University School of Medicine, 1-1-1 Honjo,Kumamoto 860-0811, Japan2 Department of Neuromuscular Research, National Institute of Neuroscience,Tokyo, Japan3 Fujirebio Central Research Institute, Tokyo, Japan

Accepted 5 May 2000

Oculopharyngeal muscular dystrophy (OPMD:OMIM 164300)15 is an autosomal dominant disorder

characterized by ptosis and dysphagia resulting fromsevere dystrophic involvement of the levator palpe-brae superioris and pharyngeal muscles.22,25,29 Clini-cal symptoms in most heterozygous cases25,26 mani-fest gradually after age 40, but those in homozygouscases start usually 10 years earlier.2 During thecourse of the latter disorder, extraocular and limbmuscles are also frequently affected, but less severelyso.3 Recently, mutational expansion of trinucleotide(GCG)6 repeat in the first exon of the poly(A) bind-ing protein 2 gene (PABP2) on chromosome 14q11to (GCG)8–13 has been identified in all OPMD pa-tients from 15 countries.5 PABP2 is known to bind

Abbreviations: AR-OPDM, autosomal recessively inherited oculopharyn-godistal myopathy; COMP, cartilage oligomeric matrix protein; DMRV,distal myopathy with rimmed vacuoles; DRPLA, dentatorubral-pallidoluysian atrophy; IBM, inclusion body myositis; ITFI, intranucleartubulofilamentous inclusions; KLH, keyhole lympet hemocyanin; PABP2,poly(A) binding protein 2 gene; PAIP, PABP-interacting protein; PSACH,pseudoachondroplasia; OMIM, Online Mendelian Inheritance in Man;OPMD, oculopharyngeal muscular dystrophyKey words: nuclear aggregates; oculopharyngeal muscular dystrophy;PABP2 immunoreactivity; poly(A) binding protein 2 gene; GCG repeatsCorrespondence to: E. Uyama; e-mail: [email protected]

© 2000 John Wiley & Sons, Inc.

PABP2 in Muscular Dystrophy MUSCLE & NERVE October 2000 1549

the pre-mRNA polyadenylation site in the 38 poly(A)tail, and regulates polyadenylation reaction andtranslational initiation of mRNA.13,14,17 The PABP2mRNA shows ubiquitous tissue distribution, withhigher expression in the skeletal muscle.5 The sub-cellular localization of PABP2 has been shown im-munocytochemically in the nucleus of HeLa cellsand in mouse hepatocytes as a diffuse or speckledstaining pattern, in the main associated with peri-chromatin fibrils, using native calf thymus PABP2 asan antigen.14

In skeletal muscle of patients with OPMD, char-acteristic intranuclear tubulofilamentous inclusions(ITFI) are found in 2.5% of the nuclei.24 The ITFI inOPMD have filaments of 8.5 nm outer diameter andthis is considered a morphological hallmark of thedisease.27 These ITFI are clearly different from thoseof inclusion body myositis (IBM),8 distal myopathywith rimmed vacuoles (DMRV),18 and autosomal re-cessively inherited oculopharyngodistal myopathy(AR-OPDM)28 that show filaments with a larger (15–18 nm) outer diameter.

To address the role of PABP2 in neuromusculardiseases, and to elucidate the molecular mechanism

underlying OPMD, we raised a specific antiserumagainst a synthetic peptide fragment predicted fromPABP2 cDNA, and examined the subcellular local-ization of PABP2 in skeletal muscle.

MATERIALS AND METHODS

Subjects. Five affected individuals from four unre-lated Japanese families with OPMD, 14 patients withother neuromuscular disorders, and three normalcontrols were studied with their informed consentand approval of our local ethics committee (Table1). OPMD was diagnosed based on the followingfindings: (1) drooping of the eyelids (ptosis) anddifficulty in swallowing (dysphagia) beginning afterthe age of 40; (2) normal function of the extraocularmuscles at an early stage; (3) ultrastructural obser-vations of biopsied skeletal muscle demonstratingITFI of 8.5 nm in outer diameter; (4) mutationalexpansion of a (GCG)n repeat in PABP2; and (5)inheritance as an autosomal dominant trait, or spo-radic occurrence. All five affected individuals sharedthese five cardinal features of OPMD, and cardiacmuscles were spared. Patient 2 was a daughter of asister of Patient 1, and both families lived in Kuma-

Table 1. Summary of the clinical and molecular genetic analyses of PABP2 in neuromuscular disease.*

Clinical diagnosis Age SexMode of

inheritanceMutation in

PABP2† MusclePABP2

expression

OPMD (Patient 126, 27) 62 F AD (GCG)11 Deltoid +++Sternohyoid +++Cricopharyngeal +

OPMD (Patient 227) 47 F AD (GCG)11 Biceps +OPMD (Patient 3) 61 F AD (GCG)11 Gastrocnemius ++OPMD (Patient 4) 81 M AD (GCG)8 Biceps +++OPMD (Patient 5) 63 M Sporadic (GCG)9 Gastrocnemius +++Distal myopathy with RV28 35 M AR Biceps −AR-OPDM28 48 M AR Biceps −Duchenne MD 10 M XR Biceps −Becker MD 33 M XR Quadriceps −Facioscapulohumeral MD 18 M AD Biceps −Emery-Dreifuss MD 41 M XR Quadriceps −Myotonic dystrophy 25 M AD Biceps −MELAS 17 M Biceps −Nemaline myopathy 20 F AD Biceps −Kennedy-Alter-Sung 45 M XR Biceps −DRPLA 62 M AD Biceps −Polymyositis 76 F Biceps −IBM 65 M Gastrocnemius −Hypothyroidism 44 F Biceps −Normal control 19 F Deltoid −Normal control 66 M Gastrocnemius −Normal control 61 F Biceps −

AD, autosomal dominant; AR, autosomal recessive; MD, muscular dystrophy; MELAS, mitochondrial encephalomyopathy, lactic acidosis, andstroke-like episodes; OPDM, oculopharyngodistal myopathy; OPMD, oculopharyngeal muscular dystrophy; RV, rimmed vacuole; XR, X-linkedrecessive.*Nuclear immunoreactivity in muscle fibers; positive with frequency indicated as follows: +++, 2.5–1.5%; ++, 1.5–0.5%; +, <0.5%; −, negative.†50 Japanese control individuals had (GCG)6/(GCG)6.

1550 PABP2 in Muscular Dystrophy MUSCLE & NERVE October 2000

moto Prefecture. Other patients originated from un-related families in Kumamoto (Patient 3), Tokyo(Patient 4), and Oita (Patient 5) Prefectures.

Mutation Analysis of PABP2 and Sequencing. Ge-nomic DNA was isolated from either peripheralblood lymphocytes or skeletal muscle samples, bystandard techniques. We analyzed PCR amplifiedproducts for PABP2 in each case, and additionally inanother 50 separate Japanese control individuals.PCR reactions contained PABP2 primers8 (0.1 µM),dNTPs (0.2 mM each), 2 × GC Buffer I (TaKaRa,Tokyo, Japan), 0.04 unit of Taq polymerase, and ge-nomic DNA (100 ng each) in a 25-µl reaction vol-ume. The forward primer was labeled with fluores-cent dye, Cy5 (Amersham Pharmacia Biotech Inc.,Piscataway, New Jersey). The amplified PCR prod-ucts were sequenced using a Thermo Sequence™Cycle sequence kit (Amersham) and ALFed Autose-quencer (Amersham). The number of the (GCG)nrepeats were also counted using a Fragment Man-ager Version 1.2 (Amersham).10

Development of PABP2 Antiserum. A peptide frag-ment (TNYNSSRSRFYSGFNSRPRGR) was synthe-sized using standard procedures,12 and conjugatedwith KLH (keyhole lympet hemocyanin). The pep-tide corresponded to amino acids 271–291 of thehuman PABP2 sequence where a cluster of post-translational arginine methylation occurs. Extra Cyswas added to the N-terminus. The peptide did notshow homology to any other known proteins. Poly-clonal antiserum was raised in a New Zealand whiterabbit in the usual way. The antiserum was affinity-purified from the serum using the peptide antigen.

Production of a PABP2 Fusion Protein. Full-lengthhuman PABP2 cDNA was made by PCR amplificationof an adult human skeletal muscle cDNA library atour laboratory through two steps. A 58 amplificationproduct (fusion protein 1: F-AAGAATTCGATGGC-GGCGGCGGCGGCGGCG, R-GCTTCTCTACCT-CGTTCTGTAGCT) and a 38 product (fusion pro-te in 2 : F -CCCGGAGCTGGAAGCTATCAA,R-CCTCCTCAGCAGTTAGTTATGG) were ligatedat the central overlapping sequences using an XhoIrestriction enzyme site, and was cloned into the GSTexpression vector pGEX-6P system (Amersham) us-ing EcoRI and NotI sites. The GST/PABP2 fusion pro-tein was produced in E. coli (BL21), and was followedby affinity purification for GST through a glutathi-one sepharose column.

Immunocytochemical Analysis of PABP2 Expres-sion. Frozen skeletal muscle biopsy specimens wereprocessed for cryosectioning (4 or 10 µm). In Patient1, bilateral sternohyoid and cricopharyngeal muscleswere obtained during cricopharyngeus myotomyprocedures. Indirect immunocytochemical stainingof biopsied human skeletal muscles with the antiserawas performed as described previously.1,9 Cryosec-tion samples were reacted for immunostaining toPABP2, or double reacted for immunostaining toPABP2 (polyclonal rabbit antiserum) and emerin(monoclonal mouse antibody). Emerin was used as aspecific marker for the inner nuclear mem-brane.16,31

Immunoblotting. Frozen muscle samples and affin-ity-purified PABP2 fusion protein were solubilized in20 volumes of SDS sample buffer (2% SDS, 0.125MTris-HCL buffer, pH6.8, 5% 2-mercaptoethanol,10% glycerol), and boiled for 5 min. After centrifu-gation, samples were loaded onto 8% polyacryl-amide gel for SDS-PAGE, and were electroblottedonto a nitrocellulose membrane, and incubated withaffinity-purified PABP2 antiserum. The blots werethen incubated with biotinylated secondary antibodyand developed in substrate solution using an ABCKit (Vector Laboratories, Burlingame, California).

RESULTS

A summary of our results is shown in Table 1. In allfive patients with OPMD, (GCG)n repeats encodinga poly(A) tract at the N-terminus of PABP2 wereheterozygously expanded to (GCG)6/(GCG)8,9,or 11.All of the 50 separate Japanese controls had homo-zygous (GCG)6 alleles. There were three differentgenotypes identified in the Japanese OPMD patients.

Immunocytochemical analysis of PABP2 expres-sion on skeletal muscle revealed a clearly positiveintranuclear immunoreaction against the PABP2 an-tiserum in the myonuclei in all five genetically-confirmed OPMD patients. In contrast, the nuclearimmunoreaction was not detected at all in samplesfrom the normal controls or the 14 other patientswith neuromuscular diseases which included IBM,DMRV, AR-OPDM, and triplet expansion diseasessuch as myotonic muscular dystrophy (CTG),Kennedy-Alter-Sung disease (CAG), and dentatoru-bral-pallidoluysian atrophy (DRPLA) (CAG) (Fig. 1,Table 1). The presence of PABP2 positive intra-nuclear aggregates was unexpectedly rare in severelyaffected cricopharyngeal muscle compared to that inmildly affected limb muscle. Furthermore, therewere no significant morphological differences be-tween the PABP2-positive and PABP2-negative


muscle fibers, even at an electron-microscopic level(data not shown).

On immunoblotting, fusion protein for PABP2showed the expected immunoreactive band (80kDa) against the anti-PABP2 antiserum. Preabsorp-tion of the antiserum with the peptide antigen andthe GST fusion protein abolished the immunoreac-tion, but GST alone did not (Fig 2). Immunoblottingfor skeletal muscle samples did not show any detect-able band in either OPMD patients or controls (datanot shown).

DISCUSSION

The discovery of the expanded trinucleotide repeatsin the PABP2 gene from all confirmed OPMD pa-tients5 implies a novel behavior different from pre-viously known triplet-repeats disorders characterizedby dynamic mutations.19 The GCG-repeat expansion(normal size: 6, mutant size: 8–13) encoding poly-alanine tract in OPMD is quite short and meioticallystable, contrasting with the CAG-repeat expansion(normal size: 10–35, mutant size: 40–120) diseases

such as Huntington’s disease, dentatorubral-pallidoluysian atrophy, and spinocerebellar ataxiatypes 1–3, 6, and 7.30 Recently, other short trinucleo-tide expansion mutations have been identified in thecartilage oligomeric matrix protein (COMP) gene inpatients with multiple epiphyseal dysplasia (MED:OMIM 132400) and pseudoachondroplasia(PSACH: OMIM 177170).7 In these autosomal dom-inant disorders, the common (GAC)5 wild-type se-quence is expanded to pathological (GAC)6 in pa-tients with MED, and that of patients with PSACH isexpanded to (GAC)7. The mutational mechanismfor short expansion is undetermined, but the occur-rence of slippage during replication has been sug-gested.5,7 Thus, there are at least three distinct dis-orders caused by short trinucleotide expansionmutations. Certainly, we identified three different(GCG)n short expansions of PABP2 in all affectedindividuals in the four Japanese OPMD families; two(GCG)6/(GCG)11, one (GCG)6/(GCG)9 and one(GCG)6 /(GCG)8 mutation. None of the samplesfrom the 50 Japanese controls showed mutationalexpansion of the (GCG)n repeat. The occurrence ofseveral different genotypes in OPMD patients in asingle community has not been clarified. In both theFrench-Canadian and Bukhara Jewish clusters,(GCG)9 mutations are causal, but haplotype analysis

FIGURE 1. Indirect immunocytochemical analysis of biopsied hu-man skeletal muscle for PABP2. Cryosections were double im-munoreacted for anti-PABP2 rabbit antiserum (green FITC) andanti-emerin monoclonal antibody (red rhodamin). Emerin wasused as a specific marker for the inner nuclear membrane(B,E,H). Note the clearly positive green immunostaining of intra-nuclear aggregates (arrows) in the myonuclei of patients withgenetically confirmed OPMD (A,C,G,I). (C) and (I) are doubleexposures of (A,B) and (G,H), respectively. In contrast, there wasno immunostaining of myonuclei for PABP2 in IBM (D,F) or otherneuromuscular diseases. (F) is a double exposure of (D) and (E).Coarse yellowish granules in (A), (D), and (G) are autofluores-cent materials in muscle. Preabsorption of the primary antiserumwith synthetic peptide or fusion protein for PABP2 abolished theimmunoreaction, and preimmune rabbit serum showed negativestaining (data not shown). Bar = 40 µm (A–F); 10 µm (G–I).

FIGURE 2. Immunoblot analysis for PABP2 fusion protein. Anti-PABP2 antiserum (crude PABP2) showed an expected 80 kDaimmunoreactive band, while preabsorption of the antiserum withantigen peptide (Abs. peptide) and GST fusion protein (Abs.GST-PABP2) abolished the immunoreaction, but GST alone(Abs. GST) did not.


indicates the different historical origins of thesepopulations.2 Our results also indicate the presenceof independent mutations of PABP2 in JapaneseOPMD families. Although a relatively small numberof French-Canadian founders were estimated in thatresearch, we suggest that there are more founders inthe Japanese situation.

In the present study, we have demonstrated thesubcellular localization of a mutated PABP2 productin the skeletal muscle from patients with OPMD, us-ing a specific antiserum raised against a syntheticpeptide fragment predicted from PABP2 cDNA.Prominent immunoreaction for PABP2 was foundwithin the myonuclei in all patients with OPMD ascaused by mutational expansion of PABP2, but noneof the patients with other neuromuscular diseases orthe normal controls showed such immunoreaction.Recent research has clarified that the C-terminus ofthe PABP2 protein of native calf thymus containedthe methylated arginine at 13 positions.21 In con-trast, in the region of the PABP2 corresponding toour synthetic antigen peptide (amino acids 271–291), all arginine residues were dimethylated. Ourantiserum raised against a synthetic peptide frag-ment may not recognize active forms of PABP2 pro-tein due to the presence of a possible posttransla-tional arginine modification.

We believe that the aggregated substances in thenuclei of OPMD muscles may contain expandedPABP2 product, which would be biochemically orstructurally altered from normal PABP2 protein. In-deed, the antiserum exclusively detects mutated anddenatured PABP2, but not the native protein, sincepositive immunoreaction as detected immunocyto-chemically by our antiserum appeared exclusively inOPMD muscle and in fusion proteins in immunob-lotting. PABP2 immunoblotting appears to be lesssensitive than immunocytochemistry in detectinglow levels of PABP2. Aggregates of the mutatedPABP2 product within the myonuclei may becomemore stable and enriched in the nuclei, which canthen be easily detected by the antiserum. The fre-quency of ITFI-positive nuclei in OPMD is less than5%. Nonetheless, specificity (100%) and sensitivity(100%) of the antiserum is evident in detecting theintranuclear aggregates in patients with OPMD. Pre-viously performed immunocytochemical studies us-ing antibodies against filamentous proteins such asubiquitin, desmin, vimentin, myosin, titin, lamins,keratins, neurofilaments, nebulin, and tubulin havefailed to label ITFI.25 Subsequent immunoelectronmicroscopic studies with antibodies against lamins A,B, and C were also negative.23 To our knowledge,our study is the first demonstration of positive im-

munoreactivity for accumulated substance in the nu-clei of OPMD muscle.

In particular, the frequency of the PABP2-positive myonuclei in our confirmed OPMD muscle(2%) was similar to that of ITFI as detected by elec-tron microscopy (2.5%).26 Interestingly, homozy-gotes for OPMD have significantly larger numbers ofITFI (9.4 versus 4.9% in heterozygotes).2 Further-more, the appearance of the PABP complex forma-tion with poly(A) RNA on atomic force microscopyseems to resemble that of ITFI on electron micros-copy.20 This evidence leads us to believe that thespecific accumulation of an expanded PABP2 prod-uct in the nuclei of OPMD skeletal muscle, implies acausative role for ITFI in the generation of progres-sive muscle fiber damages. The common pathogen-esis of autosomal dominant disorders indicates that again in the function of PABP2 may cause the accu-mulation of ITFI, probably associated with a mutatedPABP2 product. Intranuclear inclusions or aggre-gates of defective gene products have already beenidentified in other hereditary neuro-degenerativediseases and are caused by unstable expansion ofCAG repeats coding for polyglutamine stretches. Evi-dence increasingly indicates that expanded polyglu-tamine tends to self-aggregate.11 It has been sug-gested that the pathologic expansions of thepolyalanine tract, from the normal 10 alanines to12–17 alanines, may lead to formation of insolublefilaments which accumulate in the nucleus.5 Mam-malian pre-mRNA 38 end-processing components in-clude poly(A) polymerase, cleavage-polyadenylationspecificity factor, cleavage-stimulatory factor, andcleavage factors in addition to PABP2. To our knowl-edge, no relation has been shown between muta-tions of the genes of these components and nuclearaggregates except for PABP2.

As shown in our results, there was no apparentrelationship between the frequency of PABP2-positive intranuclear aggregates and the severity ofmuscle fiber damage in OPMD. Furthermore, a re-cent study in French-Canadian families with OPMDfailed to explain the clinical severity-genotype rela-tionship by analyses of PABP2.4 This evidence led usto a speculation that additional factors that interactwith PABP2 and/or the PABP2 gene, such as pre-mRNA 38 end-processing complex13 or PABP-interacting protein (PAIP),6 generate the selectivityand severity of progressive dysfunction occurring inaffected muscles, and the late-onset nature of thedisease. Further attention must be paid to molecularbiological effects of the intranuclear aggregates inOPMD muscle as caused by the abnormal (GCG)nexpansion of PABP2.


We thank Dr. Fernando M.S. Tome for helpful comments and Ms.Shizuno Okamoto for excellent assistance in preparing musclespecimens. We also thank Dr. Kerry Greer for reviewing the manu-script. This work was supported, in part, by Grants-in-Aid for Sci-entific Research (C), to E.U., and for a Center of Excellence(COE) to K.A. from the Ministry of Health and Welfare, Japan.

REFERENCES

1. Arahata K, Ishiura S, Ishiguro T, Tsukahara T, Suhara Y, Egu-chi C, Ishihara T, Nonaka I, Ozawa E, Sugita H. Immuno-staining of skeletal and cardiac muscle surface membranewith antibody against Duchenne muscular dystrophy peptide.Nature 1988;333:861–863.

2. Blumen SC, Brais B, Korczyn AD, Medinsky S, Chapman J,Asherov A, Nisipeanu P, Codere F, Bouchard JP, Fardeau M,Tome FM, Rouleau GA. Homozygotes for oculopharyngealmuscular dystrophy have a severe form of the disease. AnnNeurol 1999;46:115–118.

3. Bouchard J-P, Brais B, Brunet D, Gould PV, Rouleau GA.Recent studies on oculo-pharyngeal muscular dystrophy inQuebec. Neuromuscul Disord 1997;7(suppl 1):S22–S29.

4. Brais B, Bouchard J-P, Tome FMS, Fardeau M, Codere F,Duranceau A, Rouleau GA. Genetic evidence for the involve-ment of other genes in modulating the severity of oculopha-ryngeal muscular dystrophy. Ann Neurol 1998;44:455.

5. Brais B, Bouchard JP, Xie YG, Rochefort DL, Chretien N,Tome FMS, Lafreniere RG, Rommens JM, Uyama E, NohiraO, Blumen S, Korczyn AD, Heutink P, Mathieu J, DuranceauA, Codere F, Fardeau M, Rouleau GA. Short expansions in thePABP2 gene cause oculopharyngeal muscular dystrophy. NatGenet 1998;18:164–167.

6. Craig AWB, Haghighat A, Yu ATK, Sonenberg N. Interactionof polyadenylate-binding protein with the elF4G homologuePAIP enhances translation. Nature 1998;392:520–523.

7. Delot E, King LM, Briggs MD, Wilcox WR, Cohn DH. Tri-nucleotide expansion mutations in the cartilage oligomericmatrix protein (COMP) gene.Hum Mol Genet 1999;8:123–128.

8. Griggs RC, Askanas V, DiMauro S, Engel A, Karpati G, Men-dell JR, Rowland LP. Inclusion body myositis and myopathies.Ann Neurol 1995;38:705–713.

9. Hayashi YK, Chou FL, Engvall E, Ogawa M, Matsuda C, Hi-rabayashi S, Yokochi K, Ziober BL, Kramer RH, Kaufman SJ,Ozawa E, Goto Y, Nonaka I, Tsukahara T, Wang JZ, HoffmanEP, Arahata K. Mutations in the integrin a7 gene cause con-genital myopathy. Nat Genet 1998;19:94–97.

10. He L, Mansfield DC, Brown AF, Green DK, Morris SW, StClair DM, Muir WJ, Maclean A, Wright AF, Blackwood DH.Automated linkage analysis in psychiatric disorders. Am J MedGenet 1995;60:192–198.

11. Huq AM, Hackam A, Graham RK, Wellington CCL, HaydenMR. Molecular pathogenesis of Huntington’s disease: bio-chemical studies of Huntington. In: Wells RD, Warren ST,editors. Genetic instabilities and hereditary neurological dis-eases. San Diego: Academic Press; 1998. p 325–354.

12. Ishiura S, Arahata K, Tsukahara T, Koga R, Anraku H,Yamaguchi M, Kikuchi T, Nonaka I, Sugita H. Antibodyagainst the C-terminal protein of dystrophin crossreacts withthe 400 kDa protein in the pia mater of dystrophin-deficientmdx mouse brain. J Biochem 1990;107:510–513.

13. Keller W. No end yet messenger RNA 38 processing! Cell1995;81:829–832.

14. Krause S, Fakan S, Weis K, Wahle E. Immunodetection ofpoly(A) binding protein II in the cell nucleus. Exp Cell Res1994;214:75–82.

15. McKusick-Nathans Institute for Genetic Medicine, Johns Hop-kins University (Baltimore, MD) and National Center for Bio-technology Information, National Library of Medicine(Bethesda, MD). Online Mendelian inheritance in man,OMIM. 2000; World Wide Web URL: http://www.ncbi.nlm.nih.gov/omim/

16. Nagano A, Koga R, Ogawa M, Kurano Y, Kawada J, Okada R,Hayashi YK, Tsukahara T, Arahata K. Emerin deficiency at thenuclear membrane in patients with Emery-Dreifuss musculardystrophy. Nat Genet 1996;12:254–259.

17. Nemeth A, Krause S, Blank D, Jenny A, Jeno P, Lustig A,Wahle E. Isolation of genomic and cDNA clones encodingbovine poly(A) binding protein II. Nucleic Acids Res 1995;23:4031–4041.

18. Nonaka I, Sunohara N, Ishiura S, Satoyoshi E. Familial distalmyopathy with rimmed vacuoles and lamellar (myeloid) bodyformation. J Neurol Sci 1981;51:141–155.

19. Richards RI, Sutherland GR. Dynamic mutations: a new classof mutations causing human disease. Cell 1992;70:709–712.

20. Smith BL, Gallie DR, Le H, Hansma PK. Visualization ofpoly(A)-binding protein complex formation with poly(A)RNA using atomic force microscopy. J Struct Biol 1997;119:109–117.

21. Smith JJ, Rucknagel KP, Schierhorn A, Tang J, Nemeth A,Linder M, Herschman HR, Wahle E. Unusual sites of argininemethylation in poly(A)-binding protein II and in vitro meth-ylation by protein arginine methyltransferases PRMT1 andPRMT3. J Biol Chem 1999;274:13229–13234.

22. Taylor EW. Progressive vagus-glossopharyngeal paralysis withptosis: a contribution to the group of family diseases. J NervMent Dis 1915;42:129–139.

23. Tome FMS, Chateau D, Helbling-Leclerc A, Fardeau M. Mor-phological changes in muscle fibers in oculopharyngeal mus-cular dystrophy. Neuromuscul Disord 1997;7(suppl 1):S63–S69.

24. Tome FMS, Fardeau M. Nuclear inclusions in oculopharyn-geal dystrophy. Acta Neuropathol 1980;49:85–87.

25. Tome FMS, Fardeau M. Oculopharyngeal muscular dystro-phy. In: Engel AG, Franzini-Armstrong C, editors. Myology,2nd ed. New York: McGraw-Hill; 1994. p 1233–1245.

26. Uyama E, Nohira O, Chateau D, Tokunaga M, Uchino M,Okabe T, Ando M, Tome FMS. Oculopharyngeal musculardystrophy in two unrelated Japanese families. Neurology1996;46:773–778.

27. Uyama E, Nohira O, Tome FMS, Chateau D, Tokunaga M,Ando M, Maki M, Okabe T, Uchino M. Oculopharyngeal mus-cular dystrophy in Japan. Neuromuscul Disord 1997;7(suppl1):S41–S49.

28. Uyama E, Uchino M, Chateau D, Tome FMS. Autosomal re-cessive oculopharyngodistal myopathy in light of distal myop-athy with rimmed vacuoles and occulopharyngeal musculardystrophy. Neuromuscul Disord 1998;8:119–125.

29. Victor M, Hayes R, Adams RD. Oculopharyngeal muscle dys-trophy: a familial disease of late life characterized by dyspha-gia and progressive ptosis of the eyelids. N Engl J Med 1962;267:1267–1272.

30. Wilmot GR, Warren S. A new mutational basis for disease. In:Wells RD, Warren ST, editors. Genetic instabilities and he-reditary neurological diseases. San Diego: Academic Press;1998. p 3–12.

31. Yorifuji H, Tadano Y, Tsuchiya Y, Ogawa M, Goto K, UmetaniA, Asaka Y, Arahata K. Emerin, deficiency of which causesEmery-Dreifuss muscular dystrophy, is located at the innernuclear membrane. Neurogenetics 1997;1:135–140.


nuclear accumulation of expanded pabp2 gene product in oculopharyngeal muscular dystrophy

Documents