Journal of Clinical Neuroscience 18 (2011) 674–677

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Journal of Clinical Neuroscience

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Clinical Study

Mutation and haplotype analysis of oculopharyngeal muscular dystrophy inThai patients

T. Pulkes ⇑, C. Papsing, M. Busabaratana, C. Dejthevaporn, R. WitoonpanichDivision of Neurology, Department of Medicine, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, 270 Rama VI Road, Bangkok 10400, Thailand

a r t i c l e i n f o a b s t r a c t

Article history:Received 6 March 2010Accepted 2 August 2010

Keywords:Oculopharyngeal muscular dystrophyPABPN1 geneTrinucleotide repeat

0967-5868/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.jocn.2010.08.020

⇑ Corresponding author. Tel./fax: +66 22011386.E-mail address: ratpk@mahidol.ac.th (T. Pulkes).

Oculopharyngeal muscular dystrophy (OPMD) is an inherited neuromuscular disease associated with ashort trinucleotide repeat expansion in Exon 1 of the PABPN1 gene. OPMD is uncommon in East Asianpopulations, and there have been no previous reports of Thai patients. We studied clinical and moleculargenetic features of six unrelated Thai patients with autosomal dominant OPMD. All patients had expan-sions of the guanine–cytosine–guanine (GCG) repeat ranging from three to seven additional repeats inthe PABPN1 gene. Haplotype analysis showed that these mutations might have originated independently.Analysis of the size of the GCG repeat in the PABPN1 gene in 200 Thai control patients showed that 0.5% ofthe control subjects possessed (GCG)7, thereby suggesting that the prevalence of autosomal recessiveOPMD in the Thai population was approximately 1 in 160,000. In conclusion, our data suggest that OPMDin Thailand may be more common than previously thought.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Oculopharyngeal muscular dystrophy (OPMD) is an inheritedneuromuscular disease that typically manifests with late-onsetprogressive ptosis, dysphagia, ophthalmoparesis and proximallimb weakness.1 Symptoms often develop in the fifth or sixth dec-ade of life. Most patients have a normal life expectancy. However,almost one-third of patients have significant disabilities later in lifeand usually require esophageal dilatation or feeding through a gas-trostomy tube.1,2 A characteristic pathological feature of OPMD isthe presence of intranuclear inclusions in some muscle fibres.These inclusions are accumulations of tubular filaments (�8.5 nmin diameter).3 An underlying genetic cause of OPMD is a shortGCG repeat expansion in Exon 1 of the polyadenylate bindingprotein nuclear 1 gene (PABPN1, previously called poly(A) bindingprotein 2, or PABP2) on chromosome 14q11.4 Early reports showedthat the normal (GCG)6 allele expanded to a pathological (GCG)8–13

allele. These expansions are different from the triplet repeatexpansions of other inherited neurological diseases in that theGCG repeat expansions are short and stable through meiosis andmitosis. Further reports subsequently identified that OPMD wasnot only associated with expansions of the GCG repeat, but alsowith several different mutations consisting of additional guan-ine–cytosine–adenine (GCA) interspersions.5,6 These data suggestthat the molecular pathogenesis of OPMD might be related tohomologous unequal crossing-over or replication slippage.4–6

ll rights reserved.

Although most patients inherit OPMD through autosomal dom-inant transmission, autosomal recessive OPMD has occasionallybeen described in association with a homozygous single GCGexpansion, (GCG)7.4,6,7 Prevalence of the (GCG)7 allele is estimatedto be about 1% to 2% in French Canadians and European popula-tions, which is theoretically equivalent to an incidence of 0.25–1in 10,000 of autosomal recessive OPMD in those populations. Therelative rarity of autosomal recessive OPMD may result from thelack of family history or atypical phenotypes.7

OPMD has been identified in patients worldwide. However,there is a vast diversity of prevalence between different popula-tions. The highest prevalence has been reported in French Canadi-ans in Quebec, Bukhara Jews in Israel and Hispanic Americans inthe southwest of the USA.2,8,9 The common occurrence of OPMDamong these populations is suggested to be caused by a founder ef-fect within their populations.10,11 In contrast to the French Cana-dian population, OPMD disease in Caucasian patients from othercountries were likely to originate independently.1,12

OPMD appears to be less prevalent among people in Asia.13 Onlya few patients have been reported from families in Japan, Korea,Taiwan and Malaysia, suggesting that OPMD is rare in East Asianpopulations.5,13–17 To our knowledge, OPMD has not been de-scribed in the Thai population. Herein, we report the first six unre-lated Thai families with OPMD. We studied clinical features,electrophysiological findings and the GCG repeat length of the PAB-PN1 gene in these patients. We also analysed the GCG repeat alleleof the PABPN1 gene in a large number of control samples to esti-mate the prevalence of autosomal recessive OPMD in the Thaipopulation.

T. Pulkes et al. / Journal of Clinical Neuroscience 18 (2011) 674–677 675

2. Patients and methods

2.1. Patients

For this study, we recruited 13 unrelated patients with a prob-able clinical diagnosis of OPMD from the neurology clinic at Rama-thibodi Hospital, Mahidol University. The diagnosis of thesepatients was subsequently confirmed by PABPN1 analysis. Clinicaldetails of the OPMD patients were reviewed from their medical re-cords. For screening of the size of the triplet repeats of the PABPN1gene, blood samples were taken from 200 control Thai patientsfrom an outpatient clinic at the Department of Medicine, Ramathi-bodi Hospital, who had no genetic or neurological disease. Thisstudy was approved by the Ethical Clearance Committee on HumanRights Related to Research Involving Human Subjects, Faculty ofMedicine, Ramathibodi Hospital, Mahidol University (ID 03-51-49). All participants gave both verbal and written informedconsent.

2.2. Genetic analysis

Genomic DNA samples were extracted from peripheral bloodsamples by standard techniques. Fluorescent polymerase chainreaction (PCR)-based screening of the expanded GCG repeats ofthe PABPN1 gene was performed as previously described.1 The oli-gonucleotide primer pairs included: fluorescently labelled forwardprimer 50-CCAGTGCCCCGCCTTAGA-30; and the reverse primer 50-ACAAGATGGCGCCGCCGCCCCGGC-30. We analysed the size of thePCR products using an Applied Biosystems ABI 3100 DNA sequen-cer and Genescan software (Applied Biosystems, Foster City. CA,USA).

To determine the expanded sequence of the mutant alleles, weperformed automated direct sequencing of expanded alleles afterobtaining a larger PCR product from a 3% NuSieve� gel (Lonza,Rockland, ME, USA). The forward primer for sequencing was not la-belled with fluorescent material. Direct sequencing was performedusing a Bigdye Terminator sequencing kit (Applied Biosystems)and with an ABI 3100 DNA sequencer.

Table 1Clinical features, electrophysiological findings, PABPN1 analysis and haplotype analysis of

Patient no.: 1 2

Age at onset (yrs) 53 60Sex M FDuration (yrs) 7 6Clinical manifestations

Ptosis Mild MildOphthalmoparesis – +Diplopia – –Dysphagia – +Facial weakness – –Limb weakness – –Family history AD –Swallowing time (s)� 7 16

Electrophysiological findingsSingle fibre EMG NA NARepetitive nerve stimulation NA +b

PABPN1 analysis* 10 (4) 12 (6)Haplotype analysis

D14S283 135, 143 129, 137D14S990 137, 145 147, 149D14S1041 108, 112 108, 110

A = adenine, AD = autosomal dominant, C = cytosine, EMG = electromyogram, F = female,+a = increased jitter in >10% of the potential pairs and the mean of mean consecutive di+b = decremental response.� Swallowing time is the length of time required to drink 80 mL of ice cold water.4* Numbers show the expansion from the normal six GCG of the (GCG)6 repeat segment iexpressed as the additional numbers of triplet repeats.

All patients were genotyped using three microsatellite markerslinked to the OPMD locus including D14S283, D14S990 andD14S1041.18 The allele sizes were determined by fluorescently la-belled PCR amplification. PCR product size analysis was performedwith a Beckman CEQ 8800 genetic analysis system (Beckman Coul-ter, Brea, CA, USA).

3. Results

3.1. Clinical and electrophysiological features

In six out of the 13 patients studied, short expansions of thetriplet repeat in the PABPN1 gene were identified. The sevenremaining patients were reviewed and further investigated. Theirfinal diagnoses were myasthenia gravis (two patients), mitochon-drial myopathy (chronic progressive external ophthalmoplegia,two patients), autosomal dominant progressive external ophthal-moplegia (one patient), oculopharyngodistal myopathy (one pa-tient) and one unclassified diagnosis. In the patient with theunclassified diagnosis, Exon 1 of the PABPN1 gene was also ana-lysed by direct sequencing because a missense mutation in thisexon has been identified in association with typical OPMD.19 How-ever, we could not identify any point mutations in this patient.

Clinical features of all patients with OPMD are summarised inTable 1. Most patients had a family history of marked ptosis intwo or more additional family members in at least two genera-tions, which is consistent with autosomal dominant inheritance.Only one patient (patient 2) was unable to provide a positive fam-ily history. Her parents died at the age of about 60 years and 65years without symptoms of obvious ptosis or dysphagia. She wasunmarried and her only brother possessed normal homozygous(GCG)6 alleles. The disease onset usually commenced at 40 yearsof age. The earliest-onset patient (19 years old) had the largestexpansion of GCG repeats. All patients initially developed chronicprogressive ptosis often followed by dysphagia. Although two pa-tients did not experience difficulty swallowing, one of them hadprolonged swallowing time upon examination. Extraocular muscle

patients with oculopharyngeal muscular dystrophy.

3 4 5 6

40 55 54 19M M M M28 5 10 7

Severe Moderate Mild Moderate– –– + – –+ – + –+ + – –+ + – –AD AD AD AD24 18 15 12

+a +a NA +a

+b Normal NA Normal10 (4) 10 (4) 9 (3) 13 (7)

129, 143 133, 135 123, 143 133, 135131, 149 143, 149 131, 137 147, 147108, 108 108, 114 108, 108 108, 114

G = guanine, M = male, NA = not available, + = present, – = absent.fference >37 ls.

n the (GCG)6(GCA)3(GCG)1 sequence of the PABPN1 gene, and, in parentheses, this is

676 T. Pulkes et al. / Journal of Clinical Neuroscience 18 (2011) 674–677

weakness was observed in one-third of patients. However, thosepatients did not experience diplopia. Facial weakness was alsocommon, and it was identified in one-third of the patients. Onlyone of our patients (patient 3) had a severe phenotype with somelimitation of daily activities. At 68 years of age, this patient devel-oped marked dysphagia, dysphonia and proximal limb weakness.However, he did not require feeding via a gastrostomy tube or cri-copharyngeal myotomy. He had suffered from the disease for 28years, whereas other patients had disease durations of 10 yearsor fewer. All other patients were able to independently maintaintheir daily activities. Only one of these patients required blepharo-plasty due to ptosis, which interfered with his vision.

Single fibre electromyograms (EMG) in the extensor digitorumcommunis muscle was carried out in three patients. All of these pa-tients demonstrated increased jitter in over 10% (65%, 45% and20%) of the potential pairs, and an increased mean of the meanconsecutive differences over 37 ls (65, 56 and 54 ls). A repetitivenerve stimulation test at 3 Hz was performed in four patients, twoof whom had a decremental response.

3.2. Genetic analysis

PABPN1 analysis showed that six patients had trinucleotide re-peat expansions in Exon 1, ranging from three to seven additionalrepeats (Table 1). Sequencing analysis revealed that the mutationsof all of the patients possessed (GCG) repeat expansions with no(GCA) interspersion. The most common mutation was a (GCG)10 re-peat expansion, which was identified in four of six patients (two-thirds). Haplotype analysis suggested that there might be at leastfive different haplotypes. Only patients 2 and 3 may theoreticallyshare the same haplotype (Table 1). However, they had differentmutations [(GCG)12 and (GCG)10], suggesting that their diseasedid not originate from the same founder.

Screening of the single GCG repeat expansion in 200 controlThai subjects revealed that only one (0.5%) had a (GCG)7 repeat.

4. Discussion

Six unrelated Thai patients were newly diagnosed by geneticanalysis as having OPMD. This finding suggests that OPMD maybe more prevalent than previously thought. OPMD is often a late-onset disorder with slowly progressive ptosis followed by difficultyswallowing. These symptoms may lead to a misdiagnosis for otherneuromuscular diseases such as myasthenia gravis or chronic pro-gressive external ophthalmoplegia. Furthermore, electrophysiolog-ical studies, including single fibre EMG and a repetitive nervestimulation test, may not be able to distinguish OPMD from morecommon neuromuscular diseases.20 Unusual features such asyoung onset, profound ophthalmoplegia, a high serum level of cre-atine kinase and the lack of a family history may also be factorsthat lead to misdiagnosis.1 Some OPMD patients have relativelymild symptoms that do not affect routine daily activities. Those pa-tients may not seek medical advice, and thus many more patientswith OPMD have yet to be diagnosed. Recognition of classical clin-ical features, paired with the availability of genetic testing for thePABPN1 gene, may help to identify these undiagnosed patients.

OPMD was thought to be a rare neuromuscular disease in EastAsian populations.13,15–17 In contrast to Thailand, OPMD has beenwell studied in Japan for over one decade. To our knowledge, therehave been only seven Japanese families with OPMD reported todate, suggesting that OPMD in Japanese society is likely to berare5,13,14,21,22 There were only a few families with OPMD reportedfrom Malaysia, Korea and Taiwan.15–17 All patients in this studylived in Bangkok or a nearby province. None of the studied patientswere referred from other hospitals. There were about 200,000

people registered to use our hospital’s medical services in theNational Health Service system. However, these people came fromseveral different areas in Bangkok and nearby provinces. Moreover,people in a small neighborhood may register to several differenthospitals. Therefore, we made no attempt to calculate the preva-lence of OPMD from our hospital-based data. Our finding of sixnew unrelated Thai patients with OPMD in a small population ofBangkok and nearby provinces possibly suggests that OPMD is morecommon in Thailand than other countries in the East Asian region.

Although half of the studied patients harboured the same(GCG)10 mutation, data from the haplotype analysis suggestedthat all patients were more likely to inherit these mutationsindependently from different ancestors. This is in contrast tocountries where there is a high prevalence of OPMD becausemost patients from those countries often shared a commonancestral founder for the disease.2,8,9 The GCG repeat expansionin the PABPN1 gene is short and stable during meiosis and mito-sis. Mutations sometimes consist of GCA interspersions, suggest-ing that unequal crossing-over might lead to molecularpathogenesis.4–6 These data led us to directly sequence Exon 1of the PABPN1 gene. We did not identify GCA interspersions inthe expanded alleles in any of the studied patients. However,the sequencing data confirmed the accuracy of the Genescantesting for OPMD in our laboratory.

We have shown that 0.5% of the studied Thai subpopulationpossesses an expanded (GCG)7 in the PABPN1 gene. Therefore,prevalence of autosomal recessive OPMD (a homozygous (GCG)7

repeat carrier) in this Thai subpopulation was 1 in 160,000. Be-cause autosomal recessive OPMD with a homozygous (GCG)7

expansion often exhibits mild phenotypes with a later age of on-set,6,7 it is not surprising that autosomal recessive OPMD is rareamong Thais.

In summary, we have described the molecular genetic analysisof OPMD in six unrelated Thai patients and suggest that OPMDmay be more common in Thailand than previously thought.

Acknowledgements

This study was supported by the Neurogenetics Fund (3001180)from the Ramathibodi Foundation.

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