Oculopharyngeal Muscular Dystrophy: Phenotypic and Genotypic Studies in a Chinese Population

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  • ORIGINAL PAPER

    Oculopharyngeal Muscular Dystrophy: Phenotypic and GenotypicStudies in a Chinese Population

    Jingli Shan Bin Chen Pengfei Lin

    Duoling Li Yuebei Luo Kunqian Ji

    Jinfan Zheng Yun Yuan Chuanzhu Yan

    Received: 26 April 2014 / Accepted: 9 September 2014 / Published online: 5 October 2014

    Springer Science+Business Media New York 2014

    Abstract Oculopharyngeal muscular dystrophy (OPMD)

    is an autosomal dominant late-onset neuromuscular

    degenerative disease characterized by ptosis, dysphagia,

    and proximal muscle weakness. The genetic basis has been

    identified as an abnormal (GCN) expansion encoding the

    polyalanine tract in exon 1 of the polyadenylate-binding

    protein nuclear 1 gene (PABPN1). OPMD is worldwide

    distributed, but has rarely been reported in East Asians. In

    this study, we summarized the clinical and genetic char-

    acteristics of 34 individuals from 13 unrelated families in

    Chinese population. In our cohort, the mean age at onset

    was 47.2 years. Dysphagia, rather than ptosis, was the most

    common initial symptom. Genetically, we identified seven

    genotypes in our patients, including one compound het-

    erozygote of (GCN)11/(GCN)12. The genetic heterogeneity

    implies that there is no single founder effect in Chinese

    population, and our data also support that the (GCN)11

    polymorphism may have a disease-modifying effect.

    Additionally, the clinical features showed homogeneity

    within families, which suggests that other genetic factors

    apart from the already known genotype also play a role in

    modifying the phenotype.

    Keywords Oculopharyngeal muscular dystrophy OPMD PABPN1 (GCN) expansion

    Introduction

    Oculopharyngeal muscular dystrophy (OPMD) is a late-

    onset neuromuscular disorder characterized by progressive

    ptosis, dysphagia, and proximal muscle weakness, with

    unique nuclear filament inclusions in skeletal muscle fibers

    as its pathological hallmark (Tome and Fardeau 1980). The

    underlying cause is the abnormal (GCN) expansion or, more

    rarely, point mutations leading to a lengthening of the tract in

    exon 1 of the polyadenylate-binding protein nuclear 1 gene

    (PABPN1) on chromosome 14q11 (Brais et al. 1998; Rob-

    inson et al. 2006). The normal allele is (GCG)6(GCA)3(GCG)1 encoding 10 alanines (Ala10) (Brais et al. 1998).

    Jingli Shan and Bin Chen have contributed equally to this work.

    Electronic supplementary material The online version of thisarticle (doi:10.1007/s12017-014-8327-5) contains supplementarymaterial, which is available to authorized users.

    J. Shan P. Lin D. Li K. Ji J. Zheng C. YanLaboratory of Neuromuscular Disorders and Department of

    Neurology, Qilu Hospital, Shandong University, Jinan 250012,

    China

    B. Chen

    Department of Neurology, Beijing Tiantan Hospital,

    Beijing 100050, China

    Y. Luo

    Center for Neruomuscular and Neurological Disorders,

    Australian Neuro-Muscular Research Institute, University of

    Western Australia, Perth 6007, Australia

    Y. Yuan (&)Department of Neurology, Peking University First Hospital,

    8 Xishiku St, Xicheng District, Beijing 100034, China

    e-mail: yuanyun2002@126.com

    C. Yan (&)Key Laboratory for Experimental Teratology of the Ministry of

    Education, Brain Science Research Institute, Shandong

    University, Jinan 250012, China

    e-mail: chuanzhuyan@163.com

    123

    Neuromol Med (2014) 16:782786

    DOI 10.1007/s12017-014-8327-5

    http://dx.doi.org/10.1007/s12017-014-8327-5

  • PABPN1 variants were first described as pure (GCG)

    expansions of the normal (GCG)6 stretch (Brais et al. 1998).

    Subsequently, additional (GCA) interspersions within

    (GCG) expansions were reported in various populations

    (Muller et al. 2006; Nakamoto et al. 2002). Now, (GCN)1217has been widely accepted as the autosomal dominant form

    (Brais 2009). The extremely uncommon autosomal recessive

    form of OPMD has been shown to result from the homozy-

    gosity of (GCN)11 expansion, which was referred to as

    (GCG)7 previously (Brais et al. 1998). This (GCN)11 poly-

    morphism, with a prevalence of 12 % in North America,

    Europe, and Japan, is also considered as a phenotype modi-

    fier (Brais 2009; Brais et al. 1998).

    OPMD has a worldwide distribution, particularly pre-

    valent in the French Canadian population (1:1,000) and in

    Bukhara Jews living in Israel (1:600) (Brais 2009). The

    clusters of OPMD cases identified in various populations

    recently make it possible to explore the phenotypegeno-

    type correlation. However, OPMD appears to be sparse

    among East Asians, with most families being reported in

    Japan (Uyama et al. 1996, 1997; Nagashima et al. 2000;

    Nakamoto et al. 2002). Thus far, only rare cases have been

    reported in mainland China (You et al. 2010; Ye et al.

    2011). Taiwan, sharing a similar genetic background, has a

    higher prevalence rate (Kuo et al. 2009; Huang et al. 2010).

    Additionally, at least 3 Chinese immigrants, originally

    from southern China, have been reported to have OPMD

    (Lim et al. 1992; Goh et al. 2005; Ruegg et al. 2005).

    Therefore, it has been suspected that OPMD is being

    under-diagnosed in mainland China. Here, we report the

    clinical and genetic characteristics of 34 OPMD patients

    from 13 unrelated families in northern China.

    Materials and Methods

    All 34 subjects from 13 unrelated families were recruited

    through the Neurology department of Qilu Hospital of

    Shandong University and Peking University First Hospital.

    All probands and some of their relatives were analyzed for

    (GCN) expansion in exon 1 of the PABPN1 gene by

    Kingmed Diagnostics, Guangzhou, according to standard

    protocol (Jiahui et al. 2005), and each of them carried an

    expanded (GCN) repeat.

    Nerve conduction studies (NCS) and needle electromy-

    ography (EMG) were performed in 10 of 13 probands.

    Repetitive nerve stimulation (RNS) test was performed in 8

    probands. Muscle biopsy was carried out in probands from

    each family under local anesthesia. Muscle specimens for

    histological examination were frozen in isopentane that

    was precooled in liquid nitrogen and stored at -80 C.Serial frozen Sects. (8 lm) were stained with hematoxylinand eosin (H & E), modified Gomori trichrome,

    nicotinamide adenine dinucleotide tetrazolium dehydroge-

    nase, succinate dehydrogenase, cytochrome c oxidase, oil

    red O, periodic acid Schiff, and adenosine triphosphatase

    (ATPase; pH 4.3, 4.6, and 10.8). Muscle specimens from 4

    probands were prepared for electron microscopy. These

    specimens were fixed in ice-cold glutaraldehyde and

    osmium tetroxide successively. After rapid dehydration in

    graded series of acetone, tissue blocks were embedded in

    Epon. Thin sections (1 lm) of the embedded blocks werestained with uranyl acetate and lead citrate and examined

    by transmission electron microscopy.

    Results

    Genetic Analysis

    Genetic analysis of 13 unrelated families revealed pure

    (GCG) expansions of the PABPN1 gene in 10 families;

    specifically, we saw (GCG)8 in 1 family, (GCG)9 in 6

    families, (GCG)10 in 2 families, and (GCG)11 in 1 family.

    We detected (GCA) triplet interspersions in 3 families. The

    (GCG)6(GCA)1(GCG)3(GCA)3(GCG)1 allele was found in

    2 families. In another family shown in Fig. 1 (family 1),

    the proband was a compound heterozygote with (GCG)6(GCA)1(GCA)3(GCG)1/(GCG)6(GCA)1(GCG)1(GCA)3(GCG)1. Her elder sister had one normal allele and the

    mutated allele of (GCG)6(GCA)1(GCG)1(GCA)3(GCG)1.

    Her younger brother, who carried the (GCG)6(GCA)1(GCA)3(GCG)1 allele, was asymptomatic and was not

    included in subsequent phenotypic summary. All genotypes

    were summarized in Table 1.

    Clinical Presentations

    Out of 34 cases in our cohort, 19 were male and 15 were

    female. The mean age at onset was 47.2 11.2 years

    (range 2767 years). The mean disease duration was

    15.5 12.6 years (range 153 years) at the time of the last

    examination.

    Dysphagia was the most common initial symptom (18/

    34), with the mean age at the onset of 44.8 10.7 years.

    Ptosis was the initial symptom in 9 out of 34 cases, with the

    mean age at the onset of 57.8 7.7 years. Two patient

    presented with both symptoms at onset. Additionally, two

    had dysarthria, two had both dysphagia and dysarthria, and

    one had limb muscle weakness initially. There was a sig-

    nificant difference between the age at onset in the dysphagia

    group and that in the ptosis group as studied by Mann

    Whitney U test (p = 0.0036).

    During the course of the disease, 30 of 34 cases suffered

    from dysphagia, mainly presenting as prolonged meal time.

    None of our patients needed nasogastric tube feedings.

    Neuromol Med (2014) 16:782786 783

    123

  • Twenty-seven of 34 cases had ptosis as the disease progressed,

    with visual impairment caused by the ptosis being the main

    complaint in our cohort. One patient underwent levator

    resection, but ptosis recurred 2 years after the surgery. Oph-

    thalmoplegia was present in 18 out of 34 cases, with the

    involvement of superior rectus being the most common.

    Markedly limited eye movement in all directions was present

    in only one patient. Fourteen patients had predominantly

    proximal limb weakness, and 9 of them had mild distal muscle

    involvement along with the diseases progression. All these

    cases were ambulatory at the last visit. Sixteen patients aged

    between 54 and 79 years, with the mean disease duration of

    10.8 9.4 years (range 129 years), had no limb muscle

    weakness. Four patients had no limb muscle weakness until

    they died of unrelated causes, at the age of 6871.

    Four families with more than 3 patients (family 2, 3, 4,

    and 5) showed intrafamilial homogeneity to a great extent

    on initial symptom and occurrence of limb muscle weak-

    ness (Online Resource 1). As for patient IV6 from family 3

    and patient III1 and III2 from family 5, the absence of limb

    muscle weakness may be due to the early stage they were

    in (Online Resource 1). In family 1, the proband with

    (GCN)11/(GCN)12 had dysphagia and subsequently ptosis

    at the age of 53, then developed lower limb weakness at the

    age of 54, whereas her sister with (GCN)10/(GCN)12 pre-

    sented with dysphagia at the age of 57, which was the only

    symptom until the last examination (Online Resource 1).

    Other Investigations

    Serum creatine kinase levels that were measured in 21

    cases ranged from normal values to 3.7-fold of the upper

    normal limit. Electrophysiological studies were performed

    in 10 probands (Online Resource 2). Only 1 patient showed

    mildly decreased conduction velocity in NCS, with normal

    latency, compound muscle action potential, and sensory

    nerve action potential. Needle EMG revealed a normal

    pattern in 4 cases, myogenic changes in 4 cases, neurogenic

    changes in 1 case, and a myogenic/neurogenic mix pattern

    in 1 case. RNS tests in 8 of the cases were normal. Muscle

    biopsies performed in 13 probands at different disease

    Fig. 1 a The pedigree of family 1. b The genotypes of members in family1 were (GCG)6(GCA)1(GCA)3(GCG)1/(GCG)6(GCA)1(GCG)1(GCA)3(GCG)1 in the proband II2, (GCN)10/(GCG)6(GCA)1(GCG)1(GCA)3

    (GCG)1 in II1 and (GCN)10/(GCG)6(GCA)1(GCA)3(GCG)1 in II3. The

    lowest one was a normal control with (GCN)10/(GCN)10

    Table 1 Genotypes of PABPN1 in the analyzed population

    Genotype Total alanine

    codons/(GCN)

    No. of

    families

    Normal sequence

    (GCG)6(GCA)3(GCG)1 10

    Pure (GCG) expansion

    (GCG)8(GCA)3(GCG)1 12 1

    (GCG)9(GCA)3(GCG)1 13 6

    (GCG)10(GCA)3(GCG)1 14 2

    (GCG)11(GCA)3(GCG)1 15 1

    Expansion with (GCA) interspersion

    (GCG)6(GCA)1(GCA)3(GCG)1 11 1a

    (GCG)6 (GCA)1(GCG)1(GCA)3(GCG)1 12 1a

    (GCG)6 (GCA)1(GCG)3(GCA)3(GCG)1 14 2

    a This family includes one compound heterozygote with (GCG)6(GCA)1(GCA)3(GCG)1/(GCG)6(GCA)1(GCG)1(GCA)3(GCG)1

    784 Neuromol Med (2014) 16:782786

    123

  • stages revealed myopathic changes with rimmed vacuoles.

    The mean frequency of rimmed vacuoles was 1.7 %. By

    electron microscopy, intranuclear inclusions formed by

    tubular filaments were found in 3 of 4 probands (Fig. 2).

    Discussion

    Analysis of the PABPN1 gene in our cohort revealed 7

    different expansion types, with (GCG)6(GCA)1(GCA)3(GCG)1 being first described here. Although cryptic

    (GCN)11 has been widely referred to as the polymorphic

    allele, to the best of our knowledge, no (GCA) insertion has

    been reported before. The genetic heterogeneity implies

    multiple founders in Chinese population. Similar situation

    has been depicted in UK and German populations (Rob-

    inson et al. 2005; Muller et al. 2006). The genotypes with

    (GCA) interspersions, found in 3 families, further support

    the theory that unequal crossing over is the causative

    molecular mechanism leading to OPMD.

    Phenotypically, dysphagia was the most common initial

    symptom in our cohort, while ptosis was the most common

    initial symptom in the previous large-scale studies (Mira-

    bella et al. 2000; Hill et al. 2001; Tondo et al. 2012). A

    possible reason for this observation is the eyelid anatomical

    variation in different ethnic groups. Asian eyelids have a

    distinctive appearance with a narrower palpebral fissure, a

    lower or absent lid crease, and a greater fullness in upper

    lids, compared with that of the Caucasians (Liu and Hsu

    1986; Kim and Bhatki 2005). Such features in Asians may

    make ptosis less obvious, giving rise to the unawareness of

    an insidious onset of ptosis. However, further support based

    on large-scale studies in Asian groups is needed.

    Additionally, the low education level of our patients may

    also contribute to the unawareness. A simple stochastic

    incident due to the small sample is still possible. OPMD

    could not be excluded simply by the absence of ptosis, since

    Hill et al. also reported 23 % of patients presented with

    dysphagia initially (Hill et al. 2001). Another interesting

    finding in our cohort is that the patients in the dysphagia

    group had a much earlier onset compared with those in the

    ptosis group, which is in accordance with the finding in the

    Spanish population (Tondo et al. 2012). As for the corre-

    lation between severity of the disease and the length of the

    repeats, no conclusion could be drawn based on our

    patients. However, intrafamilial homogeneity was noticed,

    which further suggests that other genetic background apart

    from the already known genotype could modulate the age at

    onset and the presentation of some symptoms.

    Brais et al. first speculated that the (GCN)11 polymor-

    phism might have disease-modifying effects, in that a

    compound heterozygote with (GCN)11/(GCN)13 displayed a

    more severe disease phenotype than a sibling with (GCN)10/

    (GCN)13 (Brais et al. 1998). Subsequently, Hill et al.

    described a compound heterozygote sibling pair with

    (GCN)11/(GCN)12 who had a much earlier onset than

    unrelated subjects with (GCN)10/(GCN)12 (Hill et al. 2001).

    Such situations are in line with what we observed in our

    family 5 (Online Resource 1). However, such compound

    heterozygotes in the Italian population did not appear to

    have a more severe phenotype or an earlier onset (Mirabella

    et al. 2000). Further data are required to clarify the role of

    (GCN)11 polymorphism in modifying the phenotype.

    In conclusion, our data confirm the spectrum of geno-

    types reported in OPMD and support the theory of unequal

    crossing over. The heterogeneous genetic basis of OPMD...

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