Continuous remodeling of adult extraocular muscles as an explanation for selective craniofacial vulnerability in oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular mystrophy (OPMD) is an inherited disorder caused by mutations of the polyadenylate binding protein nuclear 1 (PABPN1) gene. While a pathogenic hypothesis has been formulated that links the genetic and molecular abnormalities to cellular abnormalities, there is no proven explanation for the targeting of the craniofacial muscles. We propose a hypothesis that bridges this gap. It is based on the phenomenon of continuous remodeling of normal adult extraocular muscles (EOMs). Unlike the EOMs, the myonuclei of other skeletal muscles are postmitotic in the adult unless the muscles are injured. Continuous myofiber remodeling most likely requires upregulation of genes involved in cell cycling, and in protein degradation and synthesis. PABPN1 is a nuclear protein that performs the essential function of controlling polyadenylation of mRNA and the fidelity of protein synthesis. In OPMD, the ongoing production of mutant PABPN1 in muscles undergoing continuous remodeling could result in a failure of accurate production of mRNA required for the maintenance of the myocytes. Over many years, this would lead to cumulative myonuclear loss and finally to myofiber loss. This hypothesis offers an explanation for the selective involvement of extraocular muscles affected in OPMD and the onset of symptoms in adulthood.     

Soluble expanded PABPN1 promotes cell death in oculopharyngeal muscular dystrophy

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disease caused by the expansion of a polyalanine repeat (GCG)(8-13) in exon 1 of the PABPN1 gene. Skeletal muscle fibers nuclei from OPMD patients contain insoluble polyalanine expanded PABPN1 (expPABPN1) nuclear aggregates that sequester different cellular components. Whether these aggregates are pathogenic, or the consequence of a molecular defense mechanism, remains controversial in the field of neurodegenerative disorders and OPMD. Our cellular model shows that interfering with the formation of expPABPN1-induced large nuclear aggregates increases the availability of nuclear expPABPN1 and significantly exacerbates cell death. Live microscopy reveals that cells harboring an increased amount of the soluble forms of expPABPN1 are significantly more prone to toxicity than those with nuclear aggregates. This is the first report directly indicating that nuclear aggregation in OPMD may reflect an active process by which cells sequester and inactivate the soluble toxic form of expPABPN1.     

Progress in understanding the pathogenesis of oculopharyngeal muscular dystrophy

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by progressive eyelid drooping (ptosis), swallowing difficulties (dysphagia), and proximal limb weakness. The autosomal dominant form of this disease is caused by expansions of a (GCG)6 repeat to (GCG)8-13 in the PABPN1 gene. These mutations lead to the expansion of a polyalanine stretch from 10 to 12-17 alanines in the N-terminal domain of PABPN1. Mutated PABPN1 (mPABPN1) induces the formation of muscle intranuclear inclusions that are thought to be the hallmark of this disease. In this review, we discuss: 1) OPMD genetics and PABPN I function studies; 2) diseases caused by polyalanine expansions and cellular polyalanine toxicity; 3) mPABPN1-induced intranuclear inclusion toxicity; 4) role of oligomerization of mPABPNI in the formation and toxicity of OPMD intranuclear inclusions and; 5) recruitment of subcellular components to the OPMD inclusions. We present a potential molecular mechanism for OPMD pathogenesis that accounts for these observations.     

Progressive myopathy in an inducible mouse model of oculopharyngeal muscular dystrophy

The genetic basis of oculopharyngeal muscular dystrophy (OPMD) is a short expansion of a polyalanine tract (normal allele: 10 alanines, mutant allele: 11-17 alanines) in the nuclear polyadenylate binding protein PABPN1 which is essential for controlling poly(A) tail length in messenger RNA. Mutant PABPN1 forms nuclear inclusions in OPMD muscle. To investigate the pathogenic role of mutant PABPN1 in vivo, we generated a ligand-inducible transgenic mouse model by using the mifepristone-inducible gene expression system. Induction of ubiquitous expression of mutant PABPN1 resulted in skeletal and cardiac myopathy. Histological changes of degenerative myopathy were preceded by nuclear inclusions of insoluble PABPN1. Downregulation of mutant PABPN1 expression attenuated the myopathy and reduced the nuclear burden of insoluble PABPN1. These results support association between mutant PABPN1 accumulation and degenerative myopathy in mice. Resolution of myopathy in mice suggests that the disease process in OPMD patients may be treatable.     

Mutation and haplotype analysis of oculopharyngeal muscular dystrophy in Thai patients

Oculopharyngeal muscular dystrophy (OPMD) is an inherited neuromuscular disease associated with a short trinucleotide repeat expansion in Exon 1 of the PABPN1 gene. OPMD is uncommon in East Asian populations, and there have been no previous reports of Thai patients. We studied clinical and molecular genetic features of six unrelated Thai patients with autosomal dominant OPMD. All patients had expansions of the guanine-cytosine-guanine (GCG) repeat ranging from three to seven additional repeats in the 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% of the control subjects possessed (GCG)₇, thereby suggesting that the prevalence of autosomal recessive OPMD in the Thai population was approximately 1 in 160,000. In conclusion, our data suggest that OPMD in Thailand may be more common than previously thought.     

HnRNP A1 and A/B interaction with PABPN1 in oculopharyngeal muscular dystrophy

BACKGROUND: Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by progressive ptosis, dysphagia and proximal limb weakness. The autosomal dominant form of this disease is caused by short expansions of a (GCG)6 repeat to (GCG) in the PABPN1 gene. The mutations lead to the expansion of a polyalanine stretch from 10 to 12-17 alanines in the N-terminus of PABPN1. The mutated PABPN1 (mPABPN1) induces the formation of intranuclear filamentous inclusions that sequester poly(A) RNA and are associated with cell death. METHODS: Human fetal brain cDNA library was used to look for PABPNI binding proteins using yeast two-hybrid screen. The protein interaction was confirmed by GST pull-down and co-immunoprecipitation assays. Oculopharyngeal muscular dystrophy cellular model and OPMD patient muscle tissue were used to check whether the PABPN1 binding proteins were involved in the formation of OPMD intranuclear inclusions. RESULTS: We identify two PABPNI interacting proteins, hnRNP A1 and hnRNP A/B. When co-expressed with mPABPN1 in COS-7 cells, predominantly nuclear protein hnRNP A1 and A/B co-localize with mPABPN1 in the insoluble intranuclear aggregates. Patient studies showed that hnRNP A1 is sequestered in OPMD nuclear inclusions. CONCLUSIONS: The hnRNP proteins are involved in mRNA processing and mRNA nucleocytoplasmic export, sequestering of hnRNPs in OPMD intranuclear aggregates supports the view that OPMD intranuclear inclusions are "poly(A) RNA traps", which would interfere with RNA export, and cause muscle cell death.     

Transgenic expression of an expanded (GCG)13 repeat PABPN1 leads to weakness and coordination defects in mice

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset disorder caused by a (GCG)n trinucleotide repeat expansion in the poly(A) binding protein nuclear-1 (PABPN1) gene, which in turn leads to an expanded polyalanine tract in the protein. We generated transgenic mice expressing either the wild type or the expanded form of human PABPN1, and transgenic animals with the expanded form showed clear signs of abnormal limb clasping, muscle weakness, coordination deficits, and peripheral nerves alterations. Analysis of mitotic and postmitotic tissues in those transgenic animals revealed ubiquitinated PABPN1-positive intranuclear inclusions (INIs) in neuronal cells. This latter observation led us to test and confirm the presence of similar INIs in postmortem brain sections from an OPMD patient. Our results indicate that expanded PABPN1, presumably via the toxic effects of its polyalanine tract, can lead to inclusion formation and neurodegeneration in both the mouse and the human.     

PABPN1 overexpression leads to upregulation of genes encoding nuclear proteins that are sequestered in oculopharyngeal muscular dystrophy nuclear inclusions

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disease caused by expanded (GCN)12-17 stretches encoding the N-terminal polyalanine domain of the poly(A) binding protein nuclear 1 (PABPN1). OPMD is characterized by intranuclear inclusions (INIs) in skeletal muscle fibers, which contain PABPN1, molecular chaperones, ubiquitin, proteasome subunits, and poly(A)-mRNA. We describe an adenoviral model of PABPN1 expression that produces INIs in most cells. Microarray analysis revealed that PABPN1 overexpression reproducibly changed the expression of 202 genes. Sixty percent of upregulated genes encode nuclear proteins, including many RNA and DNA binding proteins. Immunofluorescence microscopy revealed that all tested nuclear proteins encoded by eight upregulated genes colocalize with PABPN1 within the INIs: CUGBP1, SFRS3, FKBP1A, HMG2, HNRPA1, PRC1, S100P, and HSP70. In addition, CUGBP1, SFRS3, and FKBP1A were also found in OPMD muscle INIs. This study demonstrates that a large number of nuclear proteins are sequestered in OPMD INIs, which may compromise cellular function.     

A late-onset case of oculopharyngeal muscular dystrophy carrying a (GCG)8 repeat expansion in the PAPBN1 gene]

We report a sporadic case of a female patient with oculopharyngeal muscular dystrophy (OPMD). Her father died at age 86 and mother at age 74. There was no familial occurrence of the disease. The patient initially developed a nasal voice at age 66. Neurological examinations on admission at age 72 revealed bilateral ptosis, a limitation of ocular movement without diplopia, dysphagia, and proximal muscle weakness. Serum creatine kinase level was slightly increased. Biopsied muscle specimens showed variation in fiber size as well as the occasional presence of rimmed vacuoles. On the basis of these clinical and laboratory findings, we suspected a diagnosis of OPMD, although a family history was absent. To confirm the diagnosis of OPMD, we performed a gene analysis for poly A binding protein, nuclear 1 (PABPN1; PABP2), which revealed a mild expansion of GCG repeat (8 repeats) as a heterozygous state. Clinical features of the patient were consistent with those in a previous literature reporting that patients carrying (GCG)8 repeat as a heterozygous state show a relatively late onset and a mild phenotype. The case of this patient emphasizes the importance of the PABPN1 gene analysis for patients showing muscular weakness involving oculopharyngeal and proximal limb muscles even when a familial occurrence of the disease is not apparent.     

Identification of a novel mutation in a Korean patient with oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset muscle disorder characterized by progressive dysphagia and bilateral ptosis. Mutations in the polyadenylate binding protein nuclear 1 (PABPN1) gene have been found to cause OPMD. The typical mutation is a stable trinucleotide repeat expansion in the first exon of the PABPN1 gene, in which (GCG)(6) is the normal repeat length. We investigated a Korean patient with OPMD and identified a novel mutation: a heterozygous insertion of a 9-bp sequence [(GCG)(GCA)(GCA); c.27_28insGCGGCAGCA] instead of the (GCG) repeat expansion, resulting in an in-frame insertion of three alanines (p.A10insAAA). To the best of our knowledge, this is the first report of a genetically confirmed case of OPMD in Korea.     

The dynamism of PABPN1 nuclear inclusions during the cell cycle

Oculopharyngeal muscular dystrophy (OPMD) is caused by expansion of a (GCN)10 to a (GCN)11-17 repeat coding for a polyalanine domain at the N-terminal part of poly(A) binding protein nuclear 1 (PABPN1). OPMD is characterized by the presence of intranuclear inclusions (INIs) in skeletal muscle fibers of patients. The formation of GFP-b13AlaPABPN1 INIs and their fate through the cell cycle were followed by time-lapse imaging. Our observations demonstrated that the GFP-b13AlaPABPN1 INIs are dynamic structures that can disassemble during mitosis. However, their presence in cells occasionally led to apoptosis. The length of the polyalanine tail or the overexpression of PABPN1 did not significantly affect the percentage of soluble PABPN1 in vitro. Moreover, overexpression of either the wild type (wt) or mutant (mut) forms of PABPN1 slowed down the cell proliferation. The slowing down of proliferation together with the occasional occurrence of apoptosis could contribute in vivo to the late onset of this disease.     

Oculopharyngeal muscular dystrophy: A polyalanine myopathy

It has been 10 years since the identification of the first PABPN1 gene (GCN)_n/polyalanine mutations responsible for oculopharyngeal muscular dystrophy (OPMD). These mutations have been found in most cases of OPMD diagnosed in more than 35 countries. Sequence analyses have shown that such mutations have occurred numerous times in human history. Although PABPN1 was found early on to be a component of the classic filamentous intranuclear inclusions (INIs), mRNA and other proteins also have been found to coaggregate in the INIs. It is still unclear if the INIs play a pathologic or a protective role. The generation of numerous cell and animal models of OPMD has led to greater insight into its complex molecular pathophysiology and identified the first candidate therapeutic molecules. This paper reviews basic and clinical research on OPMD, with special emphasis on recent developments in the understanding of its pathophysiology.     

Restoration of dystrophin expression in cultured hybrid myotubes

Absence of dystrophin, as found in Duchenne boys, mdx mice and HFMD cats, leads to destabilization of the sarcolemmal-associated protein complex. Gene and cell therapy strategies aim to restore the dystrophin-associated protein complex. In order to better understand the cellular events involved in such therapy in feline and human muscular dystrophy, we asked whether dystrophin-deficient myoblasts would fuse with myoblasts expressing normal dystrophin, and whether the complex would be restored after such a fusion. Cat and human myoblasts were isolated from skeletal muscle of normal subjects and of patients with dystrophin deficiency and proliferated well. After co-culture with normal myoblasts, they fused to form hybrid myotubes. These hybrid myotubes expressed dystrophin, utrophin and dystrophin- associated proteins. Expression of these proteins were restored also in the vicinity of nuclei from dystrophin-deficient donors. These results demonstrate that dystrophin can be expressed and handled normally by hybrid myotubes. They show that myoblasts with a normal dystrophin gene can restore dystrophin expression in dystrophin-deficient myoblasts.     

Variability of the recessive oculopharyngeal muscular dystrophy phenotype

Oculopharyngeal muscular dystrophy (OPMD) is usually transmitted as an autosomal-dominant trait and characterized by an expansion from 6 to 8 or more GCG/GCA repeats in the poly-(A) binding protein nuclear 1 (PABPN1) gene on chromosome 14q11. Autosomal-recessive OPMD with a homozygous (GCG)7 expansion of PABPN1 has only been described in two Canadian patients, who showed a comparably mild phenotype, suggesting that it is less severe than the dominant form. We clinically and genetically characterized the first two reported cases of autosomal-recessive OPMD in Europe. Remarkably, both patients revealed severe and diverse phenotypes, with an unusual onset and atypical clinical course in one patient. Former studies found a 1%-2% frequency of the (GCG)7 allele, which theoretically produces an incidence of 1:10,000 of autosomal-recessive OPMD in the general population. We conclude that the apparent rarity of the autosomal-recessive form of OPMD may be due to the fact that genetic testing is generally administered only to patients with typical clinical features or a positive family history.     

Analysis of growth factor expression in affected and unaffected muscles of oculo-pharyngeal muscular dystrophy (OPMD) patients: A pilot study

Oculo-pharyngeal muscular dystrophy (OPMD) is characterised by progressive eyelid drooping (ptosis) and difficulties with swallowing (dysphagia). In order to determine the role of growth factors, cytokines and chemokines in the physiopathology of muscle disease we have compared the level of expression of 174 factors in both the affected (cricopharyngeal) and non-affected (sternocleidomastoid) muscles of 8 OPMD patients by means of antibody arrays. Despite an important inter-individual variability the expression of sixty factors was found to be persistently different between affected and non-affected muscles. Many of the differentially expressed factors in our study are known to be involved in the formation of fibrosis in both the liver and the lung, indicating the possibility that treatments such as those used in hepatic fibrosis may have a beneficial effect in OPMD patients.     

Light- and electron-microscopic studies of transplanted human dystrophic muscles to nude mice

Light-microscopic study of muscles from 8 patients with Duchenne muscular dystrophy (DMD) and 9 control cases transplanted into nude mice showed the formation of cross-striations and peripheral nuclei in regenerating muscle fibers of both DMD and control grafts up to 4 weeks. Quantitative study of the diameter of myotubes and myoblasts in grafted muscles showed that the group mean diameters of DMD myotubes and myoblasts were 13.4 ± 0.8 (SE) μm, 15.6 ± 0.7 μm, 17.1 ± 0.9 μm and 16.2 ± 1.0 μm after 1, 2, 3 and 4 weeks of transplantation, respectively, whereas those of control myotubes and myoblasts were 14.9 ± 0.6 (SE) μm, 19.9 ± 0.5 μm, 21.3 ± 0.7 μm and 20.7 ± 0.7 μm after the same intervals. The group mean diameters of the DMD regenerating myotubes and myoblasts were significantly smaller than those of control myotubes and myoblasts at 2 (P <0.001), 3 (P <0.005) and 4 (P <0.005) weeks after transplantation.Electron-microscopic findings of DMD muscle grafts were morphologically similar to those seen in control muscle grafts at all stages of this experiment.     

Dynamic restoration of dystrophin to dystrophin-deficient myotubes

Dystrophin domains are observed in myoblast transplantation experiments and in muscle fibers after somatic reversion in human Duchenne and mdx mouse muscular dystrophy. However, the formation and evolution of dystrophin-positive domains are not well established. Using a muscle satellite cell coculture system, we examined the dynamic restoration of dystrophin expression in dystrophin-deficient myotubes. The dystrophin-positive domains around source nuclei were clearly identified in hybrid myotubes. The occurrence of dystrophin domains was higher in myotubes differentiated from cocultures with a low concentration of normal wild-type satellite cells in relation to dystrophin-deficient satellite cells. At higher seeding ratios, the domain feature of dystrophin expression was more transitory and decreased as myotubes differentiated over time in culture. The average domain size initially increased with the addition of new nuclei by fusion early after differentiation of cocultures. However, separating dystrophin-positive domains according to their number of dystrophin-expressing contributory nuclei showed that diffusion of dystrophin contributed to domain elongation, even in early myotubes and later without fusion of additional nuclei. Diffusion occurred for all domains of one to six wild-type nuclei, and the diffusion rate was higher in domains with larger numbers of nuclei. This dynamic domain feature of dystrophin expression was also related to restoring the organization of dystrophin-associated proteins and acetylcholine receptors to hybrid myotubes. Factors regulating domain formation and diffusion therefore are important considerations in the design of strategies for both myoblast transplantation and gene therapy of Duchenne muscular dystrophy.     

眼咽型肌营养不良汉族六家系的临床和遗传学特点

目的 初步总结我国汉族眼咽型肌营养不良(OPMD)患者的临床和多腺苷酸结合蛋白核1(PABPN1)基因改变特点.方法 6个OPMD家系共28例患者,男性13例,女性15例,发病年龄32～70岁,平均发病年龄49.7岁.在可确定首发症状的患者中,以吞咽困难或构音障碍首发的13例、眼睑下垂首发的4例、双下肢无力首发的1例.经过3～20年均出现眼睑下垂、吞咽困难和构音障碍,其中7例出现四肢近端无力.对6例先证者做肌肉活体组织检查,标本进行常规组织病理和电镜检查.对6个家系的先证者以及部分家庭成员进行PABPN1基因检查,并对6例先证者进行单体型分析.结果 6例先证者的肌肉活体组织检查均发现肌纤维直径轻度变异加大伴随肌纤维内镶边空泡形成,4例患者经电镜检查发现OPMD典型的核内栅栏样丝状包涵体.3个家系的PABPN1基因型为(GCG)9,另外3个家系的基因型分别为(GCG)6(GCA)1(GCG)3、(GCG)10和(GCG)8.2个携带(GCG)9突变的家系存在rs2239579(C)-(GCG)9-SNP2622(C)的单倍体型.结论 吞咽异常和眼睑下垂均是我国汉族OPMD患者的首发症状.肌纤维出现镶边空泡以及核内包涵体是我国患者的常见病理改变.PABPN1基因的(GCG)异常扩增和(GCA)插入突变均出现在我国患者,起源具有多源性.携带(GCG)9突变的部分家系可能来自共同祖先.     

Muscle glucose-6-phosphate dehydrogenase deficiency: Restoration of enzymatic activity in hybrid myotubes

A high level of glucose-6-phosphate dehydrogenase (G6PD) activity was observed in myoblasts and myotubes from normal human and mouse cell cultures. However, only a residual amount of activity was observed in myoblasts and myotubes obtained from G6PD-deficient patients (G6PD Mediterranean). Hybrids were formed by the fusion of normal (from human and mouse) and G6PD-deficient myoblasts (from the patients). These hybrids contained a high level of G6PD activity. Hoechst staining permitted to confirm that the enzymatic activity was not restrained to a domain near the competent nuclei. These results suggest that myoblast transplantation could be used to restore normal enzymatic activity in metabolic myopathies.