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Oculopharyngeal Muscular Dystrophy

Bernard Brais Departments of Neurology and Neurosurgery and Human Genetics, Faculty of Medicine , McGill University, Montreal Neurological Institute , Montreal , Canada

Defi nition of e ntities

The common form of oculopharyngeal muscular dystrophy (OPMD) is a dominant late-onset myopathy associated with pro-gressive ptosis of the eyelids, dysphagia, and unique tubulofi la-mentous intranuclear inclusions (INI) (OMIM #164300) [1] . All dominant and recessively inherited mutations consist of short (GCN) 11-17 /polyalanine expansions in the polyadenylate-binding protein nuclear 1 gene ( PABPN1 ) localized on chromosome 14q11.2 (Figure 31.1 ) [2] . The mutations cause the lengthening of an N-terminal polyalanine domain. A variable degree of limb-girdle muscular involvement appears in all cases. The disease has a slowly progressive course. Life expectancy is normal [3] . Diag-nosis is presently made by genetic testing. The only clear neuro-cognitive involvement in OPMD has been documented as a late manifestation in homozygote cases for a dominant mutation [4] .

Molecular g enetics and p athophysiology

The sequence diversity of mutations supports a large number of independent mutation events. Autosomal dominant and recessive OPMD have been found to be allelic [2] . Dominant cases have been observed in more than 35 countries. A positional cloning strategy led to the identifi cation of short (GCN) 12-17 expansions of the polyadenylate-binding protein nuclear 1 ( PABPN1 , previ-ously abbreviated PABP2 ) gene in all dominant OPMD cases [2] . Dominant and recessively inherited OPMD are caused by mitoti-cally and meiotically stable short triplet repeat expansions of a cryptic (GCN) 10 /alanine 10 and, more rarely, point mutations, leading to a lengthening of a polyalanine domain (see Figure 31.1 ) [2,5,6] . Initially described as a (GCG)n/polyalanine disease, it was

later found to be often caused by cryptic GCN/alanine insertion [2,6] . Unequal cross-over is the most likely mutation mechanism with documented neomutations [6] .

Since the publication of the fi rst PABPN1 mutations in 1998, our understanding of the molecular pathogenesis of OPMD has progressed but no defi nitive mechanism has yet been established. Various nuclear inclusion-dependent and -independent mecha-nisms have been proposed (Figure 31.2 ). Though most hypoth-eses suggest that the expansion of the polyalanine stretch leads to a gain of function of the protein, there is evidence that suggests that the intranuclear inclusions may not be responsible for the disease and may even be protective [7] . PABPN1 is a ubiquitous polyadenylation factor essential for the formation of poly(A) tails of eukaryotic mRNA (see Figure 31.2 ). The protein shuttles between the nucleus and the cytoplasm where it may also play a role in transcription control [8] . The aggregative biophysical property of polyalanine stretches has been known for years [2] . Lengthening of the polyalanine domain has been shown to increase PABPN1 ’ s aggregative tendency and resistance to solvent by kinetic and structural studies.

Expansion from 10 to 17 alanines of the N-terminal domain of PABPN1 appears to decrease fi brillar formation, providing structural evidence that the mutated form may in fact have a greater negative impact on physiological interaction between PABPN1 molecules with itself and other partners [9] . It was observed that various substances infl uence a PABPN1 fi brillar formation, including doxycycline and trehalose, two molecules previously shown to diminish mutated PABPN1 toxicity in a mouse transgenic model, which increase fi bril formation [10] . This provides indirect evidence that increased aggregation may in fact be protective or alternatively that these substances, by ensuring a better conformation of the soluble mutated form, may slow the disease process.

31

Muscle Disease: Pathology and Genetics, Second Edition. Edited by Hans H. Goebel, Caroline A. Sewry, and Roy O. Weller.

© 2013 International Society of Neuropathology. Published 2013 by John Wiley & Sons, Ltd.

Oculopharyngeal Muscular Dystrophy Chapter 31

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Figure 31.1 Genomic OPMD PABPN1 dominant cryptic (GCN) n and point mutations and polyalanine domain expansions. Reproduced from Calado et al. [15] , with permission from Oxford University Press.

PABPN1 OPMD (GCN)n/(alanine)n dominant mutations ATG GCG GCGGCGGCGGCGGCG(GCN)nGCA GCAGCA GCG GGGGCT GCG GGC M A AAAAA (A)n A AAA P A A PABPN1 OPMD dominant (GCN)12 point mutations ATG GCG GCGGCGGCGGCGGCG GCA GCAGCA GCG GCGGCT GCG GGC M A AAAAAAAAAAAA

Figure 31.2 Cellular traffi cking of PABPN1 and possible sites of interference of mutated PABPN1 with normal cellular processes. PABPN1 is involved in the polyadenylation of all messenger RNAs (7 and 8). PABPN1 travels with the mRNA to the cytoplasm through nuclear pores (9). It is released from the mRNA on the initiation of translation and possible control of translation (11). It is actively transported back to the nucleus to take part in the polyadenylation of other mRNA molecules (10). Based on our understanding of the major role and cellular traffi cking and breakdown of PABPN1, mutated forms could interfere with different cellular processes (1–12). Interference could be caused by intranuclear inclusion-dependent mechanisms: 1 Physical rupture of the nuclear membrane; 2 Disruption of transcriptional domains; 3 Disruption of chromosomal domains; 4 Sequestering of mRNAs coding for proteins vital for cell survival; 5

Sequestering of PABPN1 in suffi cient quantity to interfere with normal mRNA processing; 6 Sequestering of other proteins: a proteins interacting normally with PABPN1, b proteins involved in protein folding (e.g. chaperones such as Hsp70, Hsp40), c proteins of the ubiquitin/proteasome pathway or d other proteins with polyalanine domains. On the other hand, intranuclear inclusion-independent mechanisms where the soluble expanded PABPN1 could interfere with: 7 normal function of PABPN1 in mRNA polyadenylation; 8 the mRNA processing machinery; 9 PABPN1 and mRNA exit of the nucleus; 10 PABPN1 reimport in the nucleus; 11 the initiation of translation; 12 other soluble polyalanine containing proteins that interact with PABPN1. Reproduced from Calado et al. [15] , with permission from Oxford University Press.

Highly expressed gene

PolII

CPSF5‘GpppG

5‘GpppG

5‘GpppG5‘GpppG

6 b)6 c)

6 a)

Nuclear Inclusion

12 3

6 d)

4 5 12

10

11

9

PABPN1

Nuclear Pore Complex

PolyA+ mRNA

Ribosome

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

A A

Heat Shock Proteins (Hsp40, Hsp70, etc.)Proteasome

Polyalanine containing protein

PAPPolyA+ mRNA

A

A A

AA

A

AAUAAA

AA

78

CTD

Overexpression of PABPN1 readily produces in cellular, mice, fl y, and nematode models the formation of INIs associated with cell death [11] . The PABPN1-containing INIs are usually fi lamen-tous and share features of OPMD muscle INIs though they are less well structured. In different cellular and animal models of OPMD, investigators have shown that some molecules reduced cellular toxicity. In cellular models, it was shown that inducing heat shock protein expression using ZnSO4, 8-hydroxyquinoline, ibuprofen, and indomethacin or exposing cells to anti-PABPN1 antibodies that interfere with oligomerization could prevent

cell death. In a mouse transgenic model of OPMD, investigators have reduced inclusion formation and cell death with agents that interfere with protein aggregation such as Congo red, doxycy-cline, and trehalose [10] . A ligand-inducible transgenic model has demonstrated that stopping the expression of mutated PABPN1 led to a reversal of the phenotype [11] . This further emphasizes that treatment strategies that could limit the expression or impact of the mutated allele could delay or even cure this muscular dystrophy.

Structural c hanges

Histological studies of skeletal muscle biopsy show changes that are common to many muscular dystrophies, such as loss of muscle fi bers, abnormal variation in fi ber size, an increase in the number of internal nuclei, and increased interstitial fi brous and fatty connective tissues. Fibers undergoing necrosis and phagocy-tosis are rare. Infl ammatory changes are usually absent. Histo-chemical studies reveal small angulated fi bers that often react strongly for oxidative enzymes (more frequently type 1 than type

Section 12 Muscle Diseases with DNA Expansions

286

Approximately 20% of more severe cases are compound hetero-zygotes for the dominant mutation and a (GCN) 11 polymorphism in their other copy of the PABPN1 gene [2] . This polymorphism has a1–2% prevalence in North America, Europe, and Japan. The spectrum of severity in carriers of the same size (GCN) n muta-tion, the small difference between the mutation size and the unre-liability of variable age of onset have not allowed a defi nitive conclusion as to the possible correlation between size of mutation

2) and rimmed vacuoles. Though the small angulated fi bers may suggest an underlying denervation process, their occurrence may mostly be due to the advanced age of patients. The vacuoles consist of irregularly round or polygonal clear spaces lined by a ring of material that is basophilic with the hematoxylin and eosin stain and stains red with Gomori trichrome stain [12] . Rimmed vacuoles are observed in several other disorders, and are therefore not considered specifi c for OPMD. The rimmed vacuoles are autophagic in nature and have been reported to have acid phos-phatase activity.

The most signifi cant ultrastructural change is the presence of intranuclear tubular fi laments with an outer diameter of 8.5 nm and inner diameter of 3 nm [1] . The fi laments are unbranched, often course in a rectilinear manner and are sometimes striated with 7–7.5 nm periodicity (Figure 31.3 ). The fi laments are up to 0.25 μ m in length. They are orientated in various directions and frequently form tangles or palisades. Large collections of fi la-ments appear as clear zones surrounded by chromatin in the affected nuclei on resin sections. These nuclei can often be identi-fi ed in semi-thin epoxy sections by phase-contrast microscopy, where they appear as clear zones. Studies of serial semi-thin sec-tions suggest that in some specimens, the fi lamentous inclusions occur in all muscle fi ber nuclei [12] . The highest percentage of muscle nuclei containing INI was observed in seven homozygous cases for (GCN) 13 PABPN1 mutations [13] . In these more severe OPMD cases, 9.4% of nuclei contained INI compared to 4.9% in cases heterozygous for the same mutation. The inclusions were found only in the nuclei of muscle fi bers and not in the nuclei of any other cells (including satellite cells) in muscle. Rarely, tubular fi laments, as seen in inclusion body myositis, with an external diameter of 16–18 nm have also been found in OPMD muscle. Inclusions have been documented in anterior horn cells of one autopsy case [14] .

Since the discovery that PABPN1 is the mutated gene in OPMD, considerable work has centered on the identifi cation of other molecules present in the INI. PABPN1 was shown to be an inte-gral part of the muscle OPMD inclusions [15] . The INI in muscle also contain components of the ubiquitin-proteasome pathway, including Hsp70, poly(T)RNA, transcription factors such as SNW1 (previously called SKIP), important in myogenesis, differ-ent mRNA binding proteins such as CUGP1, SFRS3, and FKBP1A [16] , and type 1 arginine methyl transferase (PRMT1) [17] .

Genotype- p henotype c orrelation

Gene dosage has a clear infl uence on the age of onset and severity of the OPMD phenotype [2] . The most severe OPMD phenotype is reported for individuals homozygous for a dominant OPMD mutation [2,13] . A study of homozygous OPMD cases reported that on average, onset of symptoms was 18 years earlier than in heterozygotes [13] . Further follow-up demonstrated that they developed cognitive impairment and reduced life expectancy [4] . Severity of the dominant OPMD phenotype is also variable [18] .

Figure 31.3 (a) The OPMD inclusions contain insoluble PABPN1 in human deltoid muscle. (b) PABPN1-containing inclusions are resistant to salt treatment ( arrow ), whereas nucleoplasmic PABPN1 in a nucleus with no detectable inclusion is completely solubilized. (c) The OPMD inclusions contain poly(A) RNA as detected by hybridizing with riboprobes complementary to either the poly(A) tail of mRNA (green staining) or To-Pro (red, C) and are shown to predominantly exclude DNA. (d) The anti-PABPN1 colocalizes with poly(A) RNA in the OPMD inclusions. (e,f) Immunoelectron microscopy of PABPN1 in OPMD nuclei of a 60-year-old patient. Subsarcolemmal nuclei contain inclusions of unique fi laments. These fi laments converge to form tangles or palisades, seen distinctly in sections prepared according to standard techniques for electron microscopy (e) . Using anti-PABPN1 antibodies, the fi laments are decorated by immuno-gold particles. The labeling is particularly intense at sites of fi lament convergence (f) . Reproduced from Calado et al. [15] , with permission from Oxford University Press.

(a) (b)

DNA

OPMD inclusion

OPMD inclusion OPMD inclusion

Nucleolus

(c)

(e)

(d)

(f)

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and clinical severity. Confl icting descriptions on a few recessive OPMD cases document either a milder or more severe phenotype [2,19] .

Future p erspectives

Despite the identifi cation of the mutations responsible for OPMD in 1998, many important questions remain unanswered. It is clear that the severity of the phenotype varies even with carriers of the same size of (GCN) n PABPN1 mutation [2] . However, no study has conclusively shown that the size of the mutation infl uences the severity of the phenotype, at least when correlated with age of onset. Only carriers of the smallest (GCN) 12 mutation appear clearly to have a milder phenotype with a later age of onset in the seventh decade with ptosis and only mild dysphagia [2] . Com-pound heterozygotes for dominant and recessive mutations have also been shown to have more severe phenotypes [2,20] . The sequencing of the mutation has demonstrated that OPMD muta-tions do not consist of pure (GCN) n repeat expansions but of (GCN) n /polyalanine insertions [6] . The mechanism responsible for the genesis of the mutations is still unknown, though unequal recombination is the most likely mechanism [2,6] .

In OPMD, as in most muscular dystrophies, the selective involvement of certain muscles is as yet understood. This is par-ticularly interesting in OPMD, because the mutated gene is ubiq-uitously expressed in all tissues. Furthermore, individuals who are homozygous for two dominant mutations have a normal devel-opment but clearly have a earlier, more diffuse muscular involve-ment and even central nervous system involvement [4,13] . Furthermore, knocking down the expression of PABPN1 in mice does not lead to disease [10] . Gene dosage in homozygotes and compound heterozygotes, as discussed previously, is in favor of a gene dosage effect. Lastly, despite our growing knowledge of the structure and function of PABPN1, we still do not know how exactly expansions of its short polyalanine domain cause muscle demise. As discussed above, even the pathogenic role of the INI still needs to be fully elucidated. A growing body of data suggests that the soluble, or nonaggregate, PABPN1 may be the most important protein species responsible for the disease. How the expanded polyalanine domain modifi es soluble PABPN1 func-tion has to be elucidated to ensure that appropriate treatments are designed. It is clear, however, that because of the increasing number of diseases caused by polyalanine expansions and the pathological overlap with CAG/polyaglutamine diseases, patho-logical insights are gained by the study of OPMD and could lead to a better understanding of a much larger group of developmen-tal and degenerative diseases.

References

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