Neuropathology and Applied Neurobiology (2015), 41, 385–390
doi: 10.1111/nan.12171
Scientific correspondence
Familial polyglucosan body myopathy with unusual phenotype Polyglucosan (PG) is an abnormal polysaccharide that, compared with glycogen, has fewer branched points and excessively long peripheral chains that structurally resemble the plant polysaccharide ‘amylopectin’. Under electron microscopy, PG bodies are round, nonmembrane-bound cytoplasmic particles with irregular branched filaments, which often displace myofibrils, leading to Z disk streaming. PG bodies react strongly to PAS stain and are partially resistant to diastase digestion. PG accumulation constitutes the histopathological manifestation of several clinically different conditions. It occurs mainly in the brain (Lafora disease, double athetosis) and in skeletal muscle (glycogenosis type IV, glycogenosis type VII, other polysaccharide storage myopathies [1–6]). Recently, recessive mutations in the RBCK1 gene, which encodes for an E3 ubiquitin ligase, have been recognized as causing two different disorders with PG accumulation in skeletal and cardiac muscle: a myopathy with cardiomyopathy [7,8], and a fatal immunodeficiency with myopathy and cardiomyopathy [9]. To date, only 10 families have been reported in which PG body myopathy is caused by RBCK1 gene mutations. Here we describe a new family (including one pair of siblings, Patient 1 and Patient 2, and their paternal female cousin, Patient 3), presenting as an autosomal recessive form of late-onset myopathy with limb girdle muscular dystrophy phenotype and PG accumulation in muscle. Patient 1. This 66-year-old man had complained for many years of back pain, progressive difficulty walking and climbing stairs and weakness in muscles of both girdles. He presented with a waddling gait, weakness in ileopsoas, leg and arm flexor muscles, calf hypertrophy, pes cavus and moderately increased creatine kinase level. At age 74 years he was unable to rise from sitting (Figure 1) and was able to walk only with the aid of a cane. Cardiac investigations revealed a bundle branch block and an ischemic cardiomyopathy. He also presented with skin lesions characteristic of generalized vitiligo © 2014 British Neuropathological Society
(Figure 1). Patient 2. At age 52 years she complained of progressive difficulty in climbing stairs and rising from a chair, and noticed asthenia in the lower limbs. Creatine kinase level was mildly increased. At age 57 years she was diagnosed with familial spastic paraparesis with axonal motor polyneuropathy, and with chronic inflammatory demyelinating polyneuropathy (CIDP). She presented with a waddling gait, inability to rise from the floor without support, weakness in proximal limb muscles, pes cavus, calf hypertrophy and hand muscle atrophy. No cardiac symptoms were reported. At age 60 years she became unable to walk unsupported. Patient 3. Since she was 50 years she had complained of limb myalgia. After an ischemic stroke at age 52, causing sensory-motor hemi-syndrome, she noticed progressive weakness in proximal limb girdle muscles. At age 63 years, when the first muscle biopsy was done, she presented with a waddling gait, Gowers sign, normal EMG and CK levels. At age 70 years, when a second muscle biopsy was performed, she was unable to raise her arms over her head, had scapular winging, and quadriceps muscle wasting. No cardiac symptoms were reported. Two out of the four muscle biopsies available for this study (Patient 1, first biopsy Patient 3) showed myopathic features but neither vacuoles nor accumulation of PASpositive material. Conversely, the other two muscles (Patient 2, second biopsy Patient 3) were characterized by violet or hyaline PG inclusions in about 10–20% of fibres (Figure 2). The vacuoles were filled with strongly PASpositive reacting material, whereas the cytoplasmic areas surrounding the inclusions were depleted in glycogen. The vacuoles were outlined by a rim of intense oxidative enzyme reaction, but their membranes were not immunolabelled by caveolin-3. The accumulated material showed absent staining with Lugol’s iodine and acid phosphatase, negative immunolabelling for desmin and myotilin; it was resistant to pre-digestion with both diastase/amylase and proteinase-k, and was strongly immunolabelled for ubiquitin and for p62/SQSTM1 (protein aggregates) (Figure 2). Scattered LC3-positive reaction was present in most fibres. Immunoblot analysis 385
386
Scientific correspondence
Figure 1. The upper panel shows the clinical phenotype of Patient 1, which is characterized by severe weakness in upper and lower girdle muscles (D, E, F), causing inability to rise from sitting without support. Note the generalized distribution of skin lesions corresponding to vitiligo, involving the head (D), the neck (B), the hands and the forearms (A, C, E), the thighs and the legs (F). The lower panel shows electron microscopy of muscle biopsies from Patient 3 (A), Patient 2 (B–E) and Patient 1 (F) showing glycogen filamentous areas without delimiting membrane, and myofibrils that appeared misaligned by the large granulofilamentous storage (A). Abundant subsarcolemmal and intermyofibrillar accumulation of filamentous material is surrounded by β-particles glycogen (B, C, F). Note an internalized nucleus and accumulation of filamentous protein material that disrupt myofibrils, dispersed vesicles and Z-line remnants (D), abundant glycogen accumulation that disrupts myofibrillar organization with Z-line fragmentation and sarcoplasmic reticulum disorganization (E). © 2014 British Neuropathological Society
NAN 2015; 41: 385–390
Scientific correspondence
387
Figure 2. The panel on the left shows muscle histopathology of Patient 2 and Patient 3. Sections stained with haematoxylin-eosin (H&E) (A), Gomori trichrome (B), NADH-TR reductase (C) and PAS (D, E) show many fibres displaying violet or hyaline inclusions localized in subsarcolemmal and intracytoplasmic position (A, B). The vacuoles are filled with strongly PAS-positive reacting material (D, E) and are outlined by a thin rim of intense oxidative enzyme reaction (C), whereas the cytoplasmic areas surrounding the inclusions often resulted depleted of glycogen (E). Microscope magnification: ×200. The panel on the right shows muscle histopathology and immunohistochemistry in Patient 2. Three sets of serial sections (sets 1, 2, 3) were routinely stained with PAS (F), PAS-diastase (G), PAS-proteinase-k (H), immunolabelled for LC3 (in red) and p62/SQSTM1 (in green) (I), and for ubiquitin (J). The accumulated material is resistant to digestion with both diastase and proteinase-k, and it is strongly labelled for ubiquitin and p62/SQSTM1 (marker of protein aggregates). Microscope magnification: ×200 (sets 1 and 2) and ×400 (set 3). The lower panel shows immunoblot analysis of MuRF-1 and LC3-II proteins: muscles from all the Patients (1, 2, 3) have increased protein levels as compared with control (C) following normalization to myosin content. © 2014 British Neuropathological Society
NAN 2015; 41: 385–390
388
Scientific correspondence
(Figure 2) showed that muscles from all three patients have 1.5- to 4-fold increased levels of MuRF-1, and 2- to 4.5-fold increased levels of LC3-II, a marker of autophagosomes. Ultrastructural analysis of muscle from Patient 2 showed subsarcolemmal and intermyofibrillar areas without delimiting membranes and filled with granular material mixed with filamentous structures surrounded by glycogen (Figure 1), as well as accumulation of filamentous protein material that disrupted myofibrillar organization, Z-line fragmentation and sarcoplasmic reticulum disorganization. Patient 3 and Patient 1 showed large non-membrane-bound areas of granular material and in Patient 3 there were filamentous structures with adjacent normal glycogen particles and myofibrils misaligned by the large amount of granulofilamentous storage (Figure 1). Targeted next-generation sequencing (NGS) technique (Table S1) covering 98 neuromuscular genes and direct Sanger sequencing analysis of the GBE1, PFKM and RBCK1 genes showed no mutations. The RBCK1 polymorphism (c.22+33C>T) was identified in all patients. In earlier observations, PG bodies were reported to vary in number in different muscles from the same patient [10], and were absent in younger cases but present in older ones, suggesting that they might form slowly, and be the cause of the onset of muscle weakness [11]. This agrees with our findings that PG bodies were undetectable in two out of four muscles (obtained from very late-onset patient and in an advanced stage from another), emphasizing the value of repeated muscle biopsy when the diagnosis has not formerly been established. Similarly to that observed in RBCK1-related myopathy [7] and in polysaccharide storage myopathy [4,11], the PG bodies in our patients reacted strongly for ubiquitin, suggesting that the abnormal stored material becomes ubiquitinated to be degraded by the ubiquitinproteasome system (UPS). Ubiquitin is known to recognize abnormal proteins but not polysaccharides; however, the presence of glycogen-related proteins (glycogenin) in PGs may result in carbohydrate-protein aggregation. A functional link between proteasomal function and PG was provided by PG storage in mammalian cultured cells following the block of proteasomal activity [12]. We offered further evidence supporting the role of the UPS in PG storage myopathy, by demonstrating an over-expression of MuRF-1, a muscle-specific © 2014 British Neuropathological Society
E3-ubiquitin ligase involved in UPS degradation. The discovery of RBCK1 as a causative gene in PG body myopathy has added further clues to the knowledge of the pathogenetic mechanism. Indeed, RBCK-1 is an E3-ubiquitin ligase that has a pivotal role in determining the specificity of the UPS by recognizing target substrates. Our results suggest that, even in the absence of an identified genetic aetiology, the lysosomal-autophagic pathway plays a role in this disorder. Indeed, we have shown that PG bodies are strongly reacting for p62/ SQSTM1 (which binds aggregates of ubiquitinated proteins with LC3 to be delivered to autophagosomes and degraded by fusion with lysosomes), and we found increased levels of the lipidated LC3-II form (a marker of autophagy). However, autophagic flux can also be monitored by the levels of other autophagy substrates (p62/SQSTM1). When either proteasomal or lysosomal degradation is impaired, or when the amount of material to be degraded exceeds normal capacity, ubiquitinated proteins may aggregate and form autophagic vacuoles. We speculate that in this disease the accumulated PG are insufficiently degraded by the UPS, and may cause both induction of autophagy and impairment of autophagic flux. A dysfunction in the UPS and/or autophagic processes has been implicated in many pathological conditions. Increased p62/SQSTM1 level and impaired autophagy caused PG accumulation in the animal model of Lafora disease, which results from mutations in the gene encoding either for the glycogen phosphatase ‘laforin’ or for the E3-ubiquitin ligase ‘malin’. In the absence of laforin, abnormal glycogen might remain hyper-phosphorylated and aggregate to form PG bodies. The fact that two E3-ubiquitin ligases are responsible for PG storage diseases, suggests that a non-canonical glycogen degradation system is implicated in such disorders. Indeed, glycogen-selective autophagy might be effective in removing PG. While it seems clear that PG accumulates because of inefficient ubiquitin-proteasome and/or autophagic degradation, the mechanism generating PG bodies remains elusive. Earlier evidence has suggested an imbalance between the activities of glycogen synthase and branching enzyme. PG accumulation in Lafora disease was attributed to a previously unknown regulatory mechanism involving the proteasomal degradation of glycogen NAN 2015; 41: 385–390
Scientific correspondence
synthase and the adaptor protein targeting to glycogen, and may result from both impaired autophagy and glycogen synthesis dysregulation. In glycogenosis type IV, ubiquitination has been demonstrated to be a novel means of regulating glycogen debranching enzyme levels. The histopathological features in our family exactly matched those in RBCK1-related patients [7,9], whereas the clinical phenotype differed because of the lack of an overt cardiac involvement. Notably, two of three patients in our family presented an associated auto-immune disease (generalized vitiligo and CIDP) similar to those observed in RBCK1-mutant patients in whom the muscle disease was associated either with an immune disorder with chronic auto-inflammation and recurrent episodes of sepsis [9], or with other auto-immune disorders with immunological disturbances (psoriasis, non-specific granulomatous disease, type 1 diabetes, sarcoidosis) [7]. This is not surprising, as RBCK1 is also implicated in the NF-κB signalling, which is an important regulatory player of the immune system. After excluding those genes already recognized as causing inherited forms of PG body myopathy, and the negative result of NGS investigation on 98 muscle genes, the genetic origin of the disease in our family could be attributed either to RBCK1 gene mutations localized in non-coding regions, to the limitations of NGS in detecting mutations (e.g. deletion mutation), or to another still unknown gene. The observation that among 32 patients with PG body myopathy, about one-third cases have RBCK1 gene mutations [7], indicates that other unknown recessive gene(s) with a common pathogenetic pathway might be implicated. Whole exome sequencing will be used to clarify this issue.
Acknowledgements This work was supported by research grants from the Association Française contre les Myopathies (13859 to MF, 14199 to ACN, 14999 and 15696 to CA) and the Comitato Telethon Fondazione Onlus (GTB12001 to CA, and TGM11Z06, GUP10006, GUP11006 to VN). The study has been conducted according to the Local Ethical Committee approval. Muscle biopsies have been conducted as part of the diagnostic procedure, and a written consent was obtained from the patients before biopsy. The patient in the photograph has given the consent for the publication of his clinical pictures. © 2014 British Neuropathological Society
389
Author contribution MF designed the study, analysed and interpreted the data, and drafted the manuscript. ACN, GC, VP and MS collected, analysed and interpreted the data. VN and CA revised the manuscript for intellectual content.
Disclosures The authors declare no conflict of interest. M. Fanin* A. C. Nascimbeni* M. Savarese‡§ V. Papa† G. Cenacchi† V. Nigro‡§ C. Angelini*¶ *Department of Neurosciences, University of Padova, Padova, †Department of Radiology and Histopathological Sciences, ‘Alma Mater’ University of Bologna, Bologna, ‡Department of Biochemistry, Biophysics and General Pathology, 2nd University of Naples, Naples, §Telethon Institute of Genetics and Medicine, Naples, and ¶Fondazione IRCCS San Camillo Hospital, Venice, Italy
References 1 Schoser B, Bruno C, Schneider HC, Shin YS, Podskarbi T, Goldfarb L, Muller-Felber W, Muller-Hocker J. Unclassified polysaccharidosis of the heart and skeletal muscle in siblings. Mol Genet Metab 2008; 95: 52–8 2 Thompson AJ, Swash M, Cox EL, Ingram DA, Gray A, Schwarts MS. Polysaccharide storage myopathy. Muscle Nerve 1988; 11: 349–55 3 Tonin P, Tomelleri G, Vio M, Rizzuto N. Polyglucosan body myopathy: a new case. Neuromusc Disord 1992; 2: 419–22 4 Goebel HH, Shin YS, Gullotta F, Yokota T, Alroy J, Voit T, Haller P, Schulz A. Adult polyglucosan body myopathy. J Neuropathol Exp Neurol 1992; 51: 24–35 5 Jeub M, Kappes-Horn K, Kornblum C, Fischer D. Polyglucosan body myopathy. Nervenarzt 2006; 77: 1487–91 6 Greene GM, Weldon DC, Ferrans VJ, Cheatham JP, McComb RD, Brown BI, Gumbiner CH, Vanderhoof JA, Itkin PG, McManus BM. Juvenile polysaccharidosis with cardioskeletal myopathy. Arch Pathol Lab Med 1987; 111: 977–82 7 Nilsson J, Schoser B, Laforet P, Kalev O, Lindberg C, Romero NB, Davila Lopez M, Akman HO, Wahbi K, Iglseder S, Eggers C, Engel AG, DiMauro S, Oldfors A. Polyglucosan body myopathy caused by defective ubiquitin ligase RBCK1. Ann Neurol 2013; 74: 914–19 NAN 2015; 41: 385–390
390
Scientific correspondence
8 Wang K, Kim C, Bradfield J, Guo Y, Toskala E, Otieno FG, Hou C, Thomas K, Cardinale C, Lyon GJ, Gohlar R, Hakonarson H. Whole-genome DNA/RNA sequencing identifies truncating mutations in RBCK1 in a novel mendelian disease with neuromuscular and cardiac involvement. Genome Med 2013; 5: 67–74 9 Boisson B, Laplantine E, Prando C, Giliani S, Israelsson E, Xu Z, Abhyankar A, Israel L, Trevejo-Nunez G, Bogunovic D, Capika AM, MacDuffa D, Chrabieh M, Hubeau M, Bajolle F, Debre M, Mazzolari E, Vairo D, Agou F, Virgin HW, Bossuyt X, Rambaud C, Facchetti F, Bonnet D, Quartier P, Fournet JC, Pascual V, Chaussabel D, Notarangelo LD, Puel A, Israel A, Casanova JL, Picard C. Immunodeficiency, autoinflammation and amylopectinosis in humans with inherited HOIL-1 and LUBAC deficiency. Nat Immunol 2012; 13: 1178–88 10 Weis J, Schroder JM. Adult polyglucosan body myopathy with subclinical peripheral neuropathy: case report and review of diseases associated with polyglucosan body accumulation. Clin Neuropathol 1988; 7: 271–9
© 2014 British Neuropathological Society
11 Valentine BA, Cooper BJ. Development of polyglucosan inclusions in skeletal muscle. Neuromusc Disord 2006; 16: 603–7 12 Puri R, Jain N, Ganesh S. Increased glucose concentration results in reduced proteasomal activity and the formation of glycogen positive aggresomal structures. FEBS J 2011; 278: 3688–98
Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Table S1. Targeted next-generation sequencing. Received 18 April 2014 Accepted after revision 6 July 2014 Published online Article Accepted on 15 July 2014
NAN 2015; 41: 385–390
doi: 10.1111/nan.12171
Scientific correspondence
Familial polyglucosan body myopathy with unusual phenotype Polyglucosan (PG) is an abnormal polysaccharide that, compared with glycogen, has fewer branched points and excessively long peripheral chains that structurally resemble the plant polysaccharide ‘amylopectin’. Under electron microscopy, PG bodies are round, nonmembrane-bound cytoplasmic particles with irregular branched filaments, which often displace myofibrils, leading to Z disk streaming. PG bodies react strongly to PAS stain and are partially resistant to diastase digestion. PG accumulation constitutes the histopathological manifestation of several clinically different conditions. It occurs mainly in the brain (Lafora disease, double athetosis) and in skeletal muscle (glycogenosis type IV, glycogenosis type VII, other polysaccharide storage myopathies [1–6]). Recently, recessive mutations in the RBCK1 gene, which encodes for an E3 ubiquitin ligase, have been recognized as causing two different disorders with PG accumulation in skeletal and cardiac muscle: a myopathy with cardiomyopathy [7,8], and a fatal immunodeficiency with myopathy and cardiomyopathy [9]. To date, only 10 families have been reported in which PG body myopathy is caused by RBCK1 gene mutations. Here we describe a new family (including one pair of siblings, Patient 1 and Patient 2, and their paternal female cousin, Patient 3), presenting as an autosomal recessive form of late-onset myopathy with limb girdle muscular dystrophy phenotype and PG accumulation in muscle. Patient 1. This 66-year-old man had complained for many years of back pain, progressive difficulty walking and climbing stairs and weakness in muscles of both girdles. He presented with a waddling gait, weakness in ileopsoas, leg and arm flexor muscles, calf hypertrophy, pes cavus and moderately increased creatine kinase level. At age 74 years he was unable to rise from sitting (Figure 1) and was able to walk only with the aid of a cane. Cardiac investigations revealed a bundle branch block and an ischemic cardiomyopathy. He also presented with skin lesions characteristic of generalized vitiligo © 2014 British Neuropathological Society
(Figure 1). Patient 2. At age 52 years she complained of progressive difficulty in climbing stairs and rising from a chair, and noticed asthenia in the lower limbs. Creatine kinase level was mildly increased. At age 57 years she was diagnosed with familial spastic paraparesis with axonal motor polyneuropathy, and with chronic inflammatory demyelinating polyneuropathy (CIDP). She presented with a waddling gait, inability to rise from the floor without support, weakness in proximal limb muscles, pes cavus, calf hypertrophy and hand muscle atrophy. No cardiac symptoms were reported. At age 60 years she became unable to walk unsupported. Patient 3. Since she was 50 years she had complained of limb myalgia. After an ischemic stroke at age 52, causing sensory-motor hemi-syndrome, she noticed progressive weakness in proximal limb girdle muscles. At age 63 years, when the first muscle biopsy was done, she presented with a waddling gait, Gowers sign, normal EMG and CK levels. At age 70 years, when a second muscle biopsy was performed, she was unable to raise her arms over her head, had scapular winging, and quadriceps muscle wasting. No cardiac symptoms were reported. Two out of the four muscle biopsies available for this study (Patient 1, first biopsy Patient 3) showed myopathic features but neither vacuoles nor accumulation of PASpositive material. Conversely, the other two muscles (Patient 2, second biopsy Patient 3) were characterized by violet or hyaline PG inclusions in about 10–20% of fibres (Figure 2). The vacuoles were filled with strongly PASpositive reacting material, whereas the cytoplasmic areas surrounding the inclusions were depleted in glycogen. The vacuoles were outlined by a rim of intense oxidative enzyme reaction, but their membranes were not immunolabelled by caveolin-3. The accumulated material showed absent staining with Lugol’s iodine and acid phosphatase, negative immunolabelling for desmin and myotilin; it was resistant to pre-digestion with both diastase/amylase and proteinase-k, and was strongly immunolabelled for ubiquitin and for p62/SQSTM1 (protein aggregates) (Figure 2). Scattered LC3-positive reaction was present in most fibres. Immunoblot analysis 385
386
Scientific correspondence
Figure 1. The upper panel shows the clinical phenotype of Patient 1, which is characterized by severe weakness in upper and lower girdle muscles (D, E, F), causing inability to rise from sitting without support. Note the generalized distribution of skin lesions corresponding to vitiligo, involving the head (D), the neck (B), the hands and the forearms (A, C, E), the thighs and the legs (F). The lower panel shows electron microscopy of muscle biopsies from Patient 3 (A), Patient 2 (B–E) and Patient 1 (F) showing glycogen filamentous areas without delimiting membrane, and myofibrils that appeared misaligned by the large granulofilamentous storage (A). Abundant subsarcolemmal and intermyofibrillar accumulation of filamentous material is surrounded by β-particles glycogen (B, C, F). Note an internalized nucleus and accumulation of filamentous protein material that disrupt myofibrils, dispersed vesicles and Z-line remnants (D), abundant glycogen accumulation that disrupts myofibrillar organization with Z-line fragmentation and sarcoplasmic reticulum disorganization (E). © 2014 British Neuropathological Society
NAN 2015; 41: 385–390
Scientific correspondence
387
Figure 2. The panel on the left shows muscle histopathology of Patient 2 and Patient 3. Sections stained with haematoxylin-eosin (H&E) (A), Gomori trichrome (B), NADH-TR reductase (C) and PAS (D, E) show many fibres displaying violet or hyaline inclusions localized in subsarcolemmal and intracytoplasmic position (A, B). The vacuoles are filled with strongly PAS-positive reacting material (D, E) and are outlined by a thin rim of intense oxidative enzyme reaction (C), whereas the cytoplasmic areas surrounding the inclusions often resulted depleted of glycogen (E). Microscope magnification: ×200. The panel on the right shows muscle histopathology and immunohistochemistry in Patient 2. Three sets of serial sections (sets 1, 2, 3) were routinely stained with PAS (F), PAS-diastase (G), PAS-proteinase-k (H), immunolabelled for LC3 (in red) and p62/SQSTM1 (in green) (I), and for ubiquitin (J). The accumulated material is resistant to digestion with both diastase and proteinase-k, and it is strongly labelled for ubiquitin and p62/SQSTM1 (marker of protein aggregates). Microscope magnification: ×200 (sets 1 and 2) and ×400 (set 3). The lower panel shows immunoblot analysis of MuRF-1 and LC3-II proteins: muscles from all the Patients (1, 2, 3) have increased protein levels as compared with control (C) following normalization to myosin content. © 2014 British Neuropathological Society
NAN 2015; 41: 385–390
388
Scientific correspondence
(Figure 2) showed that muscles from all three patients have 1.5- to 4-fold increased levels of MuRF-1, and 2- to 4.5-fold increased levels of LC3-II, a marker of autophagosomes. Ultrastructural analysis of muscle from Patient 2 showed subsarcolemmal and intermyofibrillar areas without delimiting membranes and filled with granular material mixed with filamentous structures surrounded by glycogen (Figure 1), as well as accumulation of filamentous protein material that disrupted myofibrillar organization, Z-line fragmentation and sarcoplasmic reticulum disorganization. Patient 3 and Patient 1 showed large non-membrane-bound areas of granular material and in Patient 3 there were filamentous structures with adjacent normal glycogen particles and myofibrils misaligned by the large amount of granulofilamentous storage (Figure 1). Targeted next-generation sequencing (NGS) technique (Table S1) covering 98 neuromuscular genes and direct Sanger sequencing analysis of the GBE1, PFKM and RBCK1 genes showed no mutations. The RBCK1 polymorphism (c.22+33C>T) was identified in all patients. In earlier observations, PG bodies were reported to vary in number in different muscles from the same patient [10], and were absent in younger cases but present in older ones, suggesting that they might form slowly, and be the cause of the onset of muscle weakness [11]. This agrees with our findings that PG bodies were undetectable in two out of four muscles (obtained from very late-onset patient and in an advanced stage from another), emphasizing the value of repeated muscle biopsy when the diagnosis has not formerly been established. Similarly to that observed in RBCK1-related myopathy [7] and in polysaccharide storage myopathy [4,11], the PG bodies in our patients reacted strongly for ubiquitin, suggesting that the abnormal stored material becomes ubiquitinated to be degraded by the ubiquitinproteasome system (UPS). Ubiquitin is known to recognize abnormal proteins but not polysaccharides; however, the presence of glycogen-related proteins (glycogenin) in PGs may result in carbohydrate-protein aggregation. A functional link between proteasomal function and PG was provided by PG storage in mammalian cultured cells following the block of proteasomal activity [12]. We offered further evidence supporting the role of the UPS in PG storage myopathy, by demonstrating an over-expression of MuRF-1, a muscle-specific © 2014 British Neuropathological Society
E3-ubiquitin ligase involved in UPS degradation. The discovery of RBCK1 as a causative gene in PG body myopathy has added further clues to the knowledge of the pathogenetic mechanism. Indeed, RBCK-1 is an E3-ubiquitin ligase that has a pivotal role in determining the specificity of the UPS by recognizing target substrates. Our results suggest that, even in the absence of an identified genetic aetiology, the lysosomal-autophagic pathway plays a role in this disorder. Indeed, we have shown that PG bodies are strongly reacting for p62/ SQSTM1 (which binds aggregates of ubiquitinated proteins with LC3 to be delivered to autophagosomes and degraded by fusion with lysosomes), and we found increased levels of the lipidated LC3-II form (a marker of autophagy). However, autophagic flux can also be monitored by the levels of other autophagy substrates (p62/SQSTM1). When either proteasomal or lysosomal degradation is impaired, or when the amount of material to be degraded exceeds normal capacity, ubiquitinated proteins may aggregate and form autophagic vacuoles. We speculate that in this disease the accumulated PG are insufficiently degraded by the UPS, and may cause both induction of autophagy and impairment of autophagic flux. A dysfunction in the UPS and/or autophagic processes has been implicated in many pathological conditions. Increased p62/SQSTM1 level and impaired autophagy caused PG accumulation in the animal model of Lafora disease, which results from mutations in the gene encoding either for the glycogen phosphatase ‘laforin’ or for the E3-ubiquitin ligase ‘malin’. In the absence of laforin, abnormal glycogen might remain hyper-phosphorylated and aggregate to form PG bodies. The fact that two E3-ubiquitin ligases are responsible for PG storage diseases, suggests that a non-canonical glycogen degradation system is implicated in such disorders. Indeed, glycogen-selective autophagy might be effective in removing PG. While it seems clear that PG accumulates because of inefficient ubiquitin-proteasome and/or autophagic degradation, the mechanism generating PG bodies remains elusive. Earlier evidence has suggested an imbalance between the activities of glycogen synthase and branching enzyme. PG accumulation in Lafora disease was attributed to a previously unknown regulatory mechanism involving the proteasomal degradation of glycogen NAN 2015; 41: 385–390
Scientific correspondence
synthase and the adaptor protein targeting to glycogen, and may result from both impaired autophagy and glycogen synthesis dysregulation. In glycogenosis type IV, ubiquitination has been demonstrated to be a novel means of regulating glycogen debranching enzyme levels. The histopathological features in our family exactly matched those in RBCK1-related patients [7,9], whereas the clinical phenotype differed because of the lack of an overt cardiac involvement. Notably, two of three patients in our family presented an associated auto-immune disease (generalized vitiligo and CIDP) similar to those observed in RBCK1-mutant patients in whom the muscle disease was associated either with an immune disorder with chronic auto-inflammation and recurrent episodes of sepsis [9], or with other auto-immune disorders with immunological disturbances (psoriasis, non-specific granulomatous disease, type 1 diabetes, sarcoidosis) [7]. This is not surprising, as RBCK1 is also implicated in the NF-κB signalling, which is an important regulatory player of the immune system. After excluding those genes already recognized as causing inherited forms of PG body myopathy, and the negative result of NGS investigation on 98 muscle genes, the genetic origin of the disease in our family could be attributed either to RBCK1 gene mutations localized in non-coding regions, to the limitations of NGS in detecting mutations (e.g. deletion mutation), or to another still unknown gene. The observation that among 32 patients with PG body myopathy, about one-third cases have RBCK1 gene mutations [7], indicates that other unknown recessive gene(s) with a common pathogenetic pathway might be implicated. Whole exome sequencing will be used to clarify this issue.
Acknowledgements This work was supported by research grants from the Association Française contre les Myopathies (13859 to MF, 14199 to ACN, 14999 and 15696 to CA) and the Comitato Telethon Fondazione Onlus (GTB12001 to CA, and TGM11Z06, GUP10006, GUP11006 to VN). The study has been conducted according to the Local Ethical Committee approval. Muscle biopsies have been conducted as part of the diagnostic procedure, and a written consent was obtained from the patients before biopsy. The patient in the photograph has given the consent for the publication of his clinical pictures. © 2014 British Neuropathological Society
389
Author contribution MF designed the study, analysed and interpreted the data, and drafted the manuscript. ACN, GC, VP and MS collected, analysed and interpreted the data. VN and CA revised the manuscript for intellectual content.
Disclosures The authors declare no conflict of interest. M. Fanin* A. C. Nascimbeni* M. Savarese‡§ V. Papa† G. Cenacchi† V. Nigro‡§ C. Angelini*¶ *Department of Neurosciences, University of Padova, Padova, †Department of Radiology and Histopathological Sciences, ‘Alma Mater’ University of Bologna, Bologna, ‡Department of Biochemistry, Biophysics and General Pathology, 2nd University of Naples, Naples, §Telethon Institute of Genetics and Medicine, Naples, and ¶Fondazione IRCCS San Camillo Hospital, Venice, Italy
References 1 Schoser B, Bruno C, Schneider HC, Shin YS, Podskarbi T, Goldfarb L, Muller-Felber W, Muller-Hocker J. Unclassified polysaccharidosis of the heart and skeletal muscle in siblings. Mol Genet Metab 2008; 95: 52–8 2 Thompson AJ, Swash M, Cox EL, Ingram DA, Gray A, Schwarts MS. Polysaccharide storage myopathy. Muscle Nerve 1988; 11: 349–55 3 Tonin P, Tomelleri G, Vio M, Rizzuto N. Polyglucosan body myopathy: a new case. Neuromusc Disord 1992; 2: 419–22 4 Goebel HH, Shin YS, Gullotta F, Yokota T, Alroy J, Voit T, Haller P, Schulz A. Adult polyglucosan body myopathy. J Neuropathol Exp Neurol 1992; 51: 24–35 5 Jeub M, Kappes-Horn K, Kornblum C, Fischer D. Polyglucosan body myopathy. Nervenarzt 2006; 77: 1487–91 6 Greene GM, Weldon DC, Ferrans VJ, Cheatham JP, McComb RD, Brown BI, Gumbiner CH, Vanderhoof JA, Itkin PG, McManus BM. Juvenile polysaccharidosis with cardioskeletal myopathy. Arch Pathol Lab Med 1987; 111: 977–82 7 Nilsson J, Schoser B, Laforet P, Kalev O, Lindberg C, Romero NB, Davila Lopez M, Akman HO, Wahbi K, Iglseder S, Eggers C, Engel AG, DiMauro S, Oldfors A. Polyglucosan body myopathy caused by defective ubiquitin ligase RBCK1. Ann Neurol 2013; 74: 914–19 NAN 2015; 41: 385–390
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Scientific correspondence
8 Wang K, Kim C, Bradfield J, Guo Y, Toskala E, Otieno FG, Hou C, Thomas K, Cardinale C, Lyon GJ, Gohlar R, Hakonarson H. Whole-genome DNA/RNA sequencing identifies truncating mutations in RBCK1 in a novel mendelian disease with neuromuscular and cardiac involvement. Genome Med 2013; 5: 67–74 9 Boisson B, Laplantine E, Prando C, Giliani S, Israelsson E, Xu Z, Abhyankar A, Israel L, Trevejo-Nunez G, Bogunovic D, Capika AM, MacDuffa D, Chrabieh M, Hubeau M, Bajolle F, Debre M, Mazzolari E, Vairo D, Agou F, Virgin HW, Bossuyt X, Rambaud C, Facchetti F, Bonnet D, Quartier P, Fournet JC, Pascual V, Chaussabel D, Notarangelo LD, Puel A, Israel A, Casanova JL, Picard C. Immunodeficiency, autoinflammation and amylopectinosis in humans with inherited HOIL-1 and LUBAC deficiency. Nat Immunol 2012; 13: 1178–88 10 Weis J, Schroder JM. Adult polyglucosan body myopathy with subclinical peripheral neuropathy: case report and review of diseases associated with polyglucosan body accumulation. Clin Neuropathol 1988; 7: 271–9
© 2014 British Neuropathological Society
11 Valentine BA, Cooper BJ. Development of polyglucosan inclusions in skeletal muscle. Neuromusc Disord 2006; 16: 603–7 12 Puri R, Jain N, Ganesh S. Increased glucose concentration results in reduced proteasomal activity and the formation of glycogen positive aggresomal structures. FEBS J 2011; 278: 3688–98
Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Table S1. Targeted next-generation sequencing. Received 18 April 2014 Accepted after revision 6 July 2014 Published online Article Accepted on 15 July 2014
NAN 2015; 41: 385–390
