Autophagic Vacuolar Myopathy Research Articles

Activation of the Keap1/Nrf2 stress response pathway in autophagic vacuolar myopathies.

Nrf2 (nuclear factor [erythroid-derived 2]-like 2; the transcriptional master regulator of the antioxidant stress response) is regulated through interaction with its cytoplasmic inhibitor Keap1 (Kelch-like ECH-associated protein 1), which under basal conditions targets Nrf2 for proteasomal degradation. Sequestosome 1 (SQSTM1)/p62–a multifunctional adapter protein that accumulates following autophagy inhibition and can serve as a diagnostic marker for human autophagic vacuolar myopathies (AVMs)–was recently shown to compete with Nrf2 for Keap1 binding, resulting in activation of the Nrf2 pathway. In this study, we used 55 human muscle biopsies divided into five groups [normal control, hydroxychloroquine- or colchicine-treated non-AVM control, hydroxychloroquine- or colchicine-induced toxic AVM, polymyositis, and inclusion body myositis (IBM)] to evaluate whether Keap1-SQSTM1 interaction led to increased Nrf2 signaling in human AVMs. In toxic AVMs and IBM, but not in control muscle groups or polymyositis, Keap1 antibody labeled sarcoplasmic protein aggregates that can be used as an alternate diagnostic marker for both AVM types; these Keap1-positive aggregates were co-labeled with the antibody against SQSTM1 but not with the antibody against autophagosome marker LC3 (microtubule-associated protein 1 light chain 3). In human AVM muscle, sequestration of Keap1 into the SQSTM1-positive protein aggregates was accompanied by an increase in mRNA and protein levels of Nrf2 target genes; similarly, treatment of differentiated C2C12 myotubes with autophagy inhibitor chloroquine led to an increase in the nuclear Nrf2 protein level and an increase in expression of the Nrf2-regulated genes. Taken together, our findings demonstrate that Nrf2 signaling is upregulated in autophagic muscle disorders and raise the possibility that autophagy disruption in skeletal muscle leads to dysregulation of cellular redox homeostasis.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-016-0384-6) contains supplementary material, which is available to authorized users.

P.349 - Myofibrillar myopathies with autophagic vacuoles: Report of a case series

The presence of autophagic vacuoles containing lysosomal, autophagosomal and sarcolemmal membrane components depicts autophagic vacuolar myopathies as a spectrum of disorders unified by distinctive myopathologic features. While not being classified as autophagic vacuolar myopathies, also muscle disorders including myopathies with rimmed vacuoles, inclusion body myopathies and myofibrillar myopathies (MFM) have autophagic pathology. Here, we present the diagnostic algorithm which led us to the diagnosis of MFM in four unrelated patients with autophagic vacuolar myopathies. Patient 1 is a 39-year-old male, that came to our attention at the age of 35 for hyperCKemia (5× normal value) and a mild exercise intolerance; his clinical conditions have worsened in the last years of follow up, with the appearance of a progressive limb girdle weakness resembling a LGMD-like phenotype. Patient 2 is a 74-year-old woman with a 7-year history of a progressive axial and pelvic girdle weakness, in association with a moderate increase of blood CK (4× normal value). Patients 3 and 4 are respectively 19-year-old and 37-year-old males that present an asymptomatic hyperckemia (5× normal value), without muscle weakness. None of them is affected by cardiomyopathy. Their family history is inconsistent for neuromuscular diseases. The ultrastructural studies of the muscle biopsies have showed myofibrillar myopathy features, including progressive myofibrillar degeneration commencing at the Z-disk, accumulation of degraded filamentous material and entrapment of dislocated membranous organelles in autophagic vacuoles.

Vacuolar myopathic lesion in a Holstein-Friesian dairy cow affected with bovine dilated cardiomyopathy: a preliminary report

Occurrence of vacuolar myopathy caused by factors other than inherited glycogen storage condition is uncommon in domestic animals. This preliminary report describes the detection of a vacuolar myopathic lesion in a 13-year-old Holstein- Friesian dairy cow which showed an 8-day history of cardiac disease. Gross inspection after slaughter revealed edema in a variety of tissues, dilatation and hypertrophy of the cardiac ventricles, and hepatic congestion. Histopathology revealed degenerative myocardial lesions and passive hepatic congestion, which were identical to those previously documented in cases of bovine dilated cardiomyopathy. Skeletal muscles represented a vacuolar myopathic lesion characterized by the presence of distinctive vacuoles in atrophic myofibers, which occurred singly in the central zone of each myofiber on transverse sections and showed an irregularly elongated appearance on longitudinal sections. Such vacuoles frequently contained a small amount of weakly eosinophilic, granular, debris-like, or amorphous substances, displaying a feature of autophagic vacuoles. These vacuolated myofibers were more prominently observed in some muscle fascicles of the longissimus thoracis muscle than other muscles examined. The vacuolar myopathic lesion recognized in this cow was similar to some degree to that of autophagic vacuolar myopathy in man. Further studies are necessary to clarify a causal relationship between this vacuolar myopathic lesion and the disorder of bovine dilated cardiomyopathy.

A novel mutation of CLN3 associated with delayed-classic juvenile ceroid lipofuscinois and autophagic vacuolar myopathy

Juvenile neuronal-ceroid-lipofuscinosis (JNCL) is a lysosomal storage disease caused by mutations in CLN3. The most frequent mutation is a 1.02-kb deletion that, when homozygous, causes the classical clinical presentation.Patients harboring mutations different than the major deletion show a marked clinical heterogeneity, including protracted disease course with possible involvement of extraneuronal tissues. Cardiac involvement is relatively rare in JNCL and it is usually due to myocardial storage of ceroid-lipofuscinin. Only recently, histopathological findings of autophagic vacuolar myopathy (AVM) were detected in JNCL patients with severe cardiomyopathy.We describe a 35-year-old male showing a delayed-classic JNCL with visual loss in childhood and neurological manifestations only appearing in adult life. He had an unusual CLN3 genotype with an unreported deletion (p.Ala349_Leu350del) and the known p.His315Glnfs*67 mutation. Autophagic vacuolar myopathy was shown by muscle biopsy. At clinical follow-up, moderately increased CPK levels were detected whereas periodic cardiac assessments have been normal to date.Adult neurologists should be aware of protracted JNCL as cause of progressive neurological decline in adults. The occurrence of autophagic vacuolar myopathy necessitates periodic cardiac surveillance, which is not usually an issue in classic JNCL due to early neurological death.

Property of Lysosomal Storage Disease Associated with Midbrain Pathology in the Central Nervous System of Lamp-2–Deficient Mice

Lysosome-associated membrane protein-2 (LAMP-2) is the gene responsible for Danon disease, which is characterized by cardiomyopathy, autophagic vacuolar myopathy, and variable mental retardation. To elucidate the function of LAMP-2 in the central nervous system, we investigated the neuropathological changes in Lamp-2-deficient mice. Immunohistochemical observations revealed that Lamp-1 and cathepsin D-positive lysosomal structures increased in the large neurons of the mouse brain. Ubiquitin-immunoreactive aggregates and concanavalin A-positive materials were detected in these neurons. By means of ultrastructural studies, we found various-shaped accumulations, including lipofuscin, glycolipid-like materials, and membranous structures, in the neurons and glial cells of Lamp-2-deficient brains. In deficient mice, glycogen granules accumulated in hepatocyte lysosomes but were not observed in neurons. These pathological features indicate lysosomal storage disease; however, the findings are unlikely a consequence of deficiency of a single lysosomal enzyme. Although previous study results have shown a large amount of autophagic vacuoles in parenchymal cells of the visceral organs, these findings were rarely detected in the brain tissue except for some axons in the substantia nigra, in which abundant activated microglial cells with increased lipid peroxidation were observed. Thus, LAMP-2 in the central nervous system has a possible role in the degradation of the various macromolecules in lysosomes and an additional function concerning protection from oxidative stress, especially in the substantia nigra.

Novel CLN3 mutation causing autophagic vacuolar myopathy

In the article “Novel CLN3 mutation causing autophagic vacuolar myopathy” by A. Cortese et …

X-linked myopathy with excessive autophagy: a failure of self-eating

Autophagic vacuolar myopathies (AVMs) are a group of disorders united by shared histopathological features on muscle biopsy that include the aberrant accumulation of autophagic vacuoles. The classic conditions that compose the AVMs include Pompe Disease, Danon Disease and X-linked myopathy with excessive autophagy (XMEA). Other disorders, including acquired myopathies like chloroquine toxicity, also have features of an autophagic myopathy. This review is focused on XMEA, a myopathy with onset of slowly progressive proximal weakness and elevated serum creatine kinase (2×to 20×normal) typically in the first decade of life. However, both late-adult onset and severe, sometimes lethal, neonatal cases also occur. Skeletal muscle pathology is characterized by numerous cytoplasmic autophagic vacuoles, complex muscle fiber splitting with internalization of capillaries, and complement C5b-9 deposition within vacuoles and along the sarcolemma. The autophagic vacuoles have sarcolemmal features. Mutations in the VMA21 gene at Xq28 cause XMEA by reducing the activity of lysosomal hydrolases. The VMA21 protein regulates the assembly of the V-ATPase required to acidify the lysosome. Increased lysosomal pH and poor degradation of cellular debris may secondarily induce autophagy, the net effect being accumulation of autophagolysosomes. The relationship of XMEA to other lysosomal disorders of muscle and potential therapeutic interventions for XMEA are discussed.

New Pathogenetic Mechanisms that Link Autophagy to Pompe Disease

Skeletal muscle is one of the tissues with the highest rate of autophagosome formation and degradation. A group of congenital myopathies, characterised by lysosomal alteration, are described by massive autophagic build-up and are named Autophagic Vacuolar Myopathies (AVM). Among these disorders, Danon disease (DD) and Pompe (GSD2) are characterised by the presence of large glycogen-fi lled lysosomes in the skeletal muscle. We have recently shown that autophagy impairment contributes to disease progression in GSD2 patients. However, the mechanism that leads to autophagy inhibition, and whether this impairment is shared in other AVM in humans is an open issue.

Autophagic vacuolar pathology in desminopathies

Autophagic vacuolar myopathies are an emerging group of muscle diseases with common pathologic features. These include autophagic vacuoles containing both lysosomal and autophagosomal proteins sometimes lined with sarcolemmal proteins such as dystrophin. These features have been most clearly described in patients with Danon's disease due to LAMP2 deficiency and X-linked myopathy with excessive autophagy (XMEA) due to mutations in VMA21. Disruptions of these proteins lead to lysosomal dysfunction and subsequent autophagic vacuolar pathology. We performed whole exome sequencing on two families with autosomal dominantly inherited myopathies with autophagic vacuolar pathology and surprisingly identified a p.R454W tail domain mutation and a novel p.S6W head domain mutation in desmin, DES. In addition, re-evaluation of muscle tissue from another family with a novel p.I402N missense DES mutation also identified autophagic vacuoles. We suggest that autophagic vacuoles may be an underappreciated pathology present in desminopathy patient muscle. Moreover, autophagic vacuolar pathology can be due to genetic etiologies unrelated to primary defects in the lysosomes or autophagic machinery. Specifically, cytoskeletal derangement and the accumulation of aggregated proteins such as desmin may activate the autophagic system leading to the pathologic features of an autophagic vacuolar myopathy.

A.P.13: Myotonic discharges in autophagic vacuolar myopathies: A potential electrophysiologic marker

Disordered lysosomal function within muscle cells is the basis for a group of diseases referred to as autophagic vacuolar myopathies. These disorders include acid maltase deficiency (Pompe disease), LAMP-2 deficiency (Danon disease), and X-linked myopathy with excess autophagy (XMEA). We present a 65year old diabetic woman with bilateral leg weakness evolving over 2years to the point she was unable to ambulate without assistance. There was no family history of a similar disorder. She had prominent weakness in her legs proximally>distally while arms and bulbar muscles had normal strength. Mild percussion myotonia of forearm muscles was present but not hand grasp myotonia. CK was 914. Acid alpha-glucosidase activity was normal. Nerve conduction studies showed evidence of a largely axonal neuropathy likely related to her poorly controlled diabetes. Needle exam demonstrated fibrillations and frequent short duration myotonic discharges in arm and leg muscles. Quadriceps muscle biopsy showed basophilic cytoplasmic stippling within myofibers that corresponded to small vacuoles discernable by enzyme histochemical staining for acetylcholinesterase and immunostaining for dystrophin and other sarcolemmal proteins. Cytoplasmic clusters of lysosomes and autophagosome extrusion with duplication of basement membranes were observed with electron microscopy. The biopsy findings are those of an autophagic vacuolar myopathy, the underlying nature of which has not been defined. Frequent short duration myotonic discharges were a remarkable feature. The presence of myotonic discharges has been previously reported to occur in patients with Pompe disease, Danon disease, and XMEA. These discharges indicate hyperexcitability of the muscle membranes and may reflect structural sarcoplasmic changes. Their occurrence in the various disorders involving autophagy suggests that short myotonic discharges may be a diagnostically useful electrophysiologic marker for this group of disorde.

A.P.16: LysoPlex: a “preferential exome” strategy to clarify molecular mechanisms of autophagic vacuolar myopathies

The autophagic vacuolar myopathies (AVMs) are an emerging group of heterogeneous muscular disorders, characterized by the presence of autophagic vacuoles, whose membranes exhibit sarcolemmal features. Although several myopathies with these features have been described, the only one in which the gene defect is known is the Danon disease, due to mutations in the lysosome-associated membrane protein-2 (LAMP2). Given the pathologic features similar to those in Danon disease, the molecular defects in the remaining AVMs could involve proteins related to lysosomal function. Recent evidence has demonstrated lysosomes are important in maintaining cellular processes in skeletal muscle and autophagy acts as contributor to disease pathogenesis and progression. We have developed a "preferential exome" NGS workflow, named LysoPlex, to sequence at high coverage 12,786 human exons of 891 genes. These genes are predicted to be involved in lysosomal function, endocytosis and autophagy pathway and most of them are not yet associated to known genetic disorders. We designed the enrichment probes using a Haloplex custom platform targeting 99.48% of exons. Until now, with Lysoplex, we have been able to perform molecular diagnosis of lysosomal storage disorders (LSDs) and neuronal ceroid lipofuscinoses (NCL), identifying disease-causing mutations in 70 patients and pointing out putative novel causative genes. Moreover, we are recruiting samples from AVM patients in order to elucidate their molecular defects. By using this preferential exome, in fact, we have a complete view of sequence variants in the genes involved in the lysosomal-autophagic pathway. In conclusion, our strategy represents a powerful approach to study the role of lysosomes and autophagy in skeletal muscles and in muscular disorders.

G.P.149: Unique brick-red auto-fluorescence of reducing bodies and protein aggregates is a useful diagnostic biopsy marker for FHL1-associated myopathies

Mutations in the four and a half LIM domain protein 1 (FHL1) gene are causative of four distinct human myopathies with a wide clinicopathological spectrum. A biopsy diagnosis can be difficult due to overlap with other protein aggregation myopathies and dystrophic changes. The main aim was to assess the auto-fluorescent characteristics of inclusions in biopsies from molecularly confirmed cases of FHL1 and compare them with other protein aggregation myopathies. 14 muscle biopsies taken from patients referred to our neuromuscular centre were reviewed retrospectively (FHL1 = 4, Bag3 = 3, Desmin = 1, Myotilin = 1, Acid maltase = 1, Colchicine myopathy = 1, Autophagic vacuolar myopathy = 1, Inclusion body myositis = 2). Frozen sections stained with haematoxylin and eosin were assessed with bright field microscopy and fluorescent microscopy using BGR multi-excitation wavelength filter on a Leica DM2500 microscope except in one FHL1 case where paraffin sections were examined. Relevant histochemistry, immunostains and electron microscopy were also assessed. Unique brick-red auto-fluorescence of the inclusions including discrete reducing bodies and majority of diffuse protein aggregates was seen only in frozen sections of FHL1 cases. Non-overlapping but closely interacting brick-red auto-fluorescence of FHL1 inclusions with yellow-orange autofluorescence of desmin and myotilin was also seen. Red auto-fluorescent material was present in and around degenerate myonuclei. Reducing bodies and diffuse reducing body material was confirmed electron microscopically. Protein aggregates in all other non-FHL1 cases showed variable yellow to orange auto-fluorescence. Menadione-NBT reduction without substrate was also seen in non-FHL1 protein aggregates. Brick-red auto-fluorescence appears to be a unique property of FHL1 containing reducing bodies and protein aggregates and may be a useful biopsy marker for the diagnosis of FHL1 myopathies.

A.P.12: A novel, EPG5-related vacuolar myopathy

Autophagy is a fundamental cellular degradative pathway and involves several tightly regulated steps conserved throughout evolution. We have recently identified recessive null mutations in EPG5, encoding a protein with a crucial role in autolysosome formation, as the cause of Vici syndrome (VS), a severe multisystem disorder characterized by callosal agenesis, cataracts, cardiomyopathy, combined immunodeficiency and hypopigmentation. The consistently associated myopathy shares prominent vacuoles as the most striking histopathological feature with the primary autophagic vacuolar myopathies (AVMs), Danon disease and X-linked myopathy with excessive autophagy (MEAX). Here we report 2 sisters presenting from childhood with generalized weakness, elevated CK levels (5–10× normal) and a characteristic pattern of selective involvement on muscle MRI. Progression was relentless in the index case, resulting in almost complete paralysis requiring a gastrostomy and continuous invasive ventilation within a decade. Muscle biopsies showed increased variability in fibre size, multiple internal nuclei, fat and endomysial connective tissue increase, and abnormal glycogen content. Autophagic vacuoles with sarcolemmal features were prominent and confirmed on EM. Whole exome sequencing revealed a paternally inherited heterozygous EPG5 frame shift mutation (g.43534647G>A, c.721C>T, p.R241^∗). Investigation of the EPG5 transcript by RT-PCR of mRNA from fibroblasts revealed a rearrangement in the other transcribed allele, with non-coding X-chromosomal material inserted into EPG5 exon 23. Further functional studies in patient-derived fibroblasts suggested a moderate disturbance of autophagic flux. We speculate that the inserted sequence partially disrupts epg5 function, resulting in a "milder" phenotype compared to typical VS. Our findings define a novel EPG5-related vacuolar myopathy and indicate that EPG5 mutations ought to be considered in unresolved patients with suggestive features.

Late adult‐onset of X‐linked myopathy with excessive autophagy

X-linked myopathy with excessive autophagy (XMEA) is characterized by autophagic vacuoles with sarcolemmal features. Mutations in VMA21 result in insufficient lysosome acidification, causing progressive proximal weakness with onset before age 20 years and loss of ambulation by middle age. We describe a patient with onset of slowly progressive proximal weakness of the lower limbs after age 50, who maintains ambulation with the assistance of a cane at age 71. Muscle biopsy at age 66 showed complex muscle fiber splitting, internalized capillaries, and vacuolar changes characteristic of autophagic vacuolar myopathy. Vacuoles stained positive for sarcolemmal proteins, LAMP2, and complement C5b-9. Ultrastructural evaluation further revealed basal lamina reduplication and extensive autophagosome extrusion. Sanger sequencing identified a known pathologic splice site mutation in VMA21 (c.164-7T>G). This case expands the clinical phenotype of XMEA and suggests VMA21 sequencing be considered in evaluating men with LAMP2-positive autophagic vacuolar myopathy.

Increased autophagy accelerates colchicine-induced muscle toxicity

Colchicine treatment is associated with an autophagic vacuolar myopathy in human patients. The presumed mechanism of colchicine-induced myotoxicity is the destabilization of the microtubule system that leads to impaired autophagosome-lysosome fusion and the accumulation of autophagic vacuoles. Using the MTOR inhibitor rapamycin we augmented colchicine’s myotoxic effect by increasing the autophagic flux; this resulted in an acute myopathy with muscle necrosis. In contrast to myonecrosis induced by cardiotoxin, myonecrosis induced by a combination of rapamycin and colchicine was associated with accumulation of autophagic substrates such as LC3-II and SQSTM1; as a result, autophagic vacuoles accumulated in the center of myofibers, where LC3-positive autophagosomes failed to colocalize with the lysosomal protein marker LAMP2. A similar pattern of central LC3 accumulation and myonecrosis is seen in human patients with colchicine myopathy, many of whom have been treated with statins (HMGCR/HMG-CoA reductase inhibitors) in addition to colchicine. In mice, cotreatment with colchicine and simvastatin also led to muscle necrosis and LC3 accumulation, suggesting that, like rapamycin, simvastatin activates autophagy. Consistent with this, treatment of mice with four different statin medications enhanced autophagic flux in skeletal muscle in vivo. Polypharmacy is a known risk factor for toxic myopathies; our data suggest that some medication combinations may simultaneously activate upstream autophagy signaling pathways while inhibiting the degradation of these newly synthesized autophagosomes, resulting in myotoxicity.

LC3 and p62 as Diagnostic Markers of Drug-induced Autophagic Vacuolar Cardiomyopathy

Autophagic vacuolar cardiomyopathy is an underrecognized, but potentially fatal, complication of treatment with chloroquine (CQ) and its derivative hydroxychloroquine (HCQ), which are used as therapy for malaria and common connective tissue disorders. Currently, the diagnosis of autophagic vacuolar cardiomyopathy is established through an endomyocardial biopsy and requires electron microscopy, which is not widely available and has a significant potential for sampling error. Recently, we have reported that immunohistochemistry for autophagic markers LC3 and p62 can replace electron microscopy in the diagnosis of HCQ-induced and colchicine-induced autophagic vacuolar skeletal myopathies. In the current study, we use 3 cases of CQ-induced or HCQ-induced cardiomyopathy and 1 HCQ-treated control case to show that the same two markers can be used to diagnose autophagic vacuolar cardiomyopathies by light microscopy. CQ-induced or HCQ-induced autophagic vacuolar cardiomyopathy is not universally fatal, but successful treatment requires early detection. By lowering the barriers to diagnosis, the application of these immunohistochemical markers will decrease the number of misdiagnosed patients, thus increasing the likelihood of favorable clinical outcomes.

Teaching Neuro Images : Hydroxychloroquine-induced vacuolar myopathy

A 58-year-old woman with long-standing mixed connective tissue disorder had proximal leg weakness for 4 months. She had been treated with 400 mg/day of hydroxychloroquine and varying doses of prednisone over 15 years. Creatine kinase was 600 U/mL. MRI of quadriceps showed edema and its biopsy revealed myriad acid-phosphatase–positive autophagic vacuoles indicating increased lysosomal activity (figure). Hydroxychloroquine induces autophagy by reducing lysosomal acidity.1 Autophagic vacuolar myopathy can be seen with chloroquine or colchicine therapy or in inherited disorders (α-glucosidase deficiency, Danon disease, and X-linked myopathy with excessive autophagy).1 Hydroxychloroquine myopathy usually presents with mild to moderate proximal weakness and rarely causes severe weakness and respiratory failure.2 Our patient's weakness improved after discontinuing hydroxychloroquine.

Defects of Vps15 in skeletal muscles lead to autophagic vacuolar myopathy and lysosomal disease

The complex of Vacuolar Protein Sorting 34 and 15 (Vps34 and Vps15) has Class III phosphatidylinositol 3-kinase activity and putative roles in nutrient sensing, mammalian Target Of Rapamycin (mTOR) activation by amino acids, cell growth, vesicular trafficking and autophagy. Contrary to expectations, here we show that Vps15-deficient mouse tissues are competent for LC3-positive autophagosome formation and maintain mTOR activation. However, an impaired lysosomal function in mutant cells is traced by accumulation of adaptor protein p62, LC3 and Lamp2 positive vesicles, which can be reverted to normal levels after ectopic overexpression of Vps15. Mice lacking Vps15 in skeletal muscles, develop a severe myopathy. Distinct from the autophagy deficient Atg7−/− mutants, pathognomonic morphological hallmarks of autophagic vacuolar myopathy (AVM) are observed in Vps15−/− mutants, including elevated creatine kinase plasma levels, accumulation of autophagosomes, glycogen and sarcolemmal features within the fibres. Importantly, Vps34/Vps15 overexpression in myoblasts of Danon AVM disease patients alleviates the glycogen accumulation. Thus, the activity of the Vps34/Vps15 complex is critical in disease conditions such as AVMs, and possibly a variety of other lysosomal storage diseases.

Minocycline-associated rimmed vacuolar myopathy in a patient with rheumatoid arthritis

BackgroundThe autophagic vacuolar myopathies (AVM) are a group of inherited myopathies defined by the presence of autophagic vacuoles in pathological muscle specimens. AVM can be categorized into three groups: acid maltase deficiency, myopathies characterized by autophagic vacuoles with unique sarcolemmal features, and rimmed vacuolar myopathies (RVM). While the pathogeneses of these conditions are still being elucidated, some drugs (e.g., chloroquine, its analog, hydroxychloroquine, and colchicine) can also cause AVM. Minocycline is a disease-modifying anti-rheumatic drug that may be used in the treatment of rheumatoid arthritis (RA). Here, we describe the first case of minocycline-associated AVM with rimmed vacuole formation.Case presentationA 75-year-old woman suffering from RA has been continuously treated with minocycline (200 mg/day) for the past 7 years. During this time, she developed a myopathy that predominantly affected her lower limbs. Histological studies of biopsied muscle revealed scattered atrophic myofibers with rimmed vacuoles that contained pigment granules. Histochemical staining revealed that the pigment comprised both iron and melanin, which is consistent with type II minocycline-induced cutaneous pigmentation. Under electron microscopy, autophagic vacuoles were consistently observed in association with numerous collections of pigment granules.ConclusionsThis is the first report of minocycline-induced pigmentation in skeletal muscle. The strong association between autophagic vacuoles and the accumulation of minocycline-induced pigments suggest that long-term minocycline treatment induced pigment accumulation, leading to elevation of autophagic activity and RVM. It might also be possible that minocycline directly activated autophagy, as the observed pigments are known to form complexes containing minocycline and/or its metabolites. As long-term minocycline treatment is expected to be used more widely in the future, we must draw attention to this adverse effect.

G.P.50 A nationwide survey of autophagic vacuolar myopathies characterized by autophagic vacuoles with sarcolemmal features (AVSF) in Japan

Danon disease (DD), X-linked myopathy with excessive autophagy (XMEA), and related autophagic vacuolar myopathies (AVMs) are pathologically characterized by autophagic vacuoles with sarcolemmal features (AVSF). AVSF delineates a group of at least five clinically different myopathies, including DD and XMEA. However, the clinical features and the prevalences of these AVMs have not been well established. We sent questionnaires on these AVMs to 2617 hospitals in Japan that have departments of neurology, cardiology, or pediatrics. We reviewed clinical histories and muscle specimens provided by hospitals with AVM patients. In addition, we performed genetic analyses of the LAMP-2 and VMA21 genes. We identified 27 DD patients from 12 families, 3 XMEA patients from 1 family, 7 X-linked congenital AVM patients from 1 family, 2 infantile AVM patients, and 1 patient with adult-onset AVM with multiorgan involvement. All DD patients had LAMP-2 gene mutations. Lethal cardiomyopathy was evident in all DD patients. Hypertrophic cardiomyopathy (HCM) was documented in most men, while dilated cardiomyopathy was more common among women. All XMEA patients had VMA21 gene mutations. XMEA, infantile AVM, and congenital AVM patients had no cardiomyopathy. HCM developed in the adult with AVM. Pathologically, AVSF in all AVMs expressed virtually all sarcolemmal proteins on their vacuolar membranes. Multilayering of the basal lamina along vacuolar membranes was present in XMEA, infantile AVM, congenital AVM, and adult AVM. In addition, the locus for infantile AVM and congenital AVM was suggested to be the same region as that in XMEA. These AVMs are very rare muscular disorders and may be primarily caused by lysosomal dysfunctions in consideration of both genetically diagnosable DD and XMEA. Because infantile AVM and congenital AVM are pathologically and genetically similar to XMEA, these three diseases might be allelic.