Autophagic Vacuolar Myopathy Research Articles

590P Genetic heterogeneity in PLIN4 gene: characterization of a new pathogenic expansion causing an autophagic vacuolar myopathy

590P Genetic heterogeneity in PLIN4 gene: characterization of a new pathogenic expansion causing an autophagic vacuolar myopathy

581P Toxic autophagic vacuolar myopathies and the role of human leukocyte antigen class I molecules and membrane attack complex in its pathogenesis

581P Toxic autophagic vacuolar myopathies and the role of human leukocyte antigen class I molecules and membrane attack complex in its pathogenesis

Identification of a Novel Intronic Mutation in VMA21 Associated with a Classical Form of X-Linked Myopathy with Autophagy.

Introduction VMA21 -related myopathy is one of the rare forms of slowly progressive myopathy observed in males. Till now, there have been only a handful of reports, mainly from Europe and America, and two reports from India. Method Here, we describe a case of genetically confirmed VMA21 -associated myopathy with clinical, histopathological, and imaging features with a list of known VMA21 mutations. Results A 29-year-old man had the onset of symptoms at 18 years of age with features of proximal lower limb weakness. Muscle magnetic resonance imaging showed the preferential involvement of vasti and adductor magnus. A biopsy of the left quadriceps femoris showed features of autophagic vacuolar myopathy with vacuoles containing granular eosinophilic materials. In targeted next-generation sequencing, hemizygous mutation in the 3' splice site of intron 2 of the VMA21 gene (c.164-7 T > A) was identified and confirmed the diagnosis of X-linked myopathy with excessive autophagy. Conclusion This report expands the phenotypic and genotypic profile of VMA21 -related myopathy, with a yet unreported mutation in India.

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.

Autophagic vacuolar myopathy involving the phenotype of spinocerebellar ataxia type 3.

Spinocerebellar ataxia type 3 (SCA3) is a form of autosomal dominant cerebellar ataxia with a wide range of clinical manifestations, including ataxia and pyramidal and extrapyramidal signs. A few SCA3 patients have been noticed to be predisposed to the development of inclusion body myositis. It is still unknown whether muscle can be primarily involved in the pathogenesis of SCA3. This study reported an SCA3 family in which the index patient initially presented with parkinsonism, sensory ataxia, and distal myopathy but the absence of cerebellar and pyramidal symptoms. The clinical and electrophysiological studies implied a possible combination of distal myopathy and sensory-motor neuropathy or neuronopathy. MRI muscle showed selective fat infiltration and absence of denervated edema-like changes, indicating the distal muscle weakness had a myopathic origin. Muscle pathology showed the myopathic involvement, besides neurogenic involvement, characterized by chronic myopathic changes with multiple autophagic vacuoles. Genetic screening revealed expanded CAG of 61 repeats in the ATXN3 gene, which showed co-segregation in the family. Besides the neurogenic origin, the myopathic origin may be partly attributed to the limb weakness of SCA3 patients, which expands the spectrum of the clinical manifestation of SCA3.

Diverse myopathological features in the congenital myasthenia syndrome with GFPT1 mutation.

IntroductionMutations in the GFPT1 gene are associated with a particular subtype of congenital myasthenia syndrome (CMS) called limb‐girdle myasthenia with tubular aggregates. However, not all patients show tubular aggregates in muscle biopsy, suggesting the diversity of myopathology should be further investigated.MethodsIn this study, we reported two unrelated patients clinically characterized by easy fatigability, limb‐girdle muscle weakness, positive decrements of repetitive stimulation, and response to pyridostigmine. The routine examinations of myopathology were conducted. The causative gene was explored by whole‐exome screening. In addition, we summarized all GFPT1‐related CMS patients with muscle biopsy in the literature.ResultsPathogenic biallelic GFPT1 mutations were identified in the two patients. In patient one, muscle biopsy indicated vacuolar myopathic changes and atypical pathological changes of myofibrillar myopathy characterized by desmin deposits, Z‐disc disorganization, and electronic dense granulofilamentous aggregation. In patient two, muscle biopsy showed typical myopathy with tubular aggregates. Among the 51 reported GFPT1‐related CMS patients with muscle biopsy, most of them showed tubular aggregates myopathy, while rimmed vacuolar myopathy, autophagic vacuolar myopathy, mitochondria‐like myopathy, neurogenic myopathy, and unspecific myopathic changes were also observed in some patients. These extra‐synaptic pathological changes might be associated with GFPT1‐deficiency hypoglycosylation and altered function of muscle‐specific glycoproteins, as well as partly responsible for the permanent muscle weakness and resistance to acetylcholinesterase inhibitor therapy.ConclusionsMost patients with GFPT1‐related CMS had tubular aggregates in the muscle biopsy, but some patients could show great diversities of the pathological change. The myopathological findings might be a biomarker to predict the prognosis of the disease.

Autophagic vacuolar myopathy: Danon disease and related myopathies

AbstractAutophagic vacuolar myopathy is a subgroup of muscle diseases defined as a condition in which autophagic vacuoles are present as a muscle pathological feature and is caused by abnormality of autophagy in skeletal muscles. There is an autophagic vacuolar myopathy characterized by the presence of specific autophagic vacuoles known as autophagic vacuoles with sarcolemmal features (AVSF). It is referred to as AVSF myopathy and includes Danon disease and related myopathies. Danon disease is an X‐linked dominant disease associated with a primary deficiency of lysosomal‐associated membrane protein 2 in which severe hypertrophic cardiomyopathy and myopathy develop. Cardiomyopathy is a determinant prognostic factor of this disease, and heart transplantation is still the sole radical treatment. The pathomechanism of AVSF myopathy is considered to be caused by abnormality of autophagy resulting from primary lysosomal dysfunction.

OTHER NMDs: EP.349 GFPT1-related myopathy overlapping with autophagic vacuolar myopathy. A case report

Autophagic vacuolar myopathies are a heterogeneous group of muscle diseases with a common denominator of altered autophagy and defective protein quality control. Pathogenic variants in GFPT1 cause limb-girdle congenital myasthenic syndrome. Muscle biopsies from affected patients usually show characteristic tubular aggregates, fiber disproportion, and in limited reports, autophagic vacuoles. A 27-year-old female born to non-consanguineous parents, presented with a progressive axial and limb-girdle weakness from 22 months of age, without cognitive impairment. She developed a progressive dorsolumbar scoliosis and restrictive respiratory pattern from 4 years of age. CK levels were normal and EMG without RNS revealed a myopathic pattern. The muscle biopsy showed variable fiber size and large numbers of fibres containing single or multiple cytoplasmic vacuoles containing cellular debris. Oxidative activity was increased in those fibers. Muscle MRI revealed generalized atrophy predominantly in axial and limb-girdle muscles. Trio-WES analysis failed to find pathogenic variants in genes related to autophagic vacuolar myopathy, but research-based reanalysis identified a homozygous pathogenic variant in GFPT1 (NM_001244710.1 c.686-2A>G). RNS revealed a mild and inconstant area decrement at trapezius muscle and SFEMG a pathological jitter with blocks at extensor digitorum communis, consistent with the diagnosis of congenital myasthenic syndrome. Treatment with pyridostigmine and 2,4-diaminopyrimidine was initiated with mild improvement. She is now able to walk independently indoors holding her head, without respiratory or oropharyngeal concerns. The initial diagnosis of autophagic vacuolar myopathy based on the presence of autophagic vacuoles delayed the genetic diagnosis and treatment in this case. We propose the inclusion of GFPT1 under the umbrella of autophagic vacuolar myopathies and encourage a deeper study of the GFPT1 function in autophagy.

ATG14 and RB1CC1 play essential roles in maintaining muscle homeostasis

ABSTRACT Defects in macroautophagy/autophagy are implicated in the pathogenesis of neuromuscular and heart diseases. To precisely define the roles of autophagy-related genes in skeletal and cardiac muscles, we generated muscle-specific rb1cc1- and atg14-conditional knockout (cKO) mice by using Ckm/Ckmm2-Cre and compared their phenotypes to those of ulk1 ulk2-conditional double-knockout (cDKO) mice. atg14-cKO mice developed hypertrophic cardiomyopathy, which was associated with abnormal accumulation of autophagic cargoes in the heart and early mortality. Skeletal muscles of both atg14-cKO and rb1cc1-cKO mice showed features of autophagic vacuolar myopathy with ubiquitin+ SQSTM1+ deposits, but only those of rb1cc1-cKO mice showed TARDBP/TDP-43+ pathology and other features of the inclusion body myopathy–like disease we previously described in ulk1 ulk2-cDKO mice. Herein, we highlight tissue-specific differences between skeletal and cardiac muscles in their reliance on core autophagy proteins and unique roles for ULK1-ULK2 and RB1CC1 among these proteins in the development of TARDBP+ pathology. ABBREVIATIONS:AVM: autophagic vacuolar myopathy; cDKO: conditional double knockout; cKO: conditional knockout; H&E: hematoxylin and eosin; IBM: inclusion body myopathy; mtDNA: mitochondrial DNA; PFA: paraformaldehyde; RNP: ribonucleoprotein; TBST: Tris-buffered saline with 0.2% Triton X-100

Novel autophagic vacuolar myopathies: Phenotype and genotype features.

Autophagic vacuolar myopathies (AVMs) are an emerging group of heterogeneous myopathies sharing histopathological features on muscle pathology, in which autophagic vacuoles are the pathognomonic morphologic hallmarks. Glycogen storage disease type II (GSDII) caused by lysosomal acid α-glucosidase (GAA) deficiency is the best-characterised AVM. This study aimed to investigate the mutational profiling of seven neuromuscular outpatients sharing clinical, myopathological and biochemical findings with AVMs. We applied a diagnostic protocol, recently published by our research group for suspected late-onset GSDII (LO-GSDII), including counting PAS-positive lymphocytes on blood smears, dried blood spot (DBS)-GAA, muscle biopsy histological and immunofluorescence studies, GAA activity assay and expression studies on muscle homogenate, GAA sequencing, GAA multiplex ligation-dependent probe amplification (MLPA) and whole exome sequencing (WES). The patients had a limb girdle-like muscular pattern with persistent hyperCKaemia; vacuolated PAS-positive lymphocytes, glycogen accumulation and impaired autophagy at muscle biopsy. Decreased GAA activity was also measured. While GAA sequencing identified no pathogenic mutations, WES approach allowed us to identify for each patient an unexpected mutational pattern in genes cooperating in lysosomal-autophagic machinery, some of which have never been linked to human diseases. Our data suggest that reduced GAA activity may occur in any condition of impaired autophagy and that WES approach is advisable in all genetically undefined cases of autophagic myopathy. Therefore, deficiency of GAA activity and PAS-positive lymphocytes should be considered as AVM markers together with LC3/p62-positive autophagic vacuoles.

AUTOPHAGIC MYOPATHIES / MYOFIBRILLAR MYOPATHIES / DISTAL MYOPATHIES / POMPE DISEASE: P.14 Autophagic vacuolar myopathy with autophagic vacuoles with sarcolemmal features (AVSF myopathy) including Danon disease and XMEA: A new clinical entity

A subgroup of autophagic vacuolar myopathies (AVMs) is characterized by unusual autophagic vacuoles with sarcolemmal features (AVSFs) and includes Danon disease and X-linked myopathy with excessive autophagy (XMEA). We suggest referring to this subgroup as ‘AVSF myopathy’ as a new clinical entity. However, the features of AVSF myopathy have not been well established. Therefore, to evaluate the clinical features, we reviewed the clinical histories, muscle specimens, and genetic analyses of Japanese patients with AVSF myopathy. In addition, to address the diagnostic importance of AVSFs, we studied AVSF myopathy and other AVMs (Pompe disease and rimmed vacuolar myopathies) pathologically. We identified 39 patients with Danon disease (17 males and 22 females) from 20 families with LAMP-2 mutations, 12 XMEA male patients from 4 families with VMA21 mutations, one male patient with adult-onset AVM with multiorgan involvement, and one asymptomatic female patient. Cardiomyopathy was evident in all patients with Danon disease. Among the 20 patients who died, 19 (95%) died of cardiac failure. Cardiomyopathy was not evident in all patients with XMEA. Among the 9 patients who died, all died of respiratory failure. Pathologically, AVSFs expressed virtually all sarcolemmal proteins in addition to AChE on their vacuolar membranes in all patients with AVSF myopathy. Multilayering of the basal lamina along vacuolar membranes was present in XMEA, adult type and asymptomatic type. This unique intracytoplasmic membrane structure was not found in other AVMs. AVSFs are autolysosomes surrounded by secondarily generated intracytoplasmic sarcolemma-like structures and delineate AVSF myopathy. In conclusion, AVSF myopathy is a very rare muscular disorder and may be primarily caused by lysosomal dysfunctions in consideration of both genetically diagnosable Danon disease and XMEA. Our findings suggest that AVSF myopathy may include more clinical phenotypes apart from Danon disease and XMEA.

P902Danon disease: clinical features and outcomes

Abstract Background Danon Disease (DD) is a rare X-linked autophagic vacuolar myopathy, characterized by high penetrance and severe cardiomyopathy; cognitive, skeletal muscle and vision impairment may occur as well. Due to its rarity, clinical presentation and outcomes are still uncertain. Purpose To describe clinical features and outcomes of DD in female and male patients Methods Individuals and families from United Kingdom, Australia, and United States were recruited through via advertisements on Facebook groups related to DD. Participants completed a survey about symptoms and medical history and provided their medical records to the research team. Results A total of 44 patients (54.5% female) with positive genetic testing for DD were included. De novo mutations occurred in one out of four patients. Cardiomyopathy occurred in 86.3% of patients (18/24 females, 20/20 males) at a mean age of 7.3 years for males and 19.4 years for females (p=0.001). Females presented with either hypertrophic cardiomyopathy (HCM, 66.7%) or dilated cardiomyopathy (DCM, 8.3%) whereas males presented with HCM 90% of the time. 34.2% of patients were diagnosed with Wolff-Parkinson-White syndrome. Twelve patients (7 females, 5 males) underwent cardiac magnetic resonance (CMR) Out of the 9 cases, 8 (88.9%) exhibited extensive patchy late gadolinium-enhancement (LGE) in multiple segments of the left ventricle; 3 cases also had right ventricular LGE. Median cardiac mass index was 155 g/m2 (Q1-Q3: 70–237; v.n. 31–79 g/m2). Overall, 17 (38.6%) patients died or required or heart transplant (HTx). Median age at the time of death or HTx was 17 years and 42 years in males and females, respectively (p=0.025 by the log-rank test) Cognitive impairment, mainly described as learning disabilities, was diagnosed in 90.0% of males (18/20) and 79.2% (19/24) of females; intelligence quotient (IQ) measurement was reported in 8 patients (3 females, 5 males) and 7 of them showed IQ below the average. Symptomatic skeletal myopathy was present in 28 (63.3%) of patients, with a higher prevalence in males (85% vs. 45.8%; p<0.01). Retinopathy was reported in 14 (31.2%) patients and occurred equally in both genders (p=0.34). Conclusions DD causes significant cardiac morbidity with the need for transplant at a young age; in 25% of cases DD is due to a de novo mutation. While in males DD is more frequently multisystemic with a more rapid clinical deterioration, in females the clinical presentation is variable. However, the presence of severe cases in females warrant the clinicians to screen for DD in both sexes with clinical manifestations or positive family history Acknowledgement/Funding Rocket Pharmaceuticals

MYOFIBRILLAR AND AUTOPHAGIC MYOPATHIES: P.76Clinical features of X-linked myopathy with excessive autophagy (XMEA) in Japan: a nationwide survey

X-linked myopathy with excessive autophagy (XMEA) is a very rare X-linked recessive vacuolar skeletal myopathy and is pathologically similar to Danon disease. Causative mutations have been identified in the VMA21 gene on Xq28. However, the clinical features of XMEA have not been well established. Therefore, to evaluate the clinical features and management of XMEA, we sent questionnaires on XMEA to 2,617 hospitals in Japan that have departments of neurology, cardiology, or pediatrics. We reviewed clinical histories, muscle specimens, and genetic analyses of the VMA21 gene. As a result, we identified 12 XMEA male patients from 4 families in Japan. Onset was from birth to childhood. All patients showed progressive proximal muscle weakness of the extremities. Among the 9 patients who had died, all died of respiratory failure or pneumonia during the infantile period for 6 patients and the fifth decade for 3 patients. Cardiomyopathy was not evident in all, although 3 patients showed mild cardiomegaly. All patients showed autophagic vacuoles with sarcolemmal features (AVSF) in muscles. In addition, they showed depositions of complement C5b-9 over the surface of muscle fibers and multilayered basal lamina along the sarcolemma, which were not seen in Danon disease. All families had VMA21 gene mutations. We found c.164-6T>G in 2 families, c.161A>T in 1 family, and c.164-7T>G in the other family. Previously we reported one family with c.164-6T>G as a congenital autophagic vacuolar myopathy (CAVM) and the other as an infantile autophagic vacuolar myopathy (IAVM). In conclusion, XMEA is an extremely rare disorder characterized by strictly skeletal muscle disease and may be primarily caused by lysosomal dysfunctions. None of the XMEA patients had cardiomyopathy except for 3 patients with cardiomegaly, which is critically different from Danon disease. In addition, we conclude that CAVM and IAVM are allelic to XMEA, caused by a severe VMA21 mutation. Our findings suggest that XMEA is more clinically variable than previously thought.

Congenital autophagic vacuolar myopathy is allelic to X-linked myopathy with excessive autophagy.

In the article genetic basis of undiagnosed muscular dystrophies and myopathies: Results from 504 patients” by Savarese et al.,1 the degree listed for the eleventh author, Dr. Alessandra Ruggieri, should be MSc rather than PhD. The authors regret the error.

The Natural History of Danon Disease: Lesson Learned from a Global Registry

Introduction Danon Disease (DD) is a rare X-linked autophagic vacuolar myopathy due to LAMP-2 mutation. DD is characterized by high penetrance, severe predominantly hypertrophic cardiomyopathy, cognitive and skeletal muscle as well as vision impairment. Due to its rarity, the natural history (NH) is still uncertain and delay in diagnosis is common. Methods Individuals and families from the United Kingdom, Australia, and North America completed a survey about symptoms and medical history and shared their medical records with the research team. Results A total of 46 patients (18 families, 43.5% males) with positive genetic testing for DD were included. The majority of patients were Caucasian (93.5%) and from the United States (80.4%). Among the 46 mutations, 23 were described as “pathogenic,” 10 as “likely pathogenic,” 3 as “variant of uncertain significance” and unknown in 10 cases. De novo mutations occurred in 30% of cases. Cardiomyopathy occurred in 85% of patients (19/26 females, 20/20 males) at a median age of 7.3 years for males and 19.4 years for females (p=0.001). Females presented with either hypertrophic cardiomyopathy (HCM, 69%) or dilated cardiomyopathy (DCM, 2.2%) whereas males presented with HCM 100% of the time. Left ventricular outflow tract (LVOT) obstruction was reported in 4.3% of patients. 34.2% of patients were diagnosed with Wolff-Parkinson-White syndrome. Twelve patients (7 females, 5 males) underwent cardiac magnetic resonance (CMR) Out of the 9 cases in whom contrast was used, 8 (89%) exhibited extensive patchy late gadolinium-enhancement (LGE) in multiple segments of the left ventricle; 3 cases also had right ventricular LGE. The median cardiac mass index was 155 g/m2 (Q1-Q3: 70-237; v.n. 31-79 g/m2). Cognitive impairment, mainly described as learning disabilities, was assessed in 90% of males (18/20) and 73.1% (19/26) of females. Symptomatic skeletal myopathy was present in 28 (60.1%) of patients, with a higher prevalence in males (85% vs 45.8%; p Conclusions DD causes significant cardiac morbidity with the need for Htx at a young age in both sexes. While in males DD is more frequently multisystemic with a more rapid clinical deterioration, females may present with isolated cardiac disease. These findings increase understanding of the natural history of DD and highlight the importance of a timely diagnosis regardless of sex.

Systemic lipofuscinosis associated with a lesion of autophagic vacuolar myopathy in the diaphragmatic muscle of a cow

A 7-year-old Holstein-Friesian dairy cow that was culled because of traumatic motor dysfunction due to accidental falling down showed a variety of gross postmortem findings, such as fracture of the caudal vertebra, massive hemorrhage in the pelvic cavity, edematous swelling of the right sciatic nerve, and brown discoloration of the masseter muscle, tongue, cardiac muscle, diaphragmatic muscle, liver, adrenal cortex, and intestinal wall. Histopathologically, the discolored and some other internal organs/tissues exhibited varying degrees of intracellular deposition of lipofuscin granules. A small number of eosinophilic proteinaceous granules, along with lipofuscin granules, were found in the smooth muscle fibers of the intestinal muscular layer. The diaphragmatic muscle showed a lesion of autophagic vacuolar myopathy, representing frequent occurrence of autophagic vacuoles that contained amorphous materials, lipofuscin granules, and eosinophilic proteinaceous droplets. It is presumable that the pathogenetic mechanism of this systemic lipofuscinosis had a close relationship with ischemia due to anemic condition caused by pelvic hemorrhage. This bovine case was considered a newly identified variant of systemic lipofuscinosis associated with an autophagic vacuolar lesion in the diaphragmatic muscle.

Danon disease: Gender differences in presentation and outcomes

BackgroundDanon disease (DD) is a rare X-linked autophagic vacuolar myopathy, characterized by high penetrance and severe cardiomyopathy. Because of its rarity, the natural history (NH) is uncertain. ObjectivesWe aimed to describe disease variability and outcomes through a systematic review of all published DD cases. MethodsAmong 83 manuscripts in MEDLINE and EMBASE on DD cases published until October 2017, we identified 146 patients with positive genetic testing for DD or positive muscle biopsy in a relative of a genetically diagnosed proband. Results56 females and 90 males were identified. 92.5% of patients had cardiac abnormalities. Females presented with either hypertrophic cardiomyopathy (HCM, 70.3%) or dilated cardiomyopathy (DCM, 29.3%) whereas males presented with HCM 96.2% of the time. The composite outcome of death, heart transplant or ventricular assist devices occurred equally in both sexes (32% of females and 37% of males, p = 0.60) but later in females (median age 38 years) than in males (median age 21 years, p < 0.001). Whereas women present with isolated cardiac disease 73% of the time, in males DD was frequently multisystemic and presented as a triad of cognitive impairment, skeletal myopathy, and HCM in 42% of patients. ConclusionsIn this first systematic review of DD, we confirmed the severe morbidity and mortality associated with disease in both sexes. Women presented with both HCM and DCM and generally with isolated cardiac disease, whereas in men DD usually presented as HCM and was frequently multi-systemic. Further prospective NH studies will be required to confirm these findings.

Autophagic vacuolar myopathy is a common feature of CLN3 disease.

ObjectiveThe neuronal ceroid lipofuscinoses (NCL) are genetic degenerative disorders of brain and retina. NCL with juvenile onset (JNCL) is genetically heterogeneous but most frequently caused by mutations of CLN3. Classical juvenile CLN3 includes a rare protracted form, which has previously been linked to autophagic vacuolar myopathy (AVM). Our study investigates the association of AVM with classic, non‐protracted CLN3.MethodsEvaluation of skeletal muscle biopsies from three, non‐related patients with classic, non‐protracted and one patient with protracted CLN3 disease by histology, immunohistochemistry, electron microscopy, and Sanger sequencing of the coding region of the CLN3 gene.ResultsWe identified a novel heterozygous CLN3 mutation (c.1056+34C>A) in one of our patients with classic, non‐protracted CLN3 disease. The skeletal muscle of all CLN3 patients was homogeneously affected by an AVM characterized by autophagic vacuoles with sarcolemmal features and characteristic lysosomal pathology.InterpretationOur observations show that AVM is not an exceptional phenomenon restricted to protracted CLN3 but rather a common feature in CLN3 myopathology. Therefore, CLN3 myopathology should be included in the diagnostic spectrum of autophagic vacuolar myopathies.

Vacuolated PAS-positive lymphocytes as an hallmark of Pompe disease and other myopathies related to impaired autophagy.

Autosomal recessive Pompe disease is a lysosomal disorder caused by mutations of the acid-α-glucosidase (GAA) gene. Deficiency of GAA enzyme leads to glycogen accumulation and autophagy impairment in cardiac and skeletal muscles, but also in lymphocytes. Since an effective therapy is available, a rapid, sensitive, and specific test is crucial to early identify affected subjects. Number of lymphocytes containing PAS-positive vacuoles was evaluated on blood films from 72 consecutive adult patients with hyperckemia and/or muscle weakness, 13 genetically confirmed late-onset-Pompe-disease (LOPD) and 13 of their offspring. GAA activity, measured on dried blood spot (DBS) in all patients inversely correlated with number of PAS-positive lymphocytes. More than 4 PAS-positive lymphocytes were found in 11 out of the 72 patients (6 new diagnosis of LOPD, 3 different glycogen storage myopathies, 1 glucose-6-phosphate dehydrogenase deficiency, 1 caveolinopathy), in all 13 LOPD patients and in the 13 LOPD offspring. These latter resulted to have all a single GAA mutation but low GAA levels. Immunostaining with the autophagy markers LC3 and p62 confirmed the autophagic nature of lymphocytes vacuoles. ROC curve assessment of PAS-positive lymphocytes disclosed 100% of sensitivity and 94% of specificity in recognizing both compound heterozygous and heterozygous GAA carriers. The other myopathies with more than 4 PAS-positive lymphocytes appeared to be all related to impaired autophagy, which seems to be responsible of PAS-positive vacuolated lymphocytes formation. Quantification of PAS-positive lymphocytes in blood films is useful to identify autophagic vacuolar myopathies and should be routinely used as first level test for Pompe disease.

Autophagy dysregulation in Danon disease

The autophagy–lysosome system is critical for muscle homeostasis and defects in lysosomal function result in a number of inherited muscle diseases, generally referred to as autophagic vacuolar myopathies (AVMs). Among them, Danon Disease (DD) and glycogen storage disease type II (GSDII) are due to primary lysosomal protein defects. DD is characterized by mutations in the lysosome-associated membrane protein 2 (LAMP2) gene. The DD mouse model suggests that inefficient lysosome biogenesis/maturation and impairment of autophagosome-lysosome fusion contribute to the pathogenesis of muscle wasting. To define the role of autophagy in human disease, we analyzed the muscle biopsies of DD patients and monitored autophagy and several autophagy regulators like transcription factor EB (TFEB), a master player in lysosomal biogenesis, and vacuolar protein sorting 15 (VPS15), a critical factor for autophagosome and endosome biogenesis and trafficking. Furthermore, to clarify whether the mechanisms involved are shared by other AVMs, we extended our mechanistic study to a group of adult GSDII patients. Our data show that, similar to GSDII, DD patients display an autophagy block that correlates with the severity of the disease. Both DD and GSDII show accumulation and altered localization of VPS15 in autophagy-incompetent fibers. However, TFEB displays a different pattern between these two lysosomal storage diseases. Although in DD TFEB and downstream targets are activated, in GSDII patients TFEB is inhibited. These findings suggest that these regulatory factors may have an active role in the pathogenesis of these diseases. Therapeutic approaches targeted to normalize these factors and restore the autophagic flux in these patients should therefore be considered.