X-linked Myopathy with Excessive Autophagy - A Rare Cause of Vacuolar Myopathy in Children
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.355110
Source of Support: None, Conflict of Interest: None
Keywords: Autophagic vacuolar myopathy, Danon, lysosome, myopathy, VMA21 gene
Autophagic vacuolar myopathies (AVM) are a group of muscle diseases characterized by accumulation of autophagic vacuoles within the myofibres. The role of aberrant autophagy in the pathogenesis of AVM has been well elucidated., This group comprises of rimmed vacuolar myopathies, myopathy due to acid maltase deficiency (Pompe disease), and myopathies associated with 'autophagic vacuoles with sarcolemmal features' (AVSF). The latter group consists of genetically characterized Danon disease and X-linked myopathy with excessive autophagy (XMEA), apart from infantile AVM and adult-onset AVM with multiorgan involvement, which are considered pathogenetically linked to XMEA. However, the list of myopathies with AVSF is still emerging and AVSFs have been recently recognized in other diseases like desminopathy, neuronal ceroid lipofuschinosis (CLN3 disease), etc.
XMEA, an X-linked recessive disorder, was first described by Kalimo in 1988 in a Finnish family. It is caused by mutation in the VMA21 gene that results in reduced vacuolar ATPase levels, leading to defective acidification of lysosomes and accumulation of undigested material. Unlike Danon disease, the prototype AVM characterized by myopathy, intellectual disability, and cardiomyopathy, XMEA is strictly a skeletal muscle disease without any systemic involvement and the patients have near-normal life expectancy.
XMEA is still an under recognized disease with less than 50 affected families identified to date, most of them belonging to Europe and occasionally North America and Japan.,,,,,,,,, Recently, a single case has been reported from India but neither the immunohistochemical staining of the vacuoles by sarcolemmal proteins nor the characteristic ultrastructural findings, which are essential diagnostic features of XMEA, were illustrated. Genetic confirmation by mutation analysis was also not done. We report a case of XMEA in an 8-year-old boy without any history of muscle disease in the family, diagnosed on muscle biopsy, and confirmed by mutation analysis. To the best of our knowledge, this is the first genetically confirmed case of XMEA from India with all the characteristic features in the muscle biopsy.
An 8-year-old boy presented with a 2-year history of slowly progressive bilateral lower limb weakness, predominantly involving the proximal muscles. There was no history of myalgia, sensory symptoms, cognitive disturbance, oculobulbar involvement, respiratory distress, or cardiac dysfunction. There was no history of muscle disease in the family. An examination revealed proximal lower limb weakness (3/5) and mild proximal upper limb weakness (4/5) with normal strength in the distal limbs. He also had winging of scapula. Fasciculations, joint laxity, or intellectual disability were absent. The ECG and echocardiography revealed no abnormalities. Electromyography was suggestive of a myopathic pattern. The CPK levels were raised more than six times the normal upper limit. A muscle biopsy was performed with a clinical diagnosis of muscular dystrophy.
Muscle biopsy was rapidly processed in precooled isopentane placed in liquid nitrogen and 8μ thick sections were cut for histochemistry and immunohistochemistry. Histopathological examination [Figure 1] revealed maintained fascicular architecture with marked perimysial fat infiltration. There was moderate variation in the fibre size, some of them showing the presence of small vacuoles filled with basophilic material. Significant inflammation, necrosis, ragged red fibres, or COX negative fibres were not seen. The vacuoles were better appreciated with acid phosphatase. On immunohistochemistry, the vacuoles stained intensely with sarcolemmal membrane proteins (dystrophins, sarcoglycans, dysferlin, and alpha dystroglycan), and weakly with merosin, thus confirming that these were autophagic vacuoles with sarcolemmal features. Membrane attack complex (MAC, complement C5b-9) deposits were diffusely present over the myofibres and in the vacuoles. The ultrastructural examination [Figure 2] revealed prominent lysosomes and autophagic vacuoles filled with amorphous debris, degenerating organelles, myelin figures, and some glycogen. The vacuoles were membrane-bound, surrounded by reduplicated basal lamina at places. Some of the vacuoles opened to the extracellular space and the extruded material could be identified within the layers of reduplicated basal lamina. A final diagnosis of X-linked myopathy with excessive autophagy was rendered.
Mutation analysis by Sanger sequencing
DNA extraction from frozen muscle tissue was performed using a commercial kit (Relia Prep Blood gDNA Miniprep System from Promega). Three separate PCR reactions were set up to amplify the three exons of the VMA gene. PCR products (50 ng each) were used for Sanger sequencing, which revealed a single nucleotide substitution at the splice branch point of intron 1 (X: 150572076A > T; c. 54-27A > T), confirming the diagnosis of XMEA [Figure 3]. Unfortunately, the patient was lost to follow-up, hence, genetic screening of the family members could not be done.
Autophagy is an essential physiological process needed for recycling of various cellular components. Defects in autophagy are now widely recognized as underlying pathogenetic mechanism in various diseases like cancer, neurodegenerative diseases, Crohn's disease, certain infections, etc., XMEA is one such disease with defective autophagy manifesting as a gradually progressive myopathy.
The pathogenesis of XMEA is linked to hypomorphic mutations in the VMA21 gene which codes for an essential assembly chaperone of the vacuolar ATPase (V-ATPase). V-ATPase is a ubiquitous proton pump expressed in the lysosomal and plasma membranes. Ramachandran et al. studied 45 male patients from 14 families with XMEA and identified six different hemizygous single-nucleotide substitutions in the VMA21 gene—four intronic, one in the coding region, and one after the termination codon. Our case exhibited one of the intronic mutations (c. 54 –27A > T) at the splice branch point of intron 1. These mutations reduce the V-ATPase levels, resulting in defective acidification of lysosomes. An increase in the lysosomal pH reduces the activity of lysosomal hydrolases, hampering the autophagic degradation of the proteins. A blockage in the final steps of autophagy leads to decreased concentration of amino acids in the cytoplasm and consequently upregulation of macroautophagy, likely via the inhibition of the mTORC1 pathway. This feed forward feedback loop leads to massive accumulation of phagolysosomes with disruption of myofibrillar architecture and subsequent loss of muscle fibres. Curiously, the effects of the V-ATPase defect are preferentially seen only in the skeletal muscles albeit V-ATPase being a major housekeeping complex. Though the exact reason for skeletal muscle specific phenotype remains unclear, it may be due to a higher dependence of skeletal muscles on autophagy than other tissues for various physiological functions.
XMEA predominantly affects males. The female carriers are either asymptomatic or have mild symptoms. The usual onset of affects weakness is in the first decade of life, progressing slowly with retained ambulation till late adulthood. However, adult-onset cases and severe neonatal cases have also been described.,, Notably, some of the early-onset severe cases harbored non-coding VMA21 deletions rather than the common splice site mutations. Proximal lower limb muscles are the most commonly affected, followed by shoulder girdle, and proximal upper limb muscles. The characteristic findings in needle electromyography (EMG) include abundant myotonic discharges (despite absence of myotonia clinically) in both the affected and unaffected muscles. The myotonic potentials are postulated to be either due to multi-layered folding of the basal lamina or to excessive calcium concentration outside the sarcolemma, thereby, causing dysfunction of membrane ion channels or altered excitability, respectively. Increased occurrence of polyphasic motor unit potentials (MUPs) with increased mean amplitude is another distinct finding, reflecting fibre size variation and presence of hypertrophic fibres. MRI demonstrates atrophy and fatty degeneration of the affected muscle groups. Serum CPK levels are usually raised but can be normal in some cases., Kurashige et al. documented elevated urinary β2 microglobulin without obvious renal dysfunction in a Japanese family with XMEA.
Muscle biopsy reveals a myopathic pattern with moderate variation in fibre size, fibre-splitting, and internalization of the nuclei. The histopathological hallmark of XMEA is the presence of autophagic vacuoles with sarcolemmal features (AVSFs), where the vacuoles show immunopositivity for sarcolemmal (dystrophin–glycoprotein complex, caveolin-3, dysferlin, and spectrin) and basal lamina proteins (merosin and perlecan) and exhibit acetylcholinesterase activity., The vacuoles stain positively for acid phosphatase, non-specific esterase, and LAMP2, consistent with their lysosomal origin. Calcium, MHC class I, and MAC deposition may be noted within the vacuoles and around the affected muscle fibres. Complement deposition on muscle fibres was first noted by Villanova et al. (1995) and they postulated it to be the primary inciting event in the pathogenesis and suggested that vacuoles are formed due to secondary invagination of sarcolemma. However, these peculiar vacuolar membranes are presently hypothesized to be formed in situ in the cytoplasm, although the exact mechanism is still unknown. MAC and calcium deposition are now thought to be secondary to the debris around the affected myofibres. Glycogen accumulation is minimal to absent. The number of AVSFs increases with the patient's age. Hence, the vacuoles may not be apparent in younger children and an unwary pathologist is likely to miss the diagnosis.
Ultrastructural examination reveals numerous autophagic vacuoles containing degenerating organelles, granular material, myelin figures, and membrane whorls. Some of these vacuoles may be seen in the subsarcolemmal location exhibiting exocytosis, extruding out their contents into the extracellular compartment. The basal lamina at these places appears thickened due to reduplication and the lysosomal debris may be seen within the multiplied layers of basal lamina.
The main differential diagnoses [Table 1] include other causes of vacuolar myopathy, particularly those with AVSFs. These vacuoles were first identified by Danon in 1981 in two young boys who presented with hypertrophic cardiomyopathy (HCM), skeletal myopathy, and mental retardation. This disease, named after him, is caused by a mutation in the LAMP2 gene. LAMP2 is a lysosomal membrane protein involved in selective import and degradation of proteins, phagolysosome formation, and in the maturation of autophagic vacuoles. Sugie et al. proposed that LAMP2 deficiency results in the failure of fusion of lysosomal and plasma membranes, thereby, impairing exocytosis. The diagnosis relies on the identification of AVSFs, presence of abnormally increased glycogen, and demonstration of the absence of LAMP2 by immunohistochemistry. The absence of the following additional features like complement activation, calcium deposits, basal lamina reduplication, and exocytosis also helps to differentiate Danon disease from XMEA. AVSFs are also a feature of infantile AVM and adult-onset AVM with multiorgan involvement., However, these conditions are not characterized genetically and are thought to be allelic to XMEA in view of similar pathological findings.,,
Another diagnostic consideration is adult-onset acid maltase deficiency myopathy (Pompe disease) as this condition may show the absence of cardiac involvement, electrophysiological myotonia, and presence of vacuoles which may label for some sarcolemmal proteins, making distinction from XMEA difficult. Enzyme assays play an important role in clinching the diagnosis of this potentially treatable condition. Chloroquine myopathy also causes AVM. When taken long term, chloroquine accumulates within the lysosomes and increases the lysosomal pH, blocking autophagic degradation in a manner similar to XMEA.
Thus, XMEA is a distinct primary lysosomal myopathy with characteristic pathological features. XMEA should be considered in the differential diagnoses of vacuolar myopathies and distinction from other mimics is important due to its favorable prognosis. Several issues still remain unsolved, including why a ubiquitous protein defect affects only skeletal muscles, are there any unknown VTPase assembly chaperones that bypass VMA21 in the extraskeletal tissues, and whether lysosomal pH could be decreased therapeutically by altering mTOR signalling pathway. While XMEA is still an emerging entity, documentation of new cases is important to fully understand the disease, and thereby, develop a definitive treatment.
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Conflicts of interest
There are no conflicts of interest.
[Figure 1], [Figure 2], [Figure 3]