Clinical and biochemical footprints of inherited metabolic disorders: X. Metabolic myopathies

Abstract

Metabolic myopathies are characterized by the deficiency or dysfunction of essential metabolites or fuels to generate energy for muscle contraction; they most commonly manifest with neuromuscular symptoms due to impaired muscle development or functioning. We have summarized associations of signs and symptoms in 358 inherited metabolic diseases presenting with myopathies. This represents the tenth of a series of articles attempting to create and maintain a comprehensive list of clinical and metabolic differential diagnoses according to system involvement.

INTRODUCTION

A comprehensive overview of inherited metabolic disorders (IMDs) associated with specific organ involvement was provided in the previous issues, including IMDs associated with movement disorders, liver disorders, psychiatric presentations, cardiovascular diseases, cerebral palsy, skin manifestations, ocular phenotypes and neoplasias [1–8]. This article focuses on skeletal muscle-specific phenotypes. The list follows the classification in the knowledgebase of IMDs (IEMbase; http://www.iembase.org) with the proposed nosology of IMDs [9] and the International Classification of IMDs (ICIMD) [10]. Data source pertains to these repositories.

Structure of skeletal muscle

Skeletal muscle represents over 40% of the body and is important for metabolism, exercise, and movement. Muscle energy failure is manifested by metabolic crises with muscle weakness, sometimes associated with muscle fatigue and failure resulting in acute necrosis.

Exercise is vital for muscle maintenance and regeneration. In muscle, several organelles (sarcoplasmic reticulum, T-tubules, triads, mitochondria, etc.) contribute to fuel utilization, the regulation of calcium homeostasis, oxidative metabolism, and mitochondrial energetic function. All these organelles may be involved in tissue damage in metabolic muscle disease and are removed by autophagy controlled by FoxO transcription and other factors that control protein turnover. The myofibrils are the major cellular constituents and occupy 85% of muscle volume [11].

When the histological image of a muscle fascicle is examined in cross-section one can use histochemical techniques that recognize two main different fiber types: type 1 (slow) and type 2 fibers (fast) and observe if there is selective fiber type atrophy.

Type 1 fibers are oxidative and rich in mitochondria and react with myosin ATPase stain at pH 4.3, NADH-TR reductase, succinate dehydrogenase, and cytochrome c oxidase, while type 2 fibers are glycolytic and react with ATPase stain at pH 9.4, Periodic Acid Shiff, and phosphorylase stain.

There is an internal membrane system, the transverse Tubular system (T-system), and the sarcoplasmic reticulum (SR) that are interrelated organelles concerned with the excitation/contraction coupling of the fibers during contraction and relaxation, in response to calcium release and uptake.

Around skeletal muscle, there are the perimysium membranes where capillary and nerves are localized (Figure 1E). Myofiber nuclei are located in the subsarcolemmal areas of the muscle fibers. On the histological image of a portion of muscle cross-section, the epimysium membrane is visible. The sarcolemma is the plasma membrane of skeletal muscle, with an internal plasmalemmal membrane and an external basal lamina composed of mucopolysaccharides, glycoprotein, and sialic acid.

Figure 1.

Hierarchical structure of skeletal muscle. A) Sarcomere morphology and sliding mechanism (scalebar 0.5 nm): Actin (red), Myosin (blue) and Titin (yellow) filaments are shown in the relaxed state (I) and during the contraction (II). The jagged sides represent the Z-lines. The central space without actin filaments is the H zone. B) Transmission Electron Microscopy (TEM) image of myofibrils (scalebar = 1 nm). C) Phase Contrast Microscope (PCM) image of skeletal muscle fibers. Dark violet elliptical elements are the myocytes nuclei (scalebar = 50 μm). D) Histological image of a fascicle cross-section. Larger white bands are the perimysium membranes. Circular structures are the muscle fibers, while the darker violet dots are the myocytes nuclei (scalebar = 100 μm). E) Histological image of a portion of muscle cross-section. In the upper part, the epimysium membrane is visible (scalebar = 0.5 mm). Adapted from „Hierarchical fibrous structures for muscle-inspired soft-actuators: A review‟ Applied Materials Today 20 (2020) 100772, (reproduced under permission).

Between the internal plasmalemma and the basal lamina are localized the satellite cells, which are undifferentiated cellular elements that, under appropriate stimuli, might proliferate from quiescent cells, fuse, and become myotubes to regenerate muscle fibers. Muscle fibers are formed by a syncytium and in males have a diameter of 40–80 μm, in females are 30–70 μm, and in children 15–40 μm [12].

Figures 2A and 2B highlights the role of the sarcoplasmic reticulum, a major player in glycolytic and calcium signaling of striated skeletal muscle fibers and involved in glycogen metabolism during the excitation-contraction cycle and muscle exercise and fatigue.

Figure 2.

A) Excitation-contraction coupling requires energy from ATP and Ca2+ions released from Sarcoplasmic reticulum (SR) cisterna stored in calsequestrin. B) The SR is divided in two domains, the longitudinal and the junctional SR. The longitudinal SR is composed of numerous tubules inter-connected with each other forming a network around each myofibril. At their ends, the longitudinal tubules form a single dilated sac called terminal cisterna. Two terminal cisternae and one T-tubule form a triad. Both the longitudinal and the junctional SR show a specific spatial organization, being regularly aligned with specific regions associated with glycolytic pathway enzymes.

Skeletal muscle is a highly adaptable tissue and responds to physiological challenges by the change in the size of fibers and type of fibers; such adaptive responses are under the control of several stimuli, both of nervous and endocrine in nature. There is general agreement that the main mechanism triggering metabolic crises is lack of energy or low fuel produced by organelles and substrates. Another cause is an abnormal SR Ca²⁺ leak due to mutated receptors involved in excitation/contraction coupling such as Ryanodine Receptor (RYR1) located in the SR, causing uncontrolled contraction and rhabdomyolysis. A series of factors must be taken into consideration: Ca²⁺ leak from the SR triggers a feed-forward mechanism leading to overproduction of reactive oxygen species (ROS) by mitochondria, which in turn causes further Ca²⁺ release (through RYR1 and extracellular Ca²⁺).

In planning the study of muscle atrophy and hypertrophy at a macroscopic level, one must consider the eventual presence of substitution in pathological states of muscle fascicles by other tissues such as adipose and connective tissue.

Muscle imaging techniques are useful to detect the abnormal patterns of myofiber alteration in muscle fascicles or muscle repair; they can be performed using muscle computed tomography, echography, or muscle MRI early in the diagnostic process, especially using STIR sequences, that might show myoedema. MRI can identify structural or developmental abnormalities in lysosomal disorders and some fatty acid oxidation metabolic disorders, for example, glycogenosis type 2 might have characteristic diagnostic imaging features [13]. Note that normal muscle MRI appearance should be attained by the age of 15 years, following completion of the process of muscle development. Drawing a balance between subjecting a patient to extensive investigations, imaging techniques appear useful in metabolic patients both for diagnosis and follow-up.

SIGNS AND SYMPTOMS

Weakness

Weakness is the most common symptom of muscular metabolic disorders; the disability a patient experience depends on which muscle groups are involved and is usually prominent in proximal lower girdle with hip weakness or distal foot weakness, while proximal girdle shoulder weakness and axial muscle involvement such as head and neck muscle weakness are less frequent.

Rather profound muscle weakness is a clinical sign appearing in several genetic muscle disorders due both to enzyme or structural protein defects. Planning the study of a suspected metabolic disorder can be difficult, but the presenting age, family, and clinical history can rationalize the approach. Characteristic examination findings, such as muscle tenderness and weakness, can help to narrow the differential diagnosis.

For example, abnormal fatiguability and subsequent weakness are common symptoms of the abnormal storage or inability to use glycogen in the “glycogenosis ” group that now is composed of 15 glycogenosis subtypes. In a patient, the extensive investigations are likely to be expensive, difficult to perform, and potentially unrewarding, but making an accurate diagnosis is an important challenge. Diagnostic nihilism should be avoided, as there are rare treatable conditions, such as the classic infantile Pompe disease (IOPD) or late-onset Pompe disease (LOPD) cases.

The two most common muscle glycogenosis are Type 2 (Pompe disease) and Type 5 (McArdle disease) which have a proximal weakness as the main feature in the late stages of the disease.

Glycogenosis type II (GSD II) is an autosomal recessive disorder caused by variants in GAA. The incidence of the disease is estimated to be 1/40.000, but data from newborn screening suggest it might be higher. GAA encodes for the enzyme acid alpha-glucosidase (GAA or acid maltase) that catalyzes the intra-lysosomal degradation of glycogen. The clinical picture is essentially myopathic with proximal weakness in a continuum of forms classified in classic Pompe (infantile form), childhood, juvenile, and adult GSD II [14]. Childhood, juvenile, and adult-onset GSD II are classified as LOPD and are characterized by skeletal muscle weakness and respiratory insufficiency, with rare cardiac involvement. LOPD show a wide range of age of onset, rate of progression of muscle involvement, and various combination of alleles resulting in residual alpha-glucosidase enzyme activity. The study of ERT replacement in LOPD has given promising results [14–17] and therefore early diagnosis is required. Classic infantile, childhood, and adult-onset GSD II patients before and after ERT treatment were analyzed [18,19]. The nature and strength of the motor response such as 6 Minute Walk Test are indicative of response to ERT, which might be used to study motor function.

In Pompe patients, autophagy is blocked, and reactivation of autophagy ameliorates GAA processing [19]. Moreover, TFEB is blocked, and VPS15 is a critical protein involved in endosome trafficking and autophagosome formation, which is abnormally localized in adult Pompe patients [20–22]. These recent findings suggest that GAA deficiency causes lysosomal dysfunction, autophagy impairment, and an alteration in several signaling pathways that might contribute to muscle weakness. An increased number of tests are available in the diagnosis of GSD II, such as dried blood spots (DBS) which allowed clinicians to discover new phenotypes: several patients present with a combination of axial muscle weakness causing severe scoliosis with lumbar hyperlordosis, rigid spine syndrome, and low mean body mass index.

Hypotonia

Clinical examination appears the most useful assessment for determining the presence of hypotonia owing to the reduced presence or absence of postural tone. The most useful positions are ventral suspension of the infant and tractions of the hands in the supine position. There are several metabolic causes of muscle hypotonia, including infantile GSD II and III, lipid storage myopathies, and mitochondrial disorders. In infancy and childhood, the most frequent disorder is GSD II (both infantile and juvenile Pompe disease) caused by an acid maltase deficiency.

In mitochondrial myopathies, clinical examination appears the most useful assessment determining the presence of hypotonia owing to the reduced presence or absence of postural tone. Myopathy is a common presentation, both as an isolated symptom or as part of a multi-systemic disorder. They include mitochondrial DNA (mtDNA) variants, nuclear DNA variants with fatty acid oxidation diseases.

mtDNA myopathies include isolated myopathy caused by pathogenic mt-tRNA variants reported with many tRNA point variants listed in MITOMAP. Most of these present in adulthood, but a number of tRNA point variants have been associated with isolated myopathy presenting in childhood.

Mitochondrial thymidine kinase 2 (TK2) presents in childhood myopathy, with a typical presentation in infancy or early childhood of fatigue, proximal myopathy, and loss of previously acquired motor skills, following a period of normal development.

The fatty acid oxidation disorders can present with symptoms of muscle impairment during development of motor skills and walking, or later in life in older children, with myopathy symptoms presenting after periods of strenuous exercise, fasting or psychological stress. They include mitochondrial tri-functional protein (MTP) with pathogenic variants in HADHA and HADHB can cause isolated LCHAD deficiency and generalized MTP deficiency, multiple acyl-CoA dehydrogenase deficiency with muscle co-enzyme Q10 deficiency, carnitine palmitoyl transferase type 2 deficiency (CPT-IID) and very long-chain acyl-CoA dehydrogenase deficiency (VLCAD).

Exercise intolerance

Exercise intolerance refers to a decreased ability to engage in physical activity, usually possible at the normal level for people of that age, sex, and muscle mass. A relatively common type of metabolic disorder is glycogenosis type 5, also named McArdle disease, caused by variants in PYGM in chromosome 11, encoding muscle glycogen phosphorylase. The disease is characterized by exercise intolerance, myalgia, painful muscle cramps, fatigue with intermittent weakness, with onset in childhood or adolescence. In half of the patients, muscle exercise results in massive creatine kinase (CK) elevation with myoglobinuria (dark urine) that might lead to kidney failure. Relief of myalgia and fatigue after a few minutes of rest is observed in many patients and is called the “second wind phenomenon”. The breakdown of fatty acids, given the impossibility to mobilize muscle glycogen deposits and the consequent shift to fatty acid oxidation is the basis of the “second wind phenomenon” that allows patients to continue exercise [14]. Later in life, there might be persistent and progressive muscle weakness. The nonsense variant p.Arg50Ter may account for 40–50% of cases in Caucasians and can be screened for diagnosis. The diagnosis is based on the clinical features, the lack of lactate elevation during the forearm exercise test and, in muscle biopsies, the presence of subsarcolemmal vacuoles caused by excess glycogen due to absent myophosphorylase.

Treatment is based on controlled physical training to develop muscle oxidative mitochondrial capacity and programmed glucose intake following periods of exercise.

Fatty acid oxidation (FAO) disorders and triglyceride metabolism disorders are inborn lipid metabolism disorders that are caused by a lack or deficiency of the enzymes needed to break down fats, leading to lipid storage myopathies (LSM). They are autosomal recessive disorders [23], associated on morphological grounds to the accumulation of lipid droplets (LDs) in the muscle due to several biochemical abnormalities. These inherited disorders of lipid metabolism leading to abnormal storage of neutral lipids in muscle and myopathic symptoms include multiple acyl-coenzyme A dehydrogenase deficiency (MADD), neutral lipid storage disease with ichthyosis (NLSD-I), and neutral lipid storage disease with myopathy (NLSD-M) [24,25]. Primary carnitine deficiency (PCD) and carnitine palmitoyltransferase deficiency (CPT 2 deficiency) are included in this group although, in patients, a scanty accumulation of muscle LDs have been detected [26]. Some disorders in this group can be managed by appropriate diet and cofactors supplementation. PCD patients only require oral L-carnitine supplementation [23].

Very long-chain acyl-CoA dehydrogenase deficiency is a clinically heterogeneous metabolic disorder and can exhibit a wide range of clinical presentations, ranging from a severe neonatal-onset disease associated with high mortality, to later-onset episodic myopathic VLCAD deficiency, which is the most common phenotype, and presents with intermittent rhabdomyolysis provoked by exercise, muscle cramps and/or pain, and/or exercise intolerance [27,28]. Hypoglycemia typically is not present at the time of symptom onset in these individuals. The diagnosis of VLCAD deficiency is established in a proband with a specific pattern of abnormal acylcarnitine levels on biochemical testing and/or by identification of biallelic pathogenic variants in ACADVL on molecular genetic testing [29]. If one ACADVL pathogenic variant is found and suspicion of VLCAD deficiency is high, specialized biochemical testing using cultured fibroblasts or lymphocytes may be needed for confirmation of the diagnosis.

Low-fat formulas or low long-chain fat/high medium-chain triglyceride (MCT) medical food (with 13%–39% of calories as total fat), total dietary protein above the dietary reference intake for age, MCT oil, carnitine supplementation, and bezafibrate have all been proposed for treatment [30].

Recently, Triheptanoin (C7) an odd medium-chain fatty acid was approved by the FDA for the treatment of pediatric and adult individuals with VLCAD deficiency [31,32].

Triheptanoin acts as an anaplerotic molecule that can correct the secondary depletion of TCA cycle intermediates occurring in this disorder. Its benefits include reduction in episodes of rhabdomyolysis as well as improvement in cardiomyopathy, hepatomegaly, and hypoglycemia that was reported in those treated with triheptanoin compared to pre-treatment. Adverse events in all of the clinical trials were similar for C7-fatty acid and C8-fatty acid treatment and predominantly consisted of gastrointestinal symptoms (i.e., abdominal pain, diarrhea).

CPT2 deficiency [26] and carnitine deficiency syndrome [33] were identified on biochemical grounds. Systemic primary carnitine deficiency is caused by pathogenic recessive variants in SLC22A5 on chromosome 5, encoding the organic cation/carnitine transporter 2 (OCTN2). Defects impairing OCTN2 function were identified in the patient described by Chapoy et al., and in several other cases [34,35] resulting in a low concentration of intramuscular, plasma, and urine total carnitine. The carnitine deficiency syndrome causes defective fatty acid oxidation and is characterized by fluctuating weakness and exercise intolerance with cardiomyopathy. In several cases, the measurement of reduced carnitine transport activity in cultured fibroblasts is the most useful test to confirm the diagnosis [36]. The OCTN2 transporter is composed of 557 amino acids that include 12 transmembrane domains with numerous missense variants that reduce carnitine transport.

RR-MADD (riboflavin-responsive multiple Acyl-CoA dehydrogenase disorder) is a recessive disorder caused by variants in either one of three different genes (ETFA, ETFB, ETFDH) encoding for electron flavoproteins; in the majority of patients with muscle weakness, the disease is caused by variants in ETFDH, encoding the ETF dehydrogenase [37]. The clinical phenotype of MADD present especially in late-onset MADD patients is called riboflavin-responsive MADD (RR-MADD). The major cause of RR-MADD is often ETFDH variants [38], that encode for electron transfer flavoprotein dehydrogenase (ETFDH). ETDFH protein consists of 617 amino acids and possesses three functional regions: a flavin adenine dinucleotide (FAD)-binding domain, a 4Fe4S cluster, and a ubiquinone (UQ) binding domain [39]. Over 700 MADD patients have been reported all over the world [37,40–53], and over 600 (95%) were affected by RR-MADD. The mutational spectrum of RR-MADD is large with various types of variants identified in the ETFDH. Most alterations are missense variants (73%), the other mutations are frameshift variants (13%), splice site variations (8%), and nonsense variants (6%). Patients presenting RR-MADD carry at least one missense variation that impairs FAD binding, which plays a central role in the conformational stabilization of flavoenzymes and might change the catalytic activity and the folding of flavoproteins [40,41]. The myopathic form of RR-MADD [37] is a well-recognized entity characterized by proximal myopathy with limb and neck muscle weakness. RR-MADD has been named “glutaric aciduria type II” because there is a large urinary excretion of glutaric, lactic, ethylmalonic, butyric, isobutyric, 2-methyl butyric, and isovaleric acids.

Neutral lipid storage disease (NLSD) refers to two different inherited disorders characterized by the enzymatic inborn errors affecting the lipase ATGL and its coactivator CGI58 [54]: NLSD-I and NLSD-M both result in massive LD accumulation, notably in the leukocytes, and in different tissues including skin, muscle, liver, bone marrow, and intestine. Chanarin-Dorfman syndrome (CDS) or NLSD-I presents with early-onset ichthyosis associated with mild myopathy, spleno-hepatomegaly, exercise intolerance, mild intellectual disability, and short stature [55,56]. The cytoplasm of granulocytes of NLSD patients shows “Jordans’ anomaly” in peripheral blood smear [57,58], i.e., they have multiple vacuoles present also in bone marrow smear [57,58]. In CDS, muscle abnormalities have been detected in about 40% of patients [59]. Overt myopathy typically begins when patients are in their thirties, but it has also been described in young children [60–62].

NLSD-M has in most cases a myopathic presentation with both proximal and distal limb weakness, and exercise intolerance is accompanied by muscular atrophy in advanced cases [59]. In NLSD-M patients, LD accumulation is due to defective ATGL activity, which normally catalyzes the first step in the hydrolysis of fatty acid from triglycerides stored in the LDs. The effects of this defect are the alteration of energy production and the involvement of skeletal muscle that causes progressive myopathy and sometimes cardiomyopathy. Muscle weakness is triggered by exercise, fasting, or infections.

Long-chain fatty acid (LCFA) oxidation presents with intermittent exercise intolerance. Four acyl-CoA dehydrogenases are involved in mitochondrial FAO: short-chain, medium-chain, long-chain, and very-long-chain acyl-CoA-dehydrogenases (SCAD, MCAD, LCAD, VLCAD) [28]. As in other metabolic disorders, therapy in patients with FAO defects can be divided into two main phases: the acute phase and long-term follow-up. In patients presenting an acute clinical event with severe hypoketotic hypoglycemia (Reye-like syndromes) or myoglobinuria, the immediate treatment is aimed to prevent the symptoms related to the metabolic block. During acute metabolic decompensation, affected individuals should receive a glucose intravenous infusion rate, eventually associated with insulin therapy. In the medium-chain (MCAD) and very-long-chain fatty acid oxidation (VLCAD) defects, the use of carnitine is still controversial both for its scarce effect [63] and for the possible danger of a cardiotoxic effect caused by long-chain acylcarnitines; also bezafibrate use for CPT2 deficiency is controversial [64].

Primary carnitine deficiency (PCD) syndromes are rare biochemical disorders due to low concentration of carnitine in skeletal muscle and are classified into two different forms: muscle carnitine deficiency where muscle weakness and lipid storage myopathy correlate to low muscle carnitine but normal liver and serum carnitine [33]; and systemic carnitine deficiency that is the most typical form, characterized by low carnitine in the liver, muscle and/or plasma. Typical signs and symptoms of PCD are the following: exercise intolerance, myalgia, fluctuating weakness, hypoglycemia, with or without ketoacidosis with a Reye-like syndrome, encephalopathy, abnormal fatigability, and cardiomyopathy [35]. Systemic carnitine deficiency is characterized by attacks of hypoketotic hypoglycemia, hepatomegaly with a Reye-like syndrome, elevated transaminases, and hyperammonemia in infants; elevated CK, and progressive cardiomyopathy in childhood; or cardiac arrhythmias and fatigability in adulthood [65,66].

Rhabdomyolysis

Rhabdomyolysis can result from acute muscle destruction and causes a darkening of the urine since a pigmented protein in muscle (myoglobin) is eliminated by the kidneys. This potentially catastrophic complication might occur also by overexertion; however, it is a regular feature of several metabolic muscle disorders if the muscle is unable to derive energy from carbohydrates (phosphorylase deficiency or McArdle disease) or fat. Typically, patients experience acute aching, fatigue, and painful swelling of affected muscles along with headaches, nausea, vomiting, and weakness. During prolonged exercise, there is such occurrence in a disorder of long-chain fatty-acid oxidation (CPT2 deficiency) that is characterized by different clinical presentations: a severe infantile hepatocardiomuscular form and a more common adult myopathic form [54]. Characteristic signs and symptoms observed in young adults are episodes of recurrent muscle pain, weakness, and rhabdomyolysis triggered by prolonged exercise, fasting, a high-fat diet, cold, or a combination of these. The attacks are associated with pain, stiffness with cramps, and highly elevated CK levels (50,000 U up to 200,000 U) reflecting muscle necrosis and myoglobinuria that may lead to acute renal failure in some cases. The main differential diagnosis is McArdle disease (glycogenosis type 5) which presents early exercise intolerance and a “second wind” phenomenon possibly related to LCFA mobilization.

CPT2 is situated in the inner mitochondrial membrane involved in a homotetrameric complex. Variants in CPT2, located in chromosome 1, cause the CPT 2 muscle deficiency, the most common form of muscle fatty acid metabolism disorder characterized by rhabdomyolysis. More than 350 patients have been described all over the world [42–49]. The CPT 2 muscle deficiency is an autosomal recessive disorder that is due to a defective protein causing intermittent muscle necrosis. Molecular analysis of the CPT2 in patients with the muscle form revealed that the most common variant observed in adult cases is the p.Ser113Leu, which compromises the enzymatic stability, and it has an observed allelic frequency of 65% in both homozygous and heterozygous status. Biochemical studies demonstrate that most patients have a residual CPT2 activity. The diagnosis can be made based on acylcarnitine plasma profile or urine [37,49] or by the study of the common variant in genomic DNA.

The classical muscle form is rather mild, and it is clinically characterized by recurrent episodes of muscle pain, muscle weakness, and rhabdomyolysis triggered mostly by prolonged exercise [26,67]. Affected individuals generally do not have muscle weakness in between the attacks. Some individuals have only a few severe attacks and are asymptomatic most of their lives, whereas others have frequent myalgia, even after moderate exercise related to daily activities. End-stage renal disease due to interstitial nephritis with acute tubular necrosis requiring dialysis is also occasionally reported. Molecular genetic investigation is regarded as the gold standard in diagnosis of CPT II deficiency. A common p.Ser113Leu variant is identified in about 70% of mutant alleles [46,68]. The phenotypes of this variant are generally mild. This variant is exclusively associated with the muscle form of CPT II deficiency. Furthermore, around 100 other rare disease-causing mutations that are associated with muscle CPT II deficiency have been reported. An open-label, non-randomized trial of bezafibrate in six patients with CPT2 deficiency was first performed by a French group. That study revealed that bezafibrate improved physical activity and myopathic manifestations, suggesting its therapeutic efficacy in the muscle form of CPT2 deficiency [69,70]. Thus, bezafibrate has remained controversial as a clinical treatment for FAODs; further studies are required to elucidate the effectiveness of this drug.

Abnormal Pathology in Metabolic Myopathies

The histopathological diagnostic hallmark of GSD II is increased muscle vacuolization and autophagy. In both infant and adult patients, several immature forms of acid-glucosidase are present and are called in children’s CRIM-positive material and might give better tolerability to ERT.

Skeletal muscle uses two distinct mechanisms to form vacuoles, i.e., lysosomes and autophagosomes. The latter accumulates in long-standing Pompe disease and might account for the transformation of such fibers into p62-positive fibers [18,19]. A diagnostic hallmark in a muscle biopsy for GSD type II are vacuolated fibers and increased acid phosphatase reaction, biochemical level of acid maltase below 30% in muscle or fibroblasts, or detection of pathogenic variants of alpha-glucosidase. The muscle fiber structure is usually more affected in the classic infantile Pompe disease, whereas the degree of vacuolization is extremely variable in biopsies of late-onset patients, independent of the age of onset, disease duration, and clinical features. The vacuoles vary in shape and size and show PAS positivity with a strong reaction to acid phosphatase. The diagnosis of LOPD can be implemented by screening suspected patients with high CK and limb-girdle syndrome or respiratory insufficiency with DBS analysis followed by biochemical enzymatic test. Mutation analysis of the GAA gene is advisable. The diagnosis of LOPD might still be challenging and often delayed by over a decade if a patient had a muscle biopsy with atypical features. Eventually, NGS can be used as a last resort in undiagnosed myopathy patients. A different characteristic in the muscle biopsy between the IOPD and LOPD cases is vacuole compartmentation, evidenced by caveolin-3 stain present in adult patient biopsies but absent in IOPD muscle, which might account for a different penetration of ERT and explain the partial response in late-onset Pompe cases, besides the presence of connective tissue.

Autophagy is the main process that becomes activated in muscle during fasting, exercise, or by different stress conditions to clear damaged proteins/organelles and rejuvenate cellular components or to generate alternative energy substrates in case of the absence of nutrients.

DIFFERENTIAL DIAGNOSIS

We categorized the signs and symptoms of metabolic disease presenting with myopathies as ‘Weakness’, ‘Hypotonia’, ‘Exercise intolerance’, ‘Rhabdomyolysis’, ‘Abnormal pathology’, and ‘Other’ (Supplemental Table S2).

Weakness, hypotonia, exercise intolerance, and rhabdomyolysis, are the most frequent symptoms associated with myopathies and reported in 297/358 (~83%), 75/358 (~21%), 30/358 (~8%), 23/358 (~6%) of IMDs with myopathies, respectively (Figure 3). Of the signs and symptoms in the group ‘Other’, most frequently reported among all disorders are ‘Muscular atrophy’ (~38%), ‘Muscle cramps’ (~15%), ‘Muscle pain’ (~15%), ‘Skeletal myopathy’ (~12%), ‘Muscle-eye-brain disease’ (~6%), ‘Lipodystrophy’ (~3%), ‘Muscle hypertrophy’ (~3%) and ‘Myalgia’ (~3%) (Supplemental Table S1). While weakness is reported in all IMDs included in this study, some signs and symptoms are specific for particular disorder groups, e.g., hypotonia in peroxisomal disorders, or rhabdomyolysis in disorders of lipids, vitamins, and cofactors, in glycogenosis type 5 or glycolytic disorders.

Figure 3.

Occurrence (%) of symptoms associated with metabolic myopathies in 10 categories of IMDs. The percentages for myopathy involvement were calculated using as the denominator the total number of IMDs in each category presenting with any metabolic myopathy. Heat scale ranges from red (0%) for diseases with no particular symptoms reported to violet (100%) for diseases with particular symptoms occurring with highly frequency. For definition of 10 categories of disorders with metabolic myopathies see Supplemental Table S2. For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.

Myoglobinuria is often correlated to a sudden muscle effort, especially during an exercise in a training set without proper hydration, and therefore myoglobin and CK should be repeated after a rest period before undergoing a specific set of investigations including a muscle biopsy. The muscle biopsy is useful to differentiate patients with McArdle disease that show absent myophosphorylase with a simple histochemical stain.

Becker muscular dystrophy can also present episodes of myoglobinuria, but these patients have elevated CK also during intercritical periods.

While most suspected cases require laboratory investigations, a series of disorders with increased CK must be differentiated by the so-called “pseudo-myopathic” presentation that is found in several genetic limb-girdle myopathies such as calpainopathies, dysferlinopathies, sarcoglycanopathies, and anoctaminopathies. The muscle MRI imaging might help show proximal or distal muscle involvement, and fat and connective tissue replacement, usually absent in metabolic myopathies except for LOPD.

Diagnosis of Danon disease should be considered when there is evidence of skeletal muscle weakness, cardiomyopathy associated with muscle biopsy findings of vacuolar myopathy and normal alpha-glucosidase activity. Diagnosis is confirmed by LAMP2 protein deficiency in muscle and leukocytes [71] or identification of LAMP2 variants. Cardiac transplantation has been performed in both male and female patients.

BIOCHEMICAL DIAGNOSIS AND RED FLAGS

Basic tests, profiles, special tests for biochemical diagnosis

A list of laboratory investigations to aid the diagnosis of IEMs is summarized in Table 1. For more details see Supplemental Table 1 and IEMbase (http://www.iembase.org).

Table 1.

Biochemical investigations in metabolic myopathies.

Basic tests	Profiles	Special tests
Blood count	Amino acids (PU)	Copper(S, U)
Vacuolated lymphocytes	Organic acids (U)	Ceruloplasmin (S)
ASAT/ALAT (P)	Acylcarnitines (DBS, P)	Carnitine (P)
CK (P)	Purines and pyrimidines (U, P)	Glycogen (M)
ALP (P)	Sialotransferins (S)	Lysosomal Enzymes (S)
Lactate (P)	Porphyrines (U)	Vitamins (S)
Glucose (P)	Oligosaccharides (U)	Flavins (B)
Ammonia (B)	VLCFA (P)	Glutathione (RBC)
Bilirubin (P)	Lipid panel (S)	Myoglobin (U)
Cholesterol (S)	Biogenic amines (CSF)	Muscle biopsy
Triglyceride (S)	Pterins (CSF)
Calcium (P)	Guanidino compounds (U, P, CSF)
Magnesium (P)
Coagulation factors

The panel of diagnostic laboratory exams consists of a smear for vacuolated leukocytes, a study of lactate, ammonia, acylcarnitines, ketoacids, CK, genomic DNA, and performing a muscle or skin biopsy. Some exams will require the use of a complex technology such as gas chromatography-mass spectrometry (GC-MS) apparatus but nowadays a genetic analysis is also needed by conventional Sanger technique or next generation sequencing. A history of intermittent attacks triggered by feeding or fasting or some stress factor may suggest a disorder either of the glycogen/glycolytic (Embden-Meyerhof) pathway or fatty acid oxidation pathway. The pediatric patient with a metabolic disorder classically can present with global developmental delay and increasing disability, often with an unremarkable birth history and hypotonia. However, there has been a considerable expansion in the understanding of the metabolic basis for many muscle conditions over the past few decades, and metabolic disorders can present in several ways, including muscle weakness with acute encephalopathy, cardiomyopathy, or myoglobinuria. Many childhood hypoketotic episodes with vomiting are also associated with metabolic disorders and provide some useful management tips. When presented with such patients it is most important to “think metabolic” and do appropriate diagnostic tests. Once the diagnosis is suspected or reached, the follow-up of the metabolic myopathy is useful, and treatment might be attempted.

The main biomarkers for primary carnitine deficiency are low free carnitine and total carnitine in plasma and urine, and high CK. For RR-MADD, the characteristic acylcarnitine profile or glutaric aciduria type II pattern might be found during fasting or metabolic crisis. The gold standard diagnostic procedure is the study of acylcarnitines by tandem mass spectrometry [50–51].

Tandem mass spectrometry requires only a small amount of plasma (100 μl) or DBS (Guthrie card), allowing the diagnosis of several inborn errors of fatty acid metabolism. CPT2deficiency leads to an increase of serum palmitoylcarnitine (C16:0) and oleoylcarnitine (C18:1) a characteristic profile of blood acylcarnitines, whereas short - and medium-chain acylcarnitines might be normal during myoglobinuric attacks and free carnitine is low. The plasma and urinary acylcarnitine profile have demonstrated its high value as a fast and non-invasive method for the detection of inborn errors of fatty acid oxidation in newborn screening.

It is important to collect samples from patients during the crisis since a normal acylcarnitine profile can be observed in patients during intercritical periods. Overnight fasting is useful but should be done with caution since it may lead also to unexpected hypoglycemia and sudden death in neonates and infants. Tandem mass spectrometry of serum acylcarnitines is a rapid screening test that should be included in the diagnostic work-up of patients with recurrent myoglobinuria and performed in cases with high CK and diffuse myalgia and cramps. Particularly in young children suspected of CPT2 deficiency, one could avoid performing muscle biopsy by appropriate acylcarnitines profile studies.

Newborn screening

Since the introduction of universal newborn screening programs for inborn errors of metabolism and the use of tandem mass spectrometry, a rapid expansion of the number of metabolic disorders screened has occurred, according to the classic inclusion criteria of Wilson and Jungner [52]. GAA enzyme activity assays in DBS are used to diagnose newborns with Pompe disease in various geographical locations such as Taiwan and North-East Italy [53–54]. It is unknown if asymptomatic cases with LOPD detected on newborn screening should be followed and eventually treated. The use of mass spectrometry is performed by DBS in neonates in the US and some European countries such as Italy to detect FAO disorders and prevent sudden infant death syndromes.

Histopathology

The autophagic process targets intracellular cytosolic components for lysosomal degradation and is important for sustaining cellular energy and metabolic balance. The progressive storage of glycogen in lysosomes is responsible for damage to their membranes, causing hydrolytic material dispersion in the cytoplasm with the impairment of muscle contractile units. Autophagic pathway alteration caused further damage to muscle fibers. The relevance of autophagy to the pathogenesis of muscle damage in Pompe disease is highlighted by the evidence that glycogen enters the lysosome via the autophagic pathway. Glycogen storage in lysosomes is a red flag and, in the biopsy, this is correlated to high phosphatase stain. In Pompe disease, extensive necrosis or damage of muscle fibers induced by massive glycogen accumulation results after some years, in fat and connective tissue replacement.

Lipid storage myopathies (LSM) are associated on morphological grounds with the accumulation of lipid droplets in the muscle as well as in other tissues, such as the liver, or leukocytes revealed by ORO stain.

ATGL deficiency (NLSD-I) and ABHD5/CGI-58 deficiency (NLSD-M) are suspected when finding the Jordans’ anomaly in peripheral blood leukocytes or bone marrow megakaryocytes (white blood cell precursors). By this morphological exam, both NLSD-I and NLSD-M can be detected, and screening for genomic DNA variant is then indicated.

PROGNOSIS AND TREATMENT

Diets and gene therapy

The avoidance of fasting is the main precautionary guideline in defects of mitochondrial beta-oxidation. Since patients with fatty acid disorders cannot utilize fatty acids by beta-oxidation, the accumulation of toxic intermediate metabolites (i.e., acyl-CoAs) should be avoided to prevent the development of the crises.

In the diet, fat consumption should be restricted to 25% of total calories, and in particular, the amount of LCFA should be minimal. The increased caloric intake from carbohydrates may be necessary during intermittent myoglobinuric attacks in FAO disorders because they substitute the increased energetic request. A low-fat, high-carbohydrate diet might be beneficial in reducing the frequency of myoglobinuric attacks in several disorders of fatty acid metabolism, such as VLCAD and CPT2 deficiency. The current dietary treatment of LCFA defects (high carbohydrates with medium-chain triglycerides and reduced long-chain fats) is based on clinical evidence derived from descriptive case series.

A diet high in carbohydrates improves exercise tolerance in patients with carnitine palmitoyltransferase II (CPT2) deficiency. Frequent meals with high carbohydrate intake are recommended, especially before and after prolonged efforts. It is difficult to perform double-blind studies to prevent myoglobinuria while moderate physical activity seems to improve exercise tolerance in these patients. Delivering drug molecules to the skeletal muscle is safe and well-tolerated, although treatment with bezafibrate has given conflicting results (30).

In several patients with primary carnitine deficiency, it has been documented that the main treatment with carnitine supplementation corrects heart problems and muscle weakness [16, 33]. The L-carnitine dose may vary from 100 to 600 mg/kg per day based on the calculated carnitine depletion from muscle, liver, heart, and kidney. To adjust the dose, several plasma carnitine level measurements might be useful. Plasma carnitine levels should be monitored frequently to reduce the episodes of hypoglycemia. Side effects of L-carnitine supplementation are mild and consist of diarrhea, intestinal discomfort, or a fishy body odor. In some cases, a medium-chain triglyceride diet (MCT) may be added. Muscle carnitine deficiency is characterized by the clinical syndrome of proximal and axial weakness. The patients show normal ketogenesis on fasting or a fat-rich diet. Diagnostic biochemical features are low muscle carnitine (below 15%) and absence of organic aciduria.

The mechanisms of high-affinity carnitine transport mediated by OCTN2 transporter have been extensively studied. Muscle carnitine deficiency is differentiated from carnitine insufficiency in FAO disorders because of the absence of acylcarnitine elevation in plasma or urine.

Riboflavin supplementation often results in marked improvement of clinical weakness (50–100mg 2–3 times daily) in ETFDH deficiency. The use of a diet poor in fat and protein, with carnitine and MCT supplementation avoiding long fasting periods, can also be helpful. Riboflavin therapy has resulted in great benefit in over 400 patients with ETFDH variants leading to improvement in their clinical and metabolic symptoms. Most of these patients are homozygotes or compound heterozygotes for missense variants. Supplementation has been partially effective or ineffective in some patients. Failure to respond to treatment is probably due to the presence of variants that dramatically reduce ETFDH stability or could be due to late treatment. For a better prognosis, riboflavin treatment should be started early in patients with late-onset MADD to prevent severe metabolic crises.

Gene therapy

Currently, many genetic therapies are being investigated at preclinical and clinical levels. However, these potential gene treatments are often variable or gene-specific and their delivery aims to treat disease with only muscular manifestations. Unfortunately for most metabolic disorders with a muscular phenotype, this is often complicated by CNS involvement, and this might contribute to life-threatening manifestations such as respiratory center dysfunction, which requires urgent intervention.

CONCLUSIONS

The accurate diagnosis of metabolic myopathies is crucial both to following the patients and to offering a genetic diagnosis to the family. Multidisciplinary clinical management is advised by a team that might include metabolic specialists, dieticians, and neurologists to provide effective care. The standard neuromuscular and general neurological diagnostic approach that was done in many clinics is likely to miss a metabolic diagnosis unless special tests are added besides the usual CK and lactate; undiagnosed cases with childhood and adult-onset can be diagnosed by DBS and NGS. When screened cases present as hyperCKemias, fluctuating weakness, myoglobinuria, or proximal weakness syndromes, the use of NGS has been found viable to identify LOPD [55]. LOPD is the only myopathic form where ERT has been approved and results in a modest motor clinical improvement and respiratory stabilization. In the COVID-19 pandemic, the use of e-health applications, and telemedicine was done by metabolic patients. Ricci et al. [56] have developed an AIG-KIT that is an application for smartphones promoted by the Italian glycogenoses association (AIG) to allow interaction between patients and doctors.

Several metabolic disorder patients use e-health applications; for instance, a group of McArdle disease patients used a heart rate monitor to adjust their activity during a trail in Wales not to stress the muscle and to prevent rhabdomyolysis.

An area that will expand is newborn screening since most families want to be prepared to follow their children with diets and other precautionary management; there is an ongoing preventive program both for GSD II and FAO defects. The main precautionary guideline in FAO defects is the avoidance of fasting. Since these patients cannot utilize fatty acids by beta-oxidation, the accumulation of toxic intermediate metabolites (i.e., acyl-CoAs) should be avoided as soon as the development of the critical signs occurs. In FAO defects fat consumption should be restricted, and the amount of long-chain fatty acids (LCFA) should be minimal. The increased caloric intake from carbohydrates may be necessary during intermittent illness crises because of increased metabolic requests. A low–fat diet is used in MADD and CPT2 deficiency patients [57] and reveals that these defects are rare but treatable.

A debated issue is when to start and when to stop ERT in LOPD. Recent European guidelines have been prepared by the EPOC consortium [15].