Additional Investigations After the Muscle Biopsy Diagnosis

After the muscle biopsy diagnosis of a lipid storage myopathy was obtained, the patient had additional laboratory tests. Urine organic acids were normal. Acylcarnitine profile showed low levels of essentially all species. Plasma total and free carnitine levels were very low; the esterified carnitine level was also decreased but to a lesser degree. The total carnitine level was 5 μmol/L (normal: 25–28), free carnitine level was 3 μmol/L (normal: 19–48), and the esterified carnitine level was 2 μmol/L (normal: 4–13). The esterified carnitine/free carnitine ratio was elevated at 0.63 (normal: 0.13–0.42).

Final Diagnosis

Lipid storage myopathy with carnitine deficiency

Patient Follow-up

The patient declined genetic testing for more specific characterization of her lipid storage myopathy. She was recommended to take empiric supplementation with L-carnitine and riboflavin. She was also referred for cardiology evaluation.

Discussion

Lipid storage myopathies (LSM), a type of metabolic myopathies, are genetic disorders caused by defects in the intracellular triacylglycerol catabolism and characterized by excessive lipid accumulation mainly in the muscle fibers. Body triacylglycerol is mostly derived from dietary fat with a small portion synthesized in adipocytes and liver. It is stored in subcutaneous and visceral adipose tissue with minimal amount in muscle, in the form of lipid droplets, to provide energy for muscle activity. Triacylglycerol catabolism takes several key steps. Triacylglycerol is first hydrolysed to fatty acids by lipases. Fatty acids are then transported in circulation and taken up by target cells. Within target cells, non-esterified fatty acids couple with coenzyme A (CoA) to form short-chain (< C8), medium-chain (C8–12), long-chain (C12–24), and very long-chain (> C24) acyl-CoAs by fatty acyl-CoA synthetases. While short- and medium-chain acyl-CoAs can passively diffuse across mitochondrial membranes, long- and very long-chain acyl-CoAs need the carnitine shuttle system. They bind free carnitine catalyzed by the carnitine palmitoyltransferase I (CPT I) to form acylcarnitines, which then translocate into mitochondrial matrix, where acylcarnitines dissociate back to acyl-CoAs and free carnitine catalyzed by the carnitine palmitoyltransferase II (CPT II). Acyl-CoAs then undergo beta-oxidation catalyzed by acyl-CoA dehydrogenases to generate acetyl-CoA molecules, which subsequently undergo tricarboxylic acid (TCA) cycle and oxidative phosphorylation to generate the energy molecule adenosine triphosphate (ATP) [1].

LSM are genetically and phenotypically heterogeneous. The onset can be early or late, and the disease presentation can be acute or chronic. Clinical presentations of infant-onset are similar across different types and are severe with multisystem involvement. Patients usually present with hypotonia, hypoketotic hypoglycemic encephalopathy, hepatomegaly, and cardiomyopathy. The late-onset presentations can be acute (recurrent rhabdomyolysis) or chronic (fixed slowly progressive muscle weakness), and the disease is relatively mild [2–5].

Recurrent rhabdomyolysis has been associated with defects in mitochondrial fatty acid transport or beta-oxidation, such as deficiencies in CPT II, very-long-chain acyl-CoA dehydrogenase (VLCAD), trifunctional protein, and lipin-1. CPT II deficiency mainly causes recurrent rhabdomyolysis in adults, which is frequently triggered by strenuous exercise and/or fasting [6, 7]. Lipin-1 deficiency is one of the most common causes of severe recurrent rhabdomyolysis in children [8, 9]. Muscle biopsy in these patients usually does not show significant abnormal lipid accumulation. In patients with CPT II deficiency, neurological examination, CK, EMG, and muscle biopsy are usually normal between the rhabdomyolysis attacks. During or shortly after the attacks, muscle biopsy may show a necrotizing myopathy with the presence of necrotic and regenerating muscle fibers. Besides acute management of rhabdomyolysis , these patients should avoid triggers such as fasting, prolonged exercise (> 30 minutes), infection, fever, cold, emotional stress, and high-fat diet. They may change diet to high-carbonhydrate and low-fat diet, take extra carbohydrate before and during exercise, and take frequent meals. They may also take carnitine. Bezafibrate did not improve fatty acid oxidation in patients with CPT II or VLCAD deficiency [10].

There are four types of LSM: neutral lipid storage disease with myopathy (NLSD-M), neutral lipid storage disease with ichthyosis (NLSD-I), primary carnitine deficiency (PCD), and multiple acyl-CoA deficiency (MADD).

NLSD-M is an autosome recessive disease caused by mutations in the patatin-like phospholipase domain containing 2 (PNPLA2) gene which encodes adipose triglyceride lipase (ATGL) [11]. NLSD-M may manifest gradually progressive muscle weakness (either distal- or proximal-predominant), exercise intolerance, myalgia, and cardiomyopathy with a disease onset in childhood or early adulthood [12, 13]. NLSD-I is caused by mutations in the comparative gene identification-58 (CGI-58) gene which encodes alpha/beta-hydrolase domain-containing protein 5 (ABHD5), a co-activator of ATGL [14]. It tends to cause less muscle weakness but prominent skin involvement with non-bullous congenital ichthyosiform erythroderma as well as cognitive, ophthalmologic, and hearing deficits, hepatomegaly, and intestinal involvement [15–17]. The disease onset of NLSD-I is earlier than that of NLSD-M . CK is typically elevated in NLSD-M but may be normal in NLSD-I. Lipid accumulation is seen in muscle as well as many other tissues. Lipid accumulation seen in leukocytes on routine peripheral blood smear is called “Jordan’s anomaly ” [4, 18]. There is no specific treatment for NLSD. Topical application of urea-containing emollients may be used for skin ichthyosis. Dietary changes with high-carbonhydrate and low-fat diet supplemented with medium-chain triacylglycerols are beneficial.

PCD is caused by mutations in the solute carrier family 22 member 5 (SLC22A5) gene which encodes organic cation/carnitine transporter 2 (OCTN2) responsible for the cellular uptake of carnitine [19, 20]. The SLC22A5 mutations impair the carnitine transport into cells and carnitine reuptake by kidneys, thus causing carnitine wasting. As carnitine is required for long-chain and very long-chain fatty acid transport from cytoplasm into mitochondria, carnitine deficiency causes impaired utilization of fatty acids for energy production. PCD has a wide clinical spectrum. Infant- or childhood-onset may manifest progressive limb weakness, cardiomyopathy, hepatomegaly, and recurrent episodes of hepatic encephalopathy, while adult-onset may only show subtle fatigability, mild progressive limb weakness, or no symptoms at all (though cardiac involvement may still be present) [6, 21]. Previously asymptomatic women may decompensate during pregnancy [22]. CK may or may not be elevated, and total carnitine and acylcarnitine levels are extremely low. Urine organic acids are normal. Lipid accumulation is seen in muscle and liver. Treatment with lifelong L-carnitine supplementation, 100–400 mg/kg/day in four daily doses, titrated to normalize plasma carnitine levels, can improve skeletal and cardiac muscle function and yield a favorable prognosis [23–26]. Pivalic acid containing antibiotics should be avoided. Patients should also have regular screening for cardiac involvement with echocardiogram and electrocardiogram [18, 22].

MADD , also known as glutaric acidemia type II , is an autosomal recessive disorder caused by dysfunction of flavoproteins, which normally function to transfer electrons from acyl-coA dehydrogenases to coenzyme Q10 in the electron transport chain [18]. The disease is caused predominantly by mutations in the electron transfer flavoprotein (ETFA, ETFB) or ETF dehydrogenase (ETFDH) genes. It can also be caused by mutations in the other genes associated with riboflavin transport such as SLC52A1, SLC52A2, and SLC52A3, or flavin adenine dinucleotide (FAD) transport or synthesis such as SLC25A32 and FLAD1. Mutations in the ETFA and ETFB tend to cause the severe neonatal-onset form, while ETFDH mutations are linked to the mild adult-onset form [27]. Most late-onset cases present with a gradual onset of muscle weakness, exercise intolerance, and myalgia, although a third present with acute and episodic metabolic decompensation with lethargy, vomiting, hypoglycemia, acidosis, and liver dysfunction [28]. Cardiomyopathy can be seen in both neonatal- and late-onset forms. Lipid accumulation can be seen in muscle and liver. There may be secondary carnitine deficiency, but blood acylcarnitines of all species are usually increased. Serum CK may be normal or elevated, though note that labs may be normal if not drawn during an episode of metabolic decompensation. A subset of MADD patients respond to riboflavin very well, and all the riboflavin-responsive cases appear associated with ETFDH mutations [27–30]. Riboflavin should be initiated at 100–400 mg daily. Carnitine and coenzyme Q10 supplementation may also be considered, particularly if there is evidence of secondary deficiency [4]. The mechanism of riboflavin-responsiveness is still not entirely clear, but supplementation likely increases mitochondrial FAD concentration, which could increase FAD binding and/or increase the chaperone effect of FAD to improve flavoprotein folding [27]. Patients should also avoid fasting [18].

Diagnostic evaluation of LSM should include blood and urine biochemical analyses, muscle biopsy, and genetic testing. Biochemical analyses to measure blood and urine carnitines, plasma acylcarnitine species, and urine organic acids are helpful to distinguish different types of LSM, but the definitive diagnosis relies on the gene tests. In PCD, plasma total and free carnitine levels as well as acylcarnitine species are usually severely reduced. Urine excretion of carnitine is increased. Urine organic acids are normal. In MADD, free carnitine may be normal or low (secondary deficiency), all acylcarnitine species are elevated, and urine C5 to C10 dicarboxylic acids are elevated [4, 18]. It may also show secondary CoQ10 deficiency. In CPT II deficiency, plasma C16 and C18 acylcarnitines are increased, but the free carnitine level is normal. Our patient showed markedly reduced blood free and total carnitines as well as low acylcarnitines but normal urine organic acids. She was well-nourished, had no significant hepatic or renal dysfunction, and was not on any medications which could cause carnitine deficiency. Therefore, she was most likely to have lipid storage myopathy from primary carnitine deficiency. Unfortunately, she refused the gene test to confirm the diagnosis and the genetic cause.

Muscle biopsy plays an essential role in diagnosing LSM as seen in our case, as the clinical presentation with mild, progressive, and proximal limb muscle weakness is quite non-specific, and can be seen with inflammatory myopathies, late-onset Pompe disease, and limb-girdle muscular dystrophies. We initially suspected an inflammatory myopathy given her weakness pattern, moderate CK elevation, and irritable myopathic changes on EMG. Bilateral calf hypertrophy also raised a suspicion for a limb-girdle muscle dystrophy. But her muscle biopsy showed no inflammation or dystrophic changes; it showed prominent sarcoplasmic lipid accumulation in type 1 fibers, characteristic for a LSM.

Muscle biopsy in LSM such as PCD typically shows a vacuolar myopathy with many small vacuoles present in the sarcoplasm of predominantly type 1 fibers. These vacuoles are not red rimmed in the Gomori trichrome stain. They are not of lysosomal origin with increased acid phosphatase activity (autophagic) as seen in Pompe disease. They are not filled with excessive PAS-positive glycogen as seen in glycogen storage disease. These vacuoles are filled with excessive neutral lipids that can be readily revealed by Oil Red O or Sudan black stain. Oil Red O stain shows intense and larger red lipid droplets in the affected type 1 fibers. EM often shows prominent accumulation of lipid droplets between the myofibrils and under the sarcolemma. The lipid droplets are often adjacent to mitochondria. Mitochondrial abnormalities such as increased proliferation, subsarcolemmal accumulation, and pleomorphism are often present [31].

It is important to obtain a specific diagnosis of LSM, as some of the LSM are treatable. PCD can be successfully treated with a high-dose of L-carnitine supplementation (100–400 mg/kg/day). Early carnitine therapy not only improves muscle strength, but also prevents cardiomyopathy and other irreversible organ damage [23–26]. A subset of MADD patients respond very well to riboflavin, and all riboflavin-responsive cases are associated with ETFDH mutations [27]. Riboflavin should be initiated at 100–400 mg daily. There is still no specific effective treatment for NLSD-M or NLSD-I. Besides the specific supplementations, it is critical for patients with LSM to avoid various triggers of rhabdomyolysis and to modify lifestyle and diet.

Pearls