Neutral lipid storage disease with myopathy: A 10-year follow-up case report.

Mutations in PNPLA2 gene encoding for adipose triglyceride lipase (ATGL), involved in triglyceride degradation, lead to an inborn error of neutral lipid metabolism. The disorder that results in abnormal storage of neutral lipid is known as neutral lipid storage disease with myopathy (NLSDM). We report the follow-up of a 30-year-old woman with NLSDM, asymptomatic until age 23. At the age of 18, a high level of CPK and neutral lipid abnormal accumulation in muscle and skin cells suggested NLSDM diagnosis, afterwards confirmed by PNPLA2 analysis. After 5 years, she developed weakness in the upper and lower extremities. She was put on a low-fat diet with medium-chain triglycerides (MCT) oil supplementation but, although her CPK level decreased, myopathy continued to progress. At present, she presents severe skeletal myopathy without cardiac involvement. In this patient, no beneficial effects on progressive skeletal muscle weakness were detected after the MCT diet, probably due to complete loss of PNPLA2 expression.

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Errors in fat metabolism can induce lipid accumulation in all tissues. Abnormal lipid storage in skeletal muscle fibers determines the onset of lipid storage myopathies (LSMs), a group of metabolic disorders associated with the loss or decrease of function of different proteins.[1] Adipose triglyceride lipase (ATGL) is the rate-limiting enzyme for the hydrolysis of triglycerides (TAGs) stored into lipid droplets (LDs) (Figure 1a).[2] ATGL is a 504 amino acid-long protein characterized by two main functional regions: a patatin domain (residues I10-L178) and a hydrophobic domain (residues P315-P360). The patatin domain contains the catalytic dyad, S47 and D166, essential for lipase activity, and three LC3-interacting region (LIR) motifs involved in ATGL-LC3 interaction. The hydrophobic domain allows ATGL-LD binding.

Figure 1.

ATGL pathway and mutations in NLSDM patients. (a) Triacylglycerol hydrolysis is composed of a series of interrelated reactions. The primary reaction is performed by the adipose triglyceride lipase (ATGL). This lipase, after activation through the interaction with ABHD5, promotes the release of the first free fatty acid (FFA) from triacylglycerol (TAG), producing diacylglycerol (DAG). In the second step the hormone-sensitive lipase (HSL) leads to DAG conversion into monoacylglycerol (MAG). The lipolytic cascade culminates with the monoglyceride lipase activity (MGL), which induces third FFA and glycerol release. (b) Percentage of ATGL different variations found in NLSDM patients. Most of these mutations, such as frameshift and splice site mutations, and retrotransposon insertion, lead to a loss or truncated protein production.

ATGL mutations cause the onset of an ultra-rare autosomal recessive LSM form, called neutral lipid storage disease with myopathy (NLSDM; MIM 610717).[3] Until now, 107 NLSDM patients have been described.[4-7] All subjects develop progressive skeletal muscle myopathy, with both proximal and distal involvement. An asymmetric muscle involvement has been observed in almost 50% of patients. Muscle biopsy displays massive lipid storage and muscle atrophy. The other main clinical features are cardiomyopathy (40% of patients), and hepatomegaly with altered hepatic enzymes (20% of patients). Some patients also show diabetes, chronic pancreatitis, and hearing loss.[8-11] In general, there is great variability in NLSDM phenotype, and a correlation between the severity degree of clinical symptoms and mutations identified in ATGL-coding gene, PNPLA2, is difficult. To date, 60 different PNPLA2 mutations have been identified and most of them lead to total loss or severe impairment of ATGL activity (Figure 1b). Nevertheless, some patients carrying severe mutations present late and/or mild progressive myopathy, without cardiac involvement.[3] For this reason, NLSDM pathophysiology is still largely unknown, and, at present, no specific treatment exists.

Here we report the follow-up of an NLSDM patient who followed a restricted diet supplemented with MCT oil in the last four years.

Case report

A 30-year-old woman affected by NLSDM was described for the first time by Akman and colleagues in 2010.[12] Since the age of 10, she presented elevated muscle and liver enzymes without myopathy. At the age of 18, the first muscle and skin biopsies revealed abnormal accumulation of intracellular LDs in muscle fibers, in particular in type I fibers, and in skin fibroblasts. These results suggested an NLSDM diagnosis that was confirmed by a genetic investigation. Indeed, sequencing analysis revealed a retrotransposon insertion in exon 3 of PNPLA2 gene leading to the complete loss of PNPLA2 mRNA synthesis.[12] Therefore, it could be hypothesized that ATGL protein was completely lacking in the cells of patient. This mutation was not found in 100 healthy subjects.

The patient remained asymptomatic except for hyperCKemia until the age of 23, when she has been experiencing muscle weakness in both arms, shoulders, and hands. These symptoms have then extended to her lower extremities. A calves MRI performed in 2015 showed lipid accumulation.

At 26 years, she was put on a restrictive low-fat diet (15 gr of natural fat per day) supplemented with 30 gr of MCT oil per day. After beginning MCT diet, CPK lowered (from 2640 U/l to 1424 U/l). Nevertheless, muscle weakness did not improve. GSGC test performed in 2020 confirmed progressive myopathy: Walking 10 meters = 10 seconds, score 2 (for mild waddling); Climbing stairs = 13 steps up = 8.10 seconds, 13 step down = 6.40 seconds, score 1 (does not need assistance); Raising from seated floor position with no hands = 2.76 seconds, score 4 (left hand on left thigh); Getting up from chair = 0.61 seconds (< less than 1 second), score 1 (normal). A new MRI at the age of 27 displayed muscle atrophy as well as tissue substitution in the following muscles: bilateral in tensor fascia-lateral, bilateral in gluteus muscles, in vastus lateralis of the thigh and partially in sartorius, bilaterally in femoral biceps, and bilaterally in semimembranosus (Figure 2). Moreover, fat tissue replacement of deltoid and biceps muscles in the right upper extremity was also observed. In 2021 she has had an increased muscle weakness in both upper and lower extremities. She had trouble walking up the stairs and walking long distances, lifting her arms above her head, and grabbing moderate to heavy objects (i.e. lifting a glass to her mouth with one hand). No heart involvement was referred for cardiac MRI.

Figure 2.

MRI evaluation in lower limbs. After MCT oil treatment, muscle MRI highlighted advanced fat and connective tissue substitution (arrows) in both lower extremities

Discussion

Defects of neutral lipids metabolism can be caused by ATGL deficiency resulting in excessive storage of TAGs in cytoplasmic LDs and determining NLSDM onset. To date, it is difficult to correlate the clinical phenotype severity with genotype. Most of patients presented ATGL mutations that cause total or dramatic decrease of lipase function. In general, these subjects show an early progressive skeletal muscle myopathy with often a cardiac involvement (40% of patients). Patients carrying ATGL variants which partially maintain enzymatic activity usually develop a slowly progressive myopathy, without hearth defects.

In this report we described a patient with a severe PNPLA2 mutation causing the total lack of gene expression. This genotype correlates with an early muscle weakness onset which slowly progressed over the years, but it does not determine cardiac disfunction. Nine patients with severe PNPLA2 mutations have previously been described.[2,9,13-16] Five of them are males who develop both skeletal muscle myopathy and cardiomyopathy. In particular, Pasanisi and coll. reported the case of a man who developed skeletal myopathy at the age of 20 (similar to our patient) and cardiac dysfunction after 4 years.[15] The other patients are females presenting variable age at onset and severity of clinical manifestations. Chen and coll. reported a 40-year-old woman who presented muscle weakness since the age of 35, without cardiomyopathy. Her brother developed general muscle weakness later, at the age of 45, and after 10 years he manifested dilated cardiomyopathy.[9] The second is a Chinese female who presented progressive muscle weakness at the age of 45. In the following 3 years, skeletal myopathy worsened, but there was not cardiac involvement.[13] In 2017 Missaglia et al. described a 54-year-old woman showing progressive skeletal myopathy since the age of 39. A mild left ventricular diastolic dysfunction was detected at 53 of age, but after 12 months heart function appeared normal.[16] Finally, Tavian et al. described an Italian woman presenting progressive skeletal myopathy with onset at the age of 18. After 30 years, she required a pacemaker implantation. Her brother presented muscle weakness since the age of 30 and died at 54 years probably of cardiac disfunction.[2] These data suggest a phenotypic heterogeneity in NLSDM which is not explained by genotype but can depend on other factors. Some authors suggest the protective role of the estrogens on cardiac function.[2,7] There could be other factors capable of modulating the severity of clinical phenotype, such as the diet or exercise. It was reported that the exercise can have an anti-inflammatory effect which protects against cardiovascular diseases.[17] Long-term physical activity improves the metabolism and works as a natural, strong anti-inflammatory factor because muscles can produce different anti-inflammatory molecules, called myokines, during exercise. Myokines secreted by skeletal muscles can modulate the anti-inflammatory mechanism in other organs. A moderate increase of one of these myokines, interleukin-6 (IL-6), can activate macrophages, maintaining glucose homeostasis, and can inhibit the production of pro-inflammatory TNF-α. These actions improve and maintain cardiac function. Our patient was a ballet dancer who practiced regular exercise for several hours every week for years. This could partially explain the preservation of heart condition.

Differences in environmental factors (diet and different lifestyle) as well as gender, can easily be seen to underlie a proportion of inter-familial manifestations. However, intra-familial variability, especially in siblings, cannot be so readily accounted for these types of mechanisms.[11] Therefore, although it is known that the defect of “LD-TAG-lipolysis” mediated by ATGL is primarily implicated in the pathogenesis of the disease, the reported data suggest that the clinical manifestations of NLSDM could be influenced also by “modifier genes”.

Our patient followed a restricted diet supplemented by MCT oil for several years without beneficial effects on myopathy progression. MCT oil is a highly concentrated source of medium-chain triglycerides which plays a key role in increasing mitochondrial fatty acid oxidation (FAO) in some disorders.[18] In skeletal muscle mitochondrial FAO is activated and maintained by PPARα.[19] It has been demonstrated in mice model that ATGL provides ligands for PPARα activity and, in case of ATGL depletion, there is a decrease in PPARα function.[19] It could be hypothesized that in our NLSDM patient there is a negative loop in which ATGL is not produced, PPARα is negatively regulated and FAO is not maintained, even in the case of MCT supplementation. To date, no positive effect has been described in NLSDM patients treated with MCT. On the contrary, some beneficial effects of special diet (poor in long chain fatty acids and enriched with medium chain fatty acids) have been reported on children affected by NLSDI (Neutral Lipid Storage Disease with Ichthyosis), which is due to ABHD5 mutations. However, the molecular mechanisms explaining the different effect of MCT are completely unknown.

We reported the follow-up of an NLSDM patient with a progressive skeletal muscle myopathy, without cardiac involvement. From the genetic point of view, she carried a dramatic PNPLA2 mutation, causing the total loss of gene expression. This genotype correlates with the early onset of muscle damage. On the contrary, the preservation of heart condition is probably related to intensive previous activity performed by the patient for many times. The special diet followed by the patient did not improve muscle function.

In conclusion, as the pathophysiology of NLSDM is largely unknown, further studies are needed to clarify molecular basis of this disease and identify a specific treatment for patients. Moreover, it would be important to collect information regarding patient lifestyle to clarify hypothetical/beneficial influences on clinical phenotype.