CASE OF THE MONTH
MUSCLE MRI IN NEUTRAL LIPID STORAGE DISEASE WITH MYOPATHY CARRYING MUTATION C.18711G>A CHUNXIAO XU, MS, YAWEN ZHAO, MS, JING LIU, WEI ZHANG, MD, ZHAOXIA WANG, MD, and YUN YUAN, MD, PhD Department of Neurology, Peking University First Hospital, Beijing 100034, China Accepted 27 October 2014 ABSTRACT: Introduction: We describe the clinical and muscle MRI changes in 2 siblings with neutral lipid storage disease with myopathy (NLSDM) carrying the mutation c.18711G>A. Methods: Peripheral blood smears, genetic tests, and muscle biopsies were performed. Thigh MRI was performed to observe fatty replacement, muscle edema, and muscle bulk from axial sections. Results: Both siblings had similar fatty infiltration and edema. T1-weighted images of the gluteus maximus, adductor magnus, semitendinosus, and semimembranosus revealed marked and diffuse fatty infiltration. There was asymmetric involvement in biceps femoris and quadriceps. There was extensive fatty infiltration in the quadriceps, except for the rectus femoris. Gracilis and sartorius were relatively spared. Thigh muscle volume was decreased, while the gracilis and sartorius appeared to show compensatory hypertrophy. Conclusions: Compared with previous reports in NLSDM, MRI changes in this myopathy tended to be more severe. Asymmetry and relatively selective fatty infiltration were characteristics. Muscle Nerve 51: 922–927, 2015
Neutral lipid storage disease is an autosomal recessive genetic disease that can be classified into neutral lipid storage disease with ichthyosis and neutral lipid storage disease with myopathy (NLSDM). NLSDM was first reported by Fisher et al. in 2007.1 It is caused by mutation of the adipose triglyceride lipase gene (ATGL; also called patatin-like phospholipase domain-containing 2 [PNPLA2]). ATGL is a rate-limiting enzyme in the hydrolysis of triglycerides (TG) and plays a primary role in lipolysis. As a result, ATGL dysfunction prevents hydrolysis of TG, leading to TG accumulation in the cytoplasm of various organs.2 Clinically, patients with NLSDM show slowly progressive myopathy in adulthood, which can be proximal or distal dominant.1,2 Proximal weakness is more common in the early stages of the disease. In the middle and late stages, 50% of patients present with potentially life-threatening hyperAbbreviations: ATGL, adipose triglyceride lipase; GSD, glycogen storage disease; LGMD, limb girdle muscular dystrophy; MGT, modified Gomori trichrome; MRC, Medical Research Council Grade Scale; NLSDM, neutral lipid storage disease with myopathy; ORO, Oil Red O; PAS, periodic acidic Schiff; PNPLA2, patatin-like phospholipase domain-containing 2; RT-PCR, reverse transcription-polymerase chain reaction; STIR, short inversion time inversion-recovery; TG, triglycerides Key words: adipose triglyceride lipase gene; magnetic resonance imaging; mutation c.18711G>A; myopathy; neutral lipid storage disease; patatin-like phospholipase domain-containing 2 Correspondence to: W. Zhang; e-mail: [email protected] C 2014 Wiley Periodicals, Inc. V
Published online 31 October 2014 in Wiley Online Library (wileyonlinelibrary. com). DOI 10.1002/mus.24507
922
trophic cardiomyopathy, which may be a cause of premature death due to heart failure or arrhythmia.3 Pathologically, several lipid droplets are deposited within muscle fibers, predominantly in type I fibers, and in cells of other organs. Rimmed vacuoles may also be observed.4,5 There have been a few reports of muscle MRI changes in NLSDM over the past several years in which the gene mutation predominantly affects the C-terminal region.6–8 This type of NLSDM generally presents with a manifestation of myopathy without multi-system damage. In contrast to previously reported cases, we describe the clinical and thigh MRI findings in 2 siblings in whom the gene mutation predominantly affects the N-terminal region. This mutation often produces multi-system damage, and our aim is to better characterize the fatty replacement in skeletal muscle and its possible use as a marker in muscle MRI. PATIENTS AND METHODS
The procedures were performed in accordance with the ethical standards of the responsible committee on human experimentation and approved by the Institutional Review Board. Informed consent for all examinations was obtained from the patients Patient 1. The proband was a 44-year-old man of Han nationality. He was 175 cm tall and weighed 65 kg (body mass index: 21.2 kg/m2). Motor developmental milestones were normal. At age 31 years, he presented with right leg weakness and walked slowly. Two months later he had difficulty lifting his arms. At age 32 years, he developed left leg weakness. Muscle weakness developed slowly. Currently, he is able to walk unaided but cannot lift his arms fully and requires help ascending and descending stairs. He had a history of congenital heart disease (atrial septal defect central type) and has had hypertension for the past decade. Physical examination revealed no ichthyosis. Neurological examination showed weakness affecting the deltoids (2/5 Medical Research Council [MRC] Scale), biceps brachii (2/5 MRC), and triceps brachii (4/5 MRC). Weakness of the distal upper limbs was 4/5 MRC. Asymmetrical lower limb weakness was observed that affected predominantly MUSCLE & NERVE
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the right leg, and muscle strength was graded as: hip flexors (right 4/5 MRC and left 41/5 MRC), knee extension (right 4/5 MRC and left 5-/5 MRC), knee flexion (right 4/5 MRC and left 5-/5 MRC), and foot dorsiflexion (right 3/5 MRC and left 5-/5 MRC). He had atrophy of shoulder girdle, gluteus maximus, thigh, and gastrocnemius muscles, along with finger joint contractures. Tendon reflexes were absent, and there were no pathological reflexes. Laboratory investigation showed serum creatine kinase levels fluctuated between 564 and 1,485 IU/L (normal, 38–174 IU/L), LDH was 467 IU/L (normal, 140–271 IU/L), ALT was 53 IU/L (normal, 5–40 IU/L), AST was 65 IU/L (normal, 8–40 IU/L), TG was 1.12 mmol/L (normal, 0–1.7 mmol/L), and high-density lipoprotein-cholesterol was 0.99 mmol/L (normal, 1.04–1.99 mmol/L). Echocardiography revealed diastolic dysfunction and asymmetric hypertrophy of the left ventricle, which satisfied a diagnosis of hypertrophic cardiomyopathy. In addition there was flow from left to right at the atrial level. Abdominal ultrasound suggested a fatty liver. EMG revealed myopathic changes in bilateral biceps, abductor pollicis brevis, sternocleidomastoid, paraspinal, vastus medialis, and tibialis anterior muscles. Nerve conduction studies, H-reflexes, F-waves, and somatosensory evoked potentials were normal. Patient 2. Patient 2 was the 42-year-old younger brother of Patient 1. Early motor development was normal. He had difficulty running since childhood. At age 27 years, he developed weakness in the right arm. At age 31 years, he presented with muscle weakness of both legs with right lower leg wasting. Two years later, he exhibited a waddling gait along with muscle atrophy in his proximal limbs and shoulder girdle muscles, but was still able to descend and ascend stairs and squat without assistance. At age 37 years, he presented with severely reduced upward arm movement (less than 90 ), frequent falls, and problems descending and ascending stairs and squatting. Muscle weakness became severe, except for the distal upper limbs. At age 41 years, he was hospitalized with acute necrotizing pancreatitis. He had a 10 year history of abnormal appearing stools and a 2-year history of hypertension. On admission, physical examination revealed mitral and triscupid murmurs. Neurological examination revealed proximal arm weakness affecting the deltoids (3/5 MRC), biceps brachii (2/5 MRC), and triceps brachii (4/5 MRC). Asymmetrical leg weakness was also observed that affected predominantly the right leg. Muscle strength was graded as: hip flexors (right 4/5 MRC and left
41/5 MRC), knee extensors (right 4-/5 MRC and left 4/5 MRC), foot dorsiflexors (right 5-/5 MRC and left 5/5 MRC). Tendon reflexes were absent, and there were no pathological reflexes. There was atrophy of the thenar, hypothenar, deltoid, supraspinatus, infraspinatus, and tibialis anterior muscles, along with pseudohypertrophy of the forearms. The patient also exhibited pes cavus. Laboratory investigation showed serum creatine kinase levels that fluctuated between 1,000 and 2,000 IU/L (normal, 25–195 IU/L). Serum levels of lactate, pyruvate, TG, serum carnitine fraction, and long-chain fatty acid profile were within normal ranges. ECG examination showed premature ventricular extrasystoles (2 beats per 24 h). Echocardiogram revealed left ventricular hypertrophy and reduced left ventricular diastolic function. Head MRI showed several focal ischemic lesions mainly in the right centrum semiovale. Neurophysiological examination revealed myogenic changes in the left extensor digitorum brevis, left biceps, right quadriceps, and right tibialis anterior muscles. Nerve conduction studies and visual evoked potentials were normal. Peripheral Blood Smears and Muscle Biopsy. Finger stick blood was taken from Patient 2 following written informed consent. The smear was stained according to standard procedures with Oil Red O (ORO). The sample was fixed in 60% isopropanol for 5 min and rinsed with running water. The dried blood smear was then stained with ORO for 30 min at room temperature, followed by differentiation in 60% isopropanol for 2 min, rinse in running water, counter-stain with hematoxylin for 30 s, and thorough rinse in running water. Finally, the smear was mounted with glycerin jelly and observed under a light microscope. With written informed consent, quadriceps biopsies were obtained from both patients. Serial frozen sections were stained according to standard procedures with hematoxylin and eosin, modified Gomori trichrome (MGT), periodic acidic Schiff (PAS), ORO, adenosine triphosphatase at pH 10.5, 10.4, 10.2, 4.2, 4.3, and 4.6, NADH dehydrogenase, succinate dehydrogenase, cytochrome c oxidase, and nonspecific esterase. Sections were observed under a light microscope.
Blood from the patients and 3 additional family members was collected in EDTA tubes. Genomic DNA was extracted from peripheral blood. The sequence of PNPLA2 was obtained from the GenBank human genome database (http://www.ncbi.nlm.nih.gov). Primers for ATGL were designed using Primer 3.0 software. All exons and flanking intron sequences of ATGL were amplified using polymerase chain reaction (PCR)
Gene Sequencing.
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FIGURE 1. (A) Numerous fat droplets were observed in the cytoplasm of nucleated cells (Jordan anomaly); magnification 31,000. (B) Muscle histology shows a myopathic pattern and numerous vacuoles of varying size. (C) MGT staining did not reveal any ragged red fibers. (D) ORO staining exhibited severe fat droplet deposition in most muscle fibers.
on a 2720 Thermal Cycler. Primers for exon 2 of ATGL were: forward primer, 50 -AGCAGGCGG CTCACAGAG-30 and reverse primer, and 50 CACGGTACCCACCGACTC-30 . PCR products were identified by 1.5% agarose gel electrophoresis and were sequenced using an ABI 3730xl automated sequencer after purification. All PCR products were sequenced in both directions. Reverse Transcription-PCR. Total RNA was extracted from muscle tissue from patient 2 and stored in RNA later to prevent RNA degradation. The main procedures have been described previously.9 Briefly, cDNA was synthesized from total RNA using SuperScript First-Strand Synthesis System for reverse transcription-PCR (RT-PCR) and used as the template for PCR. Considering the mutation site, we designed forward and reverse primers at exons 2 and 3, respectively, which were: forward primer, 50 -ATGTTTCCCCGCGAGAAGA-30 and reverse primer, 50 -ACCTGGTAAAGATCATC CGCAG-30 . Gene sequencing was the same as that described above. MRI of Thigh Muscle. MRI examination was performed using a 3.0-Tesla MRI scanner (Signa Excite; GE Medical Systems, Milwaukee, Wisconsin) with an 8-channel head coil. Routine images obtained included T1-weighted axial and coronal images, T2-weighted axial and coronal images, short inversion time inversion-recovery (STIR) coronal images, and diffusion-weighted images. 924
According to the scope and extent of muscle involvement in the T1-weighted image sequence, muscle fatty infiltration was graded into 6 degrees: 0, normal muscle; 1, punctate hyperintense lesions; 2, scattered hyperintense lesions which accounted for less than 30% of muscle volume; 3, scattered fused hyperintense lesions, which accounted for 30–60% of muscle volume; 4, large sheets of fused hyperintense lesions, which accounted for over 60% of muscle volume; and 5, all muscle tissue was replaced by fat tissue.10 Two independent researchers assessed fatty infiltration, edema, and muscle bulk in 12 muscles, including gluteus maximus, lateral thigh muscles, medial thigh muscles, and posterior thigh muscles. RESULTS Detection of Jordan Anomaly and Muscle Histology Features. Numerous fat droplets were observed in
the cytoplasm of nucleated cells in peripheral blood smears, called Jordan anomaly (Fig. 1A). Muscle histology showed myopathic changes with numerous vacuoles of varying size (Fig. 1B). Patient 2 exhibited rimmed vacuoles within some muscle fibers. MGT and PAS staining did not reveal any ragged red fibers or separate glycogen storage (Fig. 1C). ORO staining in both patients exhibited extensive fat droplet deposition in most muscle fibers, predominantly affecting type I fibers (Fig. 1D). Genetic Analysis. The patients were homozygous for the mutation at intron 2 first base G>A MUSCLE & NERVE
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FIGURE 2. (A) Genetic results from proband showing homozygous mutation in intron 2 (c.18711G>A) (arrow). (B) Genetic results from a healthy control.
(c.18711G>A) (Fig. 2). The results of RT-PCR confirmed the mutation, which resulted in retention of a 97 bp sequence from intron 2 and insertion mutants (Fig. 3). There was a heterozygous mutation of G>A in the 3 other family members tested. MRI Features of Thigh Muscle. In T1-weighted MRI sequences, the thighs of Patient 1 (Fig. 4A) and Patient 2 (Fig. 4B) revealed asymmetric distribu-
tion of fatty infiltration. The degree of muscle fatty infiltration is listed in Table 1. Gluteus maximus, adductor magnus, semitendinosus, and semimembranosus muscles showed marked and diffuse fatty infiltration. Biceps femoris and quadriceps muscles showed asymmetric involvement (the right leg was more severe than the left). Fatty infiltration in the quadriceps was severe except for the rectus femoris. Gracilis and sartorius were less affected. The gracilis muscle of Patient 1 was hypertrophied.
FIGURE 3. RT-PCR showed a mutation leading to a splice site shift (the splice site is marked with arrow). (A) Normal splice site and (B) shifted splice site due to a mutation of c.18711G>A. MUSCLE & NERVE
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FIGURE 4. Muscle MRI performed on proximal lower limbs revealed asymmetric multiple fatty infiltration in T1-weighted images. (A) Patient 1. (B) Patient 2.
Gracilis and sartorius muscles of Patient 2 were hypertrophied, and the entire thigh muscle bulk was reduced. In STIR sequences, there was asymmetric slight tissue edema, mainly involving the quadriceps and biceps femoris. DISCUSSION
NLSDM is caused by a mutation in the gene encoding adipose triglyceride lipase. Patients with NLSDM have a slowly progressive myopathy in adulthood, which can be either proximal or distal.1,2 The observation of numerous fat droplets deposited in the cytoplasm of nucleated cells in a peripheral blood smear (Jordan anomaly), together with marked fat droplet deposition in type I muscle fibers and genetic tests can lead to the diagnosis. In this study, 2 siblings presented with progressive muscle weakness and cardiomyopathy. Clinical profiles, pathological examination, and genetic results suggested a diagnosis of NLSDM. The mutation of c.18711G>A, predominantly affecting the N-terminal region of ATGL, is reported rarely. We report clinical and thigh muscle MRI changes in these 2 patients. Generally, muscle fatty infiltration was observed mainly in the gluteus maximus and posterior and lateral thigh muscles. Both patients had similar patterns of fatty infiltration on muscle MRI, and the severity of muscle involvement was also similar. Gluteus maximus, semitendinosus, semimembranosus, adductor magnus, and vastus medialis muscles all exhibited large sheets of fatty infiltration, while gracilis, adductor longus, and sartorius muscles showed punctate fatty infiltration. The distribution of the fatty infiltration in the 2 patients was similar. Posterior thigh muscles were more involved than 926
lateral and medial thigh muscles. In particular, the fatty infiltration was asymmetric, involving the right thigh more than the left. It is possible that the reason for this observation was in some way related to early weakness of the right leg and preference for right limb weight bearing. Fatty infiltration also showed relative selectivity in quadriceps femoris. Vastus lateralis, vastus medialis, and vastus intermedius were all heavily involved, in contrast to the rectus femoris, which was relatively spared. Edema was mainly observed in the quadriceps and biceps femoris, and was not pronounced compared with the observed fatty infiltration, which may be related to the course of the disease. In time, edema will be gradually replaced by fatty tissue. Muscle bulk generally decreases, while gracilis and sartorius muscles, which play a secondary role in lower limb movement, show compensatory hypertrophy.
Table 1. Degree of fatty infiltration in muscles observed with MRI. Patient 1
Gluteus maximus Biceps femoris Semitendinosus Semimembranosus Vastus lateralis Vastus intermedius Vastus medialis Rectus femoris Adductor magnus Adductor longus Sartorius Gracilis
Patient 2
Left
Right
Left
Right
5 1 5 5 1 2 5 1 5 2 1 1
5 5 5 5 5 5 5 1 5 2 1 1
5 3 5 5 3 3 5 1 5 1 1 1
5 5 5 5 3 5 5 1 5 1 1 1
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Our findings suggest that MRI changes in NLSDM carrying the mutation c.18711G>A might show a severe type of myopathy with regard to degree and scope of involvement. Over the past several years, there have been a few reports of MRI changes in NLSDM with the gene mutation predominantly affecting the C-terminal region,6–8 which led to isolated myopathy compared with the gene mutation predominantly affecting the Nterminal region (our patients) resulting in multisystem abnormality. With regard to MRI changes in NLSDM predominantly affecting the C-terminal region, posterior thigh muscles are reported to be more severely involved than quadriceps,6–8 and quadriceps appears to be spared.7,8 There are, however, conflicting opinions on the involvement of semitendinosus and semimembranosus muscles. For example, Fiorillo et al.8 suggested that the semitendinosus seemed to not be involved, while Lafor^et et al.7 suggested that it was severely involved. We suggest that the difference in findings between these researchers can be explained by the onset age and duration of the condition. In the former case, the patient was a 14-year-old boy with subclinical symptoms, while in the latter case, the patients were older and showed typical clinical symptoms for a decade. This perhaps suggests that the semitendinosus muscle is involved relatively late in the condition. Our patients share similar onset ages and duration with patients in previous studies.6,7 However, our patients presented with marked fatty infiltration in the gluteus maximus, adductor magnus, and posterior thigh muscles (semitendinosus and semimembranosus), and asymmetric involvement of quadriceps. The difference observed in thigh MRI coincides with a study1 that reported that mutations at the N-terminal region always lead to severe myopathy compared with mutations at the C-terminal region. Apart from disease evaluation, thigh MRI may also inform differential diagnosis. In patients with progressive proximal limb weakness, as in this study, it is difficult to distinguish limb girdle muscular dystrophy (LGMD) and glycogen storage disease (GSD), especially late-onset GSD type 2. In contrast to NLSDM, weakness in LGMD2B affects the adductor magnus early, then the vastus lateralis, and in the final stages muscle wasting is diffuse, with relative preservation of the biceps femoris.11 Furthermore, muscle involvement can be detected as hyperintensity on STIR sequences before it is evident clinically.12,13 GSD is characterized by limbgirdle weakness and multi-system abnormality. Muscle MRI in GSD type 2 shows fatty infiltration mainly involving the adductor magnus and semimembranosus in early stages, and the long head of
biceps femoris, semitendinosus, and anterior thigh muscles at advanced stages.14 The differences in MRI manifestations between NLSDM with the mutation at the C-terminal and the N-terminal may be explained partly on a molecular basis. ATGL is the so-called patatin-like phospholipase domain-containing 2, which contains a patatin domain at the N-terminal region and a putative lipid-binding domain at the C-terminal region. Mutations in different domains of ATGL lead to different clinical profiles. The N-terminal region (encoded by 1–5 exons), containing the patatin domain and the catalytic site, is responsible for hydrolysis of TG, whereas the C-terminal region (encoded by 6–10 exons) accounts for the link between lipids and ATGL, and hydrolytic enzyme activity is not affected significantly. As a result, a mutation affecting the Nterminal region always leads to severe myopathy with multi-system involvement, while a mutation affecting the C-terminal region leads merely to muscle involvement along with lipid deposition.1 Complete deletion of PNPLA2 has not been identified, and further study is required. REFERENCES 1. Fischer J, Lefe`vre C, Morava E, Mussini JM, Lafor^ et P, Negre-Salvayre A, et al. The gene encoding adipose triglyeeride lipase (PNPLA2) is mutated in neutral lipid storage disease with myopathy. Nat Genet 2007;399:28–30. 2. Liang WC, Nishino I. Lipid storage myopathy. Curr Neurol Neurosci Rep 2011;11:97–103. 3. Hirano K, Ikeda Y, Zaima N, Sakata Y, Matsumiya G. Triglyceride deposit cardiomyovasculopathy. N Engl J Med 2008;359:2396–2398. 4. Ohkuma A, Nonaka I, Malicdan MC, Noguchi S, Ohji S, Nomura K, et al. Distal lipid storage myopathy due to PNPLA2 mutation. Neuromuscul Disord 2008;18:671–674. 5. Chen J, Hong D, Wang Z, Yuan Y. A novel PNPLA2 mutation causes neutral lipid storage disease with myopathy (NLSDM) presenting muscular dystrophic features with lipid storage and rimmed vacuoles. Clin Neuropathol 2010;29:351–356. 6. Reilich P, Horvath R, Krause S, Schramm N, Turnbull DM, Trenell M, et al. The phenotypic spectrum of neutral lipid storage myopathy due to mutations in the PNPLA2 gene. J Neurol 2011;258:1987–1997. 7. Lafor^ et P, Stojkovic T, Bassez G, Carlier PG, Cl ement K, Wahbi K, et al. Neutral lipid storage disease with myopathy: a whole-body nuclear MRI and metabolic study. Mol Genet Metab 2013;108:125– 131. 8. Fiorillo C, Brisca G, Cassandrini D, Scapolan S, Astrea G, Valle M, et al. Subclinical myopathy in a child with neutral lipid storage disease and mutations in the PNPLA2 gene. Biochem Biophys Res Commun 2013;430:241–244. 9. Kobayashi K, Inoguchi T, Maeda Y, Nakashima N, Kuwano A, Eto E, et al. The Lack of the C-terminal domain of adipose triglyceride lipase causes neutral lipid storage disease through impaired interactions with lipid droplets. J Clin Endocrinol Metab 2008;93:2877– 2884. 10. Mercuri E, Pichiecchio A, Counsell S, Allsop J, Cini C, Jungbluth H, et al. A short protocol for muscle MRI in children with muscular dystrophies [J]. Eur J Paediatr Neurol 2002;6:305–307. 11. Angelini C, Peterle E, Gaiani A, Bortolussi L, Borsato C. Dysferlinopathy course and sportive activity: clues for possible treatment. Acta Myol 2011;30:127–132. 12. Borsato C, Padoan R, Stramare R, et al. Limb-girdle muscular dystrophies type 2A and 2B: clinical and radiological aspects. Basic Appl Myol 2006;16:17–25. 13. Stramare R, Beltrame V, Dal Borgo R, Gallimberti L, Frigo AC, Pegoraro E, et al. MRI in the assessment of muscular pathology: a comparison between limb-girdle muscular dystrophies, hyaline body myopathies and myotonic dystrophies. Radiol Med 2010;115:585–599. 14. Pichiecchio A, Uggetti C, Ravaglia S, Egitto MG, Rossi M, Sandrini G, et al. Muscle MRI in adult-onset acid maltase deficiency. Neuromuscular Disorders 2004;14:51–55.
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MUSCLE MRI IN NEUTRAL LIPID STORAGE DISEASE WITH MYOPATHY CARRYING MUTATION C.18711G>A CHUNXIAO XU, MS, YAWEN ZHAO, MS, JING LIU, WEI ZHANG, MD, ZHAOXIA WANG, MD, and YUN YUAN, MD, PhD Department of Neurology, Peking University First Hospital, Beijing 100034, China Accepted 27 October 2014 ABSTRACT: Introduction: We describe the clinical and muscle MRI changes in 2 siblings with neutral lipid storage disease with myopathy (NLSDM) carrying the mutation c.18711G>A. Methods: Peripheral blood smears, genetic tests, and muscle biopsies were performed. Thigh MRI was performed to observe fatty replacement, muscle edema, and muscle bulk from axial sections. Results: Both siblings had similar fatty infiltration and edema. T1-weighted images of the gluteus maximus, adductor magnus, semitendinosus, and semimembranosus revealed marked and diffuse fatty infiltration. There was asymmetric involvement in biceps femoris and quadriceps. There was extensive fatty infiltration in the quadriceps, except for the rectus femoris. Gracilis and sartorius were relatively spared. Thigh muscle volume was decreased, while the gracilis and sartorius appeared to show compensatory hypertrophy. Conclusions: Compared with previous reports in NLSDM, MRI changes in this myopathy tended to be more severe. Asymmetry and relatively selective fatty infiltration were characteristics. Muscle Nerve 51: 922–927, 2015
Neutral lipid storage disease is an autosomal recessive genetic disease that can be classified into neutral lipid storage disease with ichthyosis and neutral lipid storage disease with myopathy (NLSDM). NLSDM was first reported by Fisher et al. in 2007.1 It is caused by mutation of the adipose triglyceride lipase gene (ATGL; also called patatin-like phospholipase domain-containing 2 [PNPLA2]). ATGL is a rate-limiting enzyme in the hydrolysis of triglycerides (TG) and plays a primary role in lipolysis. As a result, ATGL dysfunction prevents hydrolysis of TG, leading to TG accumulation in the cytoplasm of various organs.2 Clinically, patients with NLSDM show slowly progressive myopathy in adulthood, which can be proximal or distal dominant.1,2 Proximal weakness is more common in the early stages of the disease. In the middle and late stages, 50% of patients present with potentially life-threatening hyperAbbreviations: ATGL, adipose triglyceride lipase; GSD, glycogen storage disease; LGMD, limb girdle muscular dystrophy; MGT, modified Gomori trichrome; MRC, Medical Research Council Grade Scale; NLSDM, neutral lipid storage disease with myopathy; ORO, Oil Red O; PAS, periodic acidic Schiff; PNPLA2, patatin-like phospholipase domain-containing 2; RT-PCR, reverse transcription-polymerase chain reaction; STIR, short inversion time inversion-recovery; TG, triglycerides Key words: adipose triglyceride lipase gene; magnetic resonance imaging; mutation c.18711G>A; myopathy; neutral lipid storage disease; patatin-like phospholipase domain-containing 2 Correspondence to: W. Zhang; e-mail: [email protected] C 2014 Wiley Periodicals, Inc. V
Published online 31 October 2014 in Wiley Online Library (wileyonlinelibrary. com). DOI 10.1002/mus.24507
922
trophic cardiomyopathy, which may be a cause of premature death due to heart failure or arrhythmia.3 Pathologically, several lipid droplets are deposited within muscle fibers, predominantly in type I fibers, and in cells of other organs. Rimmed vacuoles may also be observed.4,5 There have been a few reports of muscle MRI changes in NLSDM over the past several years in which the gene mutation predominantly affects the C-terminal region.6–8 This type of NLSDM generally presents with a manifestation of myopathy without multi-system damage. In contrast to previously reported cases, we describe the clinical and thigh MRI findings in 2 siblings in whom the gene mutation predominantly affects the N-terminal region. This mutation often produces multi-system damage, and our aim is to better characterize the fatty replacement in skeletal muscle and its possible use as a marker in muscle MRI. PATIENTS AND METHODS
The procedures were performed in accordance with the ethical standards of the responsible committee on human experimentation and approved by the Institutional Review Board. Informed consent for all examinations was obtained from the patients Patient 1. The proband was a 44-year-old man of Han nationality. He was 175 cm tall and weighed 65 kg (body mass index: 21.2 kg/m2). Motor developmental milestones were normal. At age 31 years, he presented with right leg weakness and walked slowly. Two months later he had difficulty lifting his arms. At age 32 years, he developed left leg weakness. Muscle weakness developed slowly. Currently, he is able to walk unaided but cannot lift his arms fully and requires help ascending and descending stairs. He had a history of congenital heart disease (atrial septal defect central type) and has had hypertension for the past decade. Physical examination revealed no ichthyosis. Neurological examination showed weakness affecting the deltoids (2/5 Medical Research Council [MRC] Scale), biceps brachii (2/5 MRC), and triceps brachii (4/5 MRC). Weakness of the distal upper limbs was 4/5 MRC. Asymmetrical lower limb weakness was observed that affected predominantly MUSCLE & NERVE
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the right leg, and muscle strength was graded as: hip flexors (right 4/5 MRC and left 41/5 MRC), knee extension (right 4/5 MRC and left 5-/5 MRC), knee flexion (right 4/5 MRC and left 5-/5 MRC), and foot dorsiflexion (right 3/5 MRC and left 5-/5 MRC). He had atrophy of shoulder girdle, gluteus maximus, thigh, and gastrocnemius muscles, along with finger joint contractures. Tendon reflexes were absent, and there were no pathological reflexes. Laboratory investigation showed serum creatine kinase levels fluctuated between 564 and 1,485 IU/L (normal, 38–174 IU/L), LDH was 467 IU/L (normal, 140–271 IU/L), ALT was 53 IU/L (normal, 5–40 IU/L), AST was 65 IU/L (normal, 8–40 IU/L), TG was 1.12 mmol/L (normal, 0–1.7 mmol/L), and high-density lipoprotein-cholesterol was 0.99 mmol/L (normal, 1.04–1.99 mmol/L). Echocardiography revealed diastolic dysfunction and asymmetric hypertrophy of the left ventricle, which satisfied a diagnosis of hypertrophic cardiomyopathy. In addition there was flow from left to right at the atrial level. Abdominal ultrasound suggested a fatty liver. EMG revealed myopathic changes in bilateral biceps, abductor pollicis brevis, sternocleidomastoid, paraspinal, vastus medialis, and tibialis anterior muscles. Nerve conduction studies, H-reflexes, F-waves, and somatosensory evoked potentials were normal. Patient 2. Patient 2 was the 42-year-old younger brother of Patient 1. Early motor development was normal. He had difficulty running since childhood. At age 27 years, he developed weakness in the right arm. At age 31 years, he presented with muscle weakness of both legs with right lower leg wasting. Two years later, he exhibited a waddling gait along with muscle atrophy in his proximal limbs and shoulder girdle muscles, but was still able to descend and ascend stairs and squat without assistance. At age 37 years, he presented with severely reduced upward arm movement (less than 90 ), frequent falls, and problems descending and ascending stairs and squatting. Muscle weakness became severe, except for the distal upper limbs. At age 41 years, he was hospitalized with acute necrotizing pancreatitis. He had a 10 year history of abnormal appearing stools and a 2-year history of hypertension. On admission, physical examination revealed mitral and triscupid murmurs. Neurological examination revealed proximal arm weakness affecting the deltoids (3/5 MRC), biceps brachii (2/5 MRC), and triceps brachii (4/5 MRC). Asymmetrical leg weakness was also observed that affected predominantly the right leg. Muscle strength was graded as: hip flexors (right 4/5 MRC and left
41/5 MRC), knee extensors (right 4-/5 MRC and left 4/5 MRC), foot dorsiflexors (right 5-/5 MRC and left 5/5 MRC). Tendon reflexes were absent, and there were no pathological reflexes. There was atrophy of the thenar, hypothenar, deltoid, supraspinatus, infraspinatus, and tibialis anterior muscles, along with pseudohypertrophy of the forearms. The patient also exhibited pes cavus. Laboratory investigation showed serum creatine kinase levels that fluctuated between 1,000 and 2,000 IU/L (normal, 25–195 IU/L). Serum levels of lactate, pyruvate, TG, serum carnitine fraction, and long-chain fatty acid profile were within normal ranges. ECG examination showed premature ventricular extrasystoles (2 beats per 24 h). Echocardiogram revealed left ventricular hypertrophy and reduced left ventricular diastolic function. Head MRI showed several focal ischemic lesions mainly in the right centrum semiovale. Neurophysiological examination revealed myogenic changes in the left extensor digitorum brevis, left biceps, right quadriceps, and right tibialis anterior muscles. Nerve conduction studies and visual evoked potentials were normal. Peripheral Blood Smears and Muscle Biopsy. Finger stick blood was taken from Patient 2 following written informed consent. The smear was stained according to standard procedures with Oil Red O (ORO). The sample was fixed in 60% isopropanol for 5 min and rinsed with running water. The dried blood smear was then stained with ORO for 30 min at room temperature, followed by differentiation in 60% isopropanol for 2 min, rinse in running water, counter-stain with hematoxylin for 30 s, and thorough rinse in running water. Finally, the smear was mounted with glycerin jelly and observed under a light microscope. With written informed consent, quadriceps biopsies were obtained from both patients. Serial frozen sections were stained according to standard procedures with hematoxylin and eosin, modified Gomori trichrome (MGT), periodic acidic Schiff (PAS), ORO, adenosine triphosphatase at pH 10.5, 10.4, 10.2, 4.2, 4.3, and 4.6, NADH dehydrogenase, succinate dehydrogenase, cytochrome c oxidase, and nonspecific esterase. Sections were observed under a light microscope.
Blood from the patients and 3 additional family members was collected in EDTA tubes. Genomic DNA was extracted from peripheral blood. The sequence of PNPLA2 was obtained from the GenBank human genome database (http://www.ncbi.nlm.nih.gov). Primers for ATGL were designed using Primer 3.0 software. All exons and flanking intron sequences of ATGL were amplified using polymerase chain reaction (PCR)
Gene Sequencing.
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FIGURE 1. (A) Numerous fat droplets were observed in the cytoplasm of nucleated cells (Jordan anomaly); magnification 31,000. (B) Muscle histology shows a myopathic pattern and numerous vacuoles of varying size. (C) MGT staining did not reveal any ragged red fibers. (D) ORO staining exhibited severe fat droplet deposition in most muscle fibers.
on a 2720 Thermal Cycler. Primers for exon 2 of ATGL were: forward primer, 50 -AGCAGGCGG CTCACAGAG-30 and reverse primer, and 50 CACGGTACCCACCGACTC-30 . PCR products were identified by 1.5% agarose gel electrophoresis and were sequenced using an ABI 3730xl automated sequencer after purification. All PCR products were sequenced in both directions. Reverse Transcription-PCR. Total RNA was extracted from muscle tissue from patient 2 and stored in RNA later to prevent RNA degradation. The main procedures have been described previously.9 Briefly, cDNA was synthesized from total RNA using SuperScript First-Strand Synthesis System for reverse transcription-PCR (RT-PCR) and used as the template for PCR. Considering the mutation site, we designed forward and reverse primers at exons 2 and 3, respectively, which were: forward primer, 50 -ATGTTTCCCCGCGAGAAGA-30 and reverse primer, 50 -ACCTGGTAAAGATCATC CGCAG-30 . Gene sequencing was the same as that described above. MRI of Thigh Muscle. MRI examination was performed using a 3.0-Tesla MRI scanner (Signa Excite; GE Medical Systems, Milwaukee, Wisconsin) with an 8-channel head coil. Routine images obtained included T1-weighted axial and coronal images, T2-weighted axial and coronal images, short inversion time inversion-recovery (STIR) coronal images, and diffusion-weighted images. 924
According to the scope and extent of muscle involvement in the T1-weighted image sequence, muscle fatty infiltration was graded into 6 degrees: 0, normal muscle; 1, punctate hyperintense lesions; 2, scattered hyperintense lesions which accounted for less than 30% of muscle volume; 3, scattered fused hyperintense lesions, which accounted for 30–60% of muscle volume; 4, large sheets of fused hyperintense lesions, which accounted for over 60% of muscle volume; and 5, all muscle tissue was replaced by fat tissue.10 Two independent researchers assessed fatty infiltration, edema, and muscle bulk in 12 muscles, including gluteus maximus, lateral thigh muscles, medial thigh muscles, and posterior thigh muscles. RESULTS Detection of Jordan Anomaly and Muscle Histology Features. Numerous fat droplets were observed in
the cytoplasm of nucleated cells in peripheral blood smears, called Jordan anomaly (Fig. 1A). Muscle histology showed myopathic changes with numerous vacuoles of varying size (Fig. 1B). Patient 2 exhibited rimmed vacuoles within some muscle fibers. MGT and PAS staining did not reveal any ragged red fibers or separate glycogen storage (Fig. 1C). ORO staining in both patients exhibited extensive fat droplet deposition in most muscle fibers, predominantly affecting type I fibers (Fig. 1D). Genetic Analysis. The patients were homozygous for the mutation at intron 2 first base G>A MUSCLE & NERVE
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FIGURE 2. (A) Genetic results from proband showing homozygous mutation in intron 2 (c.18711G>A) (arrow). (B) Genetic results from a healthy control.
(c.18711G>A) (Fig. 2). The results of RT-PCR confirmed the mutation, which resulted in retention of a 97 bp sequence from intron 2 and insertion mutants (Fig. 3). There was a heterozygous mutation of G>A in the 3 other family members tested. MRI Features of Thigh Muscle. In T1-weighted MRI sequences, the thighs of Patient 1 (Fig. 4A) and Patient 2 (Fig. 4B) revealed asymmetric distribu-
tion of fatty infiltration. The degree of muscle fatty infiltration is listed in Table 1. Gluteus maximus, adductor magnus, semitendinosus, and semimembranosus muscles showed marked and diffuse fatty infiltration. Biceps femoris and quadriceps muscles showed asymmetric involvement (the right leg was more severe than the left). Fatty infiltration in the quadriceps was severe except for the rectus femoris. Gracilis and sartorius were less affected. The gracilis muscle of Patient 1 was hypertrophied.
FIGURE 3. RT-PCR showed a mutation leading to a splice site shift (the splice site is marked with arrow). (A) Normal splice site and (B) shifted splice site due to a mutation of c.18711G>A. MUSCLE & NERVE
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FIGURE 4. Muscle MRI performed on proximal lower limbs revealed asymmetric multiple fatty infiltration in T1-weighted images. (A) Patient 1. (B) Patient 2.
Gracilis and sartorius muscles of Patient 2 were hypertrophied, and the entire thigh muscle bulk was reduced. In STIR sequences, there was asymmetric slight tissue edema, mainly involving the quadriceps and biceps femoris. DISCUSSION
NLSDM is caused by a mutation in the gene encoding adipose triglyceride lipase. Patients with NLSDM have a slowly progressive myopathy in adulthood, which can be either proximal or distal.1,2 The observation of numerous fat droplets deposited in the cytoplasm of nucleated cells in a peripheral blood smear (Jordan anomaly), together with marked fat droplet deposition in type I muscle fibers and genetic tests can lead to the diagnosis. In this study, 2 siblings presented with progressive muscle weakness and cardiomyopathy. Clinical profiles, pathological examination, and genetic results suggested a diagnosis of NLSDM. The mutation of c.18711G>A, predominantly affecting the N-terminal region of ATGL, is reported rarely. We report clinical and thigh muscle MRI changes in these 2 patients. Generally, muscle fatty infiltration was observed mainly in the gluteus maximus and posterior and lateral thigh muscles. Both patients had similar patterns of fatty infiltration on muscle MRI, and the severity of muscle involvement was also similar. Gluteus maximus, semitendinosus, semimembranosus, adductor magnus, and vastus medialis muscles all exhibited large sheets of fatty infiltration, while gracilis, adductor longus, and sartorius muscles showed punctate fatty infiltration. The distribution of the fatty infiltration in the 2 patients was similar. Posterior thigh muscles were more involved than 926
lateral and medial thigh muscles. In particular, the fatty infiltration was asymmetric, involving the right thigh more than the left. It is possible that the reason for this observation was in some way related to early weakness of the right leg and preference for right limb weight bearing. Fatty infiltration also showed relative selectivity in quadriceps femoris. Vastus lateralis, vastus medialis, and vastus intermedius were all heavily involved, in contrast to the rectus femoris, which was relatively spared. Edema was mainly observed in the quadriceps and biceps femoris, and was not pronounced compared with the observed fatty infiltration, which may be related to the course of the disease. In time, edema will be gradually replaced by fatty tissue. Muscle bulk generally decreases, while gracilis and sartorius muscles, which play a secondary role in lower limb movement, show compensatory hypertrophy.
Table 1. Degree of fatty infiltration in muscles observed with MRI. Patient 1
Gluteus maximus Biceps femoris Semitendinosus Semimembranosus Vastus lateralis Vastus intermedius Vastus medialis Rectus femoris Adductor magnus Adductor longus Sartorius Gracilis
Patient 2
Left
Right
Left
Right
5 1 5 5 1 2 5 1 5 2 1 1
5 5 5 5 5 5 5 1 5 2 1 1
5 3 5 5 3 3 5 1 5 1 1 1
5 5 5 5 3 5 5 1 5 1 1 1
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Our findings suggest that MRI changes in NLSDM carrying the mutation c.18711G>A might show a severe type of myopathy with regard to degree and scope of involvement. Over the past several years, there have been a few reports of MRI changes in NLSDM with the gene mutation predominantly affecting the C-terminal region,6–8 which led to isolated myopathy compared with the gene mutation predominantly affecting the Nterminal region (our patients) resulting in multisystem abnormality. With regard to MRI changes in NLSDM predominantly affecting the C-terminal region, posterior thigh muscles are reported to be more severely involved than quadriceps,6–8 and quadriceps appears to be spared.7,8 There are, however, conflicting opinions on the involvement of semitendinosus and semimembranosus muscles. For example, Fiorillo et al.8 suggested that the semitendinosus seemed to not be involved, while Lafor^et et al.7 suggested that it was severely involved. We suggest that the difference in findings between these researchers can be explained by the onset age and duration of the condition. In the former case, the patient was a 14-year-old boy with subclinical symptoms, while in the latter case, the patients were older and showed typical clinical symptoms for a decade. This perhaps suggests that the semitendinosus muscle is involved relatively late in the condition. Our patients share similar onset ages and duration with patients in previous studies.6,7 However, our patients presented with marked fatty infiltration in the gluteus maximus, adductor magnus, and posterior thigh muscles (semitendinosus and semimembranosus), and asymmetric involvement of quadriceps. The difference observed in thigh MRI coincides with a study1 that reported that mutations at the N-terminal region always lead to severe myopathy compared with mutations at the C-terminal region. Apart from disease evaluation, thigh MRI may also inform differential diagnosis. In patients with progressive proximal limb weakness, as in this study, it is difficult to distinguish limb girdle muscular dystrophy (LGMD) and glycogen storage disease (GSD), especially late-onset GSD type 2. In contrast to NLSDM, weakness in LGMD2B affects the adductor magnus early, then the vastus lateralis, and in the final stages muscle wasting is diffuse, with relative preservation of the biceps femoris.11 Furthermore, muscle involvement can be detected as hyperintensity on STIR sequences before it is evident clinically.12,13 GSD is characterized by limbgirdle weakness and multi-system abnormality. Muscle MRI in GSD type 2 shows fatty infiltration mainly involving the adductor magnus and semimembranosus in early stages, and the long head of
biceps femoris, semitendinosus, and anterior thigh muscles at advanced stages.14 The differences in MRI manifestations between NLSDM with the mutation at the C-terminal and the N-terminal may be explained partly on a molecular basis. ATGL is the so-called patatin-like phospholipase domain-containing 2, which contains a patatin domain at the N-terminal region and a putative lipid-binding domain at the C-terminal region. Mutations in different domains of ATGL lead to different clinical profiles. The N-terminal region (encoded by 1–5 exons), containing the patatin domain and the catalytic site, is responsible for hydrolysis of TG, whereas the C-terminal region (encoded by 6–10 exons) accounts for the link between lipids and ATGL, and hydrolytic enzyme activity is not affected significantly. As a result, a mutation affecting the Nterminal region always leads to severe myopathy with multi-system involvement, while a mutation affecting the C-terminal region leads merely to muscle involvement along with lipid deposition.1 Complete deletion of PNPLA2 has not been identified, and further study is required. REFERENCES 1. Fischer J, Lefe`vre C, Morava E, Mussini JM, Lafor^ et P, Negre-Salvayre A, et al. The gene encoding adipose triglyeeride lipase (PNPLA2) is mutated in neutral lipid storage disease with myopathy. Nat Genet 2007;399:28–30. 2. Liang WC, Nishino I. Lipid storage myopathy. Curr Neurol Neurosci Rep 2011;11:97–103. 3. Hirano K, Ikeda Y, Zaima N, Sakata Y, Matsumiya G. Triglyceride deposit cardiomyovasculopathy. N Engl J Med 2008;359:2396–2398. 4. Ohkuma A, Nonaka I, Malicdan MC, Noguchi S, Ohji S, Nomura K, et al. Distal lipid storage myopathy due to PNPLA2 mutation. Neuromuscul Disord 2008;18:671–674. 5. Chen J, Hong D, Wang Z, Yuan Y. A novel PNPLA2 mutation causes neutral lipid storage disease with myopathy (NLSDM) presenting muscular dystrophic features with lipid storage and rimmed vacuoles. Clin Neuropathol 2010;29:351–356. 6. Reilich P, Horvath R, Krause S, Schramm N, Turnbull DM, Trenell M, et al. The phenotypic spectrum of neutral lipid storage myopathy due to mutations in the PNPLA2 gene. J Neurol 2011;258:1987–1997. 7. Lafor^ et P, Stojkovic T, Bassez G, Carlier PG, Cl ement K, Wahbi K, et al. Neutral lipid storage disease with myopathy: a whole-body nuclear MRI and metabolic study. Mol Genet Metab 2013;108:125– 131. 8. Fiorillo C, Brisca G, Cassandrini D, Scapolan S, Astrea G, Valle M, et al. Subclinical myopathy in a child with neutral lipid storage disease and mutations in the PNPLA2 gene. Biochem Biophys Res Commun 2013;430:241–244. 9. Kobayashi K, Inoguchi T, Maeda Y, Nakashima N, Kuwano A, Eto E, et al. The Lack of the C-terminal domain of adipose triglyceride lipase causes neutral lipid storage disease through impaired interactions with lipid droplets. J Clin Endocrinol Metab 2008;93:2877– 2884. 10. Mercuri E, Pichiecchio A, Counsell S, Allsop J, Cini C, Jungbluth H, et al. A short protocol for muscle MRI in children with muscular dystrophies [J]. Eur J Paediatr Neurol 2002;6:305–307. 11. Angelini C, Peterle E, Gaiani A, Bortolussi L, Borsato C. Dysferlinopathy course and sportive activity: clues for possible treatment. Acta Myol 2011;30:127–132. 12. Borsato C, Padoan R, Stramare R, et al. Limb-girdle muscular dystrophies type 2A and 2B: clinical and radiological aspects. Basic Appl Myol 2006;16:17–25. 13. Stramare R, Beltrame V, Dal Borgo R, Gallimberti L, Frigo AC, Pegoraro E, et al. MRI in the assessment of muscular pathology: a comparison between limb-girdle muscular dystrophies, hyaline body myopathies and myotonic dystrophies. Radiol Med 2010;115:585–599. 14. Pichiecchio A, Uggetti C, Ravaglia S, Egitto MG, Rossi M, Sandrini G, et al. Muscle MRI in adult-onset acid maltase deficiency. Neuromuscular Disorders 2004;14:51–55.
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