Sissel Løseth, Nicol C. Voermans, Torberg Torbergsen, Sue Lillis, Christoffer Jonsrud, Sigurd Lindal, Erik- Jan Kamsteeg, Martin Lammens, Marcus Broman, Gabriele Dekomien, Paul Maddison, Francesco Muntoni, Caroline Sewry, Aleksandar Radunovic, Marianne de Visser, Volker Straub, Baziel van Engelen, Heinz Jungbluth
A novel late-onset axial myopathy associated with mutations in the skeletal muscle ryanodine receptor (RYR1) gene
Journal of Neurology
SUPPLEMENTAL FILE S1
SUPPLEMENTAL FILE S1 – Case histories
Family 1 - Patient 1
This 38-year-old Norwegian farmer presented from his early 20s with slowly progressive lumbar and, to a lesser extent, pelvic girdle weakness. Early motor developmental milestones were normal. He had particular difficulties carrying objects (such as a tray) when standing or walking with his arms straightened, which would make him fall forward. However, when sitting in a chair or lying supine he had no problems raising his arms above his head, or raising his legs. In addition, from his early teens he had experienced a deep, aching pain affecting his lower back, shoulders and legs, particularly after physical exercise. The pain had increased in frequency and intensity in recent years and he also experienced pins and needles sensations in his hand, which had become a major complaint. There was no history of rhabdomyolysis or myoglobinuria. There were no symptoms suggestive of bulbar, respiratory or cardiac involvement. He was extremely myopic and had cataracts secondary to staphyloma, which also affected other family members including his mother. He had a recent anesthesia without complications.
On examination, he had a waddling gait, lumbar hyperlordosis and scapular winging. There was paraspinal muscle atrophy but muscles in the shoulder girdle appeared hypertrophic. There was profound axial weakness and he had to extend his upper limbs to maintain his centre of gravity. He could not bend forward without falling. There were no fasciculations. Tendon reflexes were present and sensory examination was normal.
CK was slightly raised (486-857 U/L; normal value <400 U/L). Other blood test were normal except from slightly elevated anti-GM1 IgG (1800 BTU) and IgM (2520 BTU) levels (normal ranges 0-800 BTU).
EMG in the lower thoracic and upper lumbar paraspinal muscles, semimembranous and semitendinous muscles showed spontaneous activity with fibrillation potentials, positive sharp waves and myotonic discharges. There was a pronounced myopathic pattern of motor unit potentials. In the lower lumbar paraspinal muscles no muscle activity was detected. In the limb muscles EMG was normal or showed only mild abnormalities with a predominant myopathic pattern, but in some muscles there were additional mild neurogenic features. Nerve conduction studies and quantitative sensory testing were normal.
Muscle MRI showed prominent involvement of the lower lumbar paraspinal muscles and, less pronounced pelvic and posterior thigh muscles. In particular, within the thigh there was marked fatty infiltration of the biceps femoris, semitendinosus and semimembranosus muscles with some edema. Lower leg muscles were normal. MRI imaging of the brain and the spinal cord were normal.
Muscle biopsy from the right quadriceps showed mildly increased fibre size variability and some uneveness of oxidative stains. There were no cores or other more overt myopathic features. There were no abnormalities on immunohistochemical stains or on electron microscopy. α-glucosidase activity in fibroblasts was normal, excluding Pompe's disease.
The patient remained essentially stable over a follow-up period of 10 years and still works full time as a farmer, although his back pain continues to be a problem and responds poorly to analgesics. In recent years he has experienced episodes of dizziness and breathlessness but further investigations including an electroencephalography, echocardiography and spirometry were normal.
Molecular genetic testing for mutations in FKRP, CAV3, MYOT and DES did not reveal any pathogenic variations. RYR1 sequencing revealed a heterozygous c.4178A>G; p.Lys1393Arg substitution in the patient and his similarly affected mother, previously reported in the Scandinavian population in association with MHS. A recent in vitro contracture test (IVCT) was positive.
More extensive genetic studies in this family confirmed the c.4178A>G; p.Lys1393Arg RYR1 mutation also in the similarly affected mother of the patient whose medical history and presenting features are outlined below (Patient 2). The c.4178A>G; p.Lys1393Arg RYR1 mutation was also identified in the patient’s two sisters, one of them currently asymptomatic and one with complaints of marked lower back pain but no clinical muscle weakness. A maternal aunt of the patient (sister of Patient 2) had muscle weakness in the lower legs, attributed to a severe axonal polyneuropathy; she could not be reassessed recently as she had died from cancer some years ago but tested positive for the familial RYR1 mutation on a stored DNA sample. The brother and son of Patient 1 tested negative for the familial RYR1 mutation and did not have any symptoms.
Family 1 - Patient 2
The mother of Patient 1 presented from her 20s with weakness and pain in the lower back, exacerbated by exercise and aggravated during pregnancies. Early motor developmental milestones had been normal. From around 50 years of age she began to feel weak also in her hips and thighs, and had to use sticks when walking outdoors.
She had been healthy apart from the extreme myopia and secondary cataracts also affecting her son and other relatives. She had one previous general anaesthesia without complications.
CK was normal at 170 IU/l. EMG at presentation time showed moderate myopathic changes in the deltoid and biceps muscles, and a mild myopathic pattern in the vastus lateralis. Subsequent EMGs revealed a moderate inactive neurogenic pattern in the quadriceps and tibialis anterior muscles, whilst there was a myopathic pattern in the gluteus medius, biceps brachii and deltoid muscles; in the latter, there were also few fibrillation potentials and positive sharp waves, but no myotonic discharges. There was no EMG detectable activity in the lumbar paraspinal muscles. Nerve conduction studies were normal.
MRI of the low back showed degenerative changes with some disc protrusion. There was complete fatty degeneration in the lumbar and the lower thoracic paravertebral muscles, as well as pronounced fat infiltration in the glutei. Muscle MRI of the lower limbs was not performed.
Serial muscle biopsies from the quadriceps muscles, gluteus maximus and biceps brachii, respectively, were either normal or showed mild abnormalities with a few necrotic fibres and some perivascular lymphocytic infiltrates suggestive of polymyositis. She was treated with steroids with no effect. The most recent muscle biopsy performed at 72 years of age revealed grouping of both type 1 and type 2 muscle fibres consistent with neurogenic changes. No overt cores or other abnormalities were seen with stains for oxidative enzymes.
Her further course was slowly progressive. On the most recent examination at 76 years of age she was still able to walk without support indoors, but had an extremely waddling gait due to hip muscle weakness. There was no camptocormia, but lumbar hyperlordosis. The distal muscles were normally strong without muscle atrophy.
Molecular genetic testing for facioscapulohumeral muscular dystrophy, dystrophia myotonica type 1 and type 2, spinal muscular atrophy and Charcot Marie Tooth (CMT) type 2A were all negative. RYR1 sequencing revealed the same heterozygous c.4178A>G; p.Lys1393Arg substitution also identified in her son (Patient 1).
Family 2 - Patient 3
This 39 year old patient presented with a 5 year history of increasing myalgias and fatigue. Myalgia was most pronounced during and following exercise in the shoulder girdle muscles. Prior to the onset of symptoms he used to be very athletic, engaging in a wide range of endurance activities including cycling, running and skating. Motor development had been normal. He worked as a dairy farmer but found certain tasks involving outstretching his arms, for example during milking, increasingly difficult and painful. After the diagnosis of a myopathy, he installed a milking robot and markedly reduced his physical activities, resulting in substantial reduction of muscle pain and cramps.
In the past medical history, he had several unrelated problems including carpal tunnel syndrome, a testicular tumor, a bone fracture and a choleostatoma, all requiring surgical intervention. Despite repeated exposure to anaesthetic agents he did not suffer a malignant hyperthermia reaction. However, he was very intolerant of high temperatures which he reported to cause him a “boiling feeling inside”. His brother suffered similar but less severe complaints and both adapted their working hours according to the outside temperatures but never sought medical attention. No family member had previous perioperative complications.
On examination there was no facial weakness and range of eye movements was full. There was axial and scapular winging but only mild weakness in the limbs. Specific MRC grades were MRC4 in hip flexion and extension and MRC3 in the rhomboid and the supraspinatus muscles.
CK levels were 213 IU/l. Muscle biopsy showed mild myopathic features with type 1 predominance and unevenness of stain but not overt cores.
RYR1 sequencing revealed a heterozygous c.7025A>G (p.Asn2342Ser) variation in exon 43 previously reported in association with MHS [Robinson 2006]. No more extensive genetic studies were performed in the family.
Family 3 - Patient 4
This 62 year old lady presented with a 2 year history of painless and progressive forward bending of her spine, resulting in a stooped posture whilst walking. Her relatives commented that she could almost be “bent double” on occasions. She did not report any double vision, limb weakness or urinary or bowel disturbance. There was no history of muscle cramps, limb posturing or tremor.
In the past medical history she had a history of Hashimoto thyroiditis and had undergone a thyroidectomy for goitre without any anaesthetic complications. There was no family history of neurological or neuromuscular disorders. Early motor developmental milestones had been normal.
On examination her bent posture manifested almost immediately when walking but could be corrected when standing still against a wall or in the supine. She could not puff her cheeks out but there was otherwise no facial weakness. Extraocular movements were normal. Muscle tone and power was normal in all four limbs. Deep tendon reflexes were relatively brisk with downgoing plantars. There was no ataxia and no bradykinesia.
CPK levels were 300 IU/l. An EMG of the upper limbs, tibialis anterior and paraspinal muscles showed excess of polyphasic units. Muscle MRI of the lower limbs showed mildly increased signal within the hamstrings and the soleus bilaterally on T1-weighted images. There was no significant oedema.
Muscle biopsy from the left quadriceps showed mildly increased variability in fibre size due to the presence of several round and angulated atrophic fibres as well as few hypertrophic fibres. There were a few basophilic fibres but no other signs of inflammation or necrosis. There was no increase in internal nuclei, fatty or connective tissue. Fibre typing showed marked type I predominance. The NADH, SDH and COX staining showed large centrally or peripherally located areas devoid of mitochondria and oxidative enzyme activity in many type I fibres, suggestive of central cores. These areas were also devoid of myophosphorylase and PAS positivity, although a peripheral PAS-positive rim was noted around the cores. Myofibrillar ATPase activity was also lost in the majority of these lesions, consistent with unstructured cores. Four COX negative and SDH positive fibers were seen but no ragged red fibres noted. Glycogen and lipid content as well as acid phosphatase activity appeared normal. Immunolabelling showed a few fibres positive for neonatal myosin. Immunostaining for CD3, CD8, CD4, CD20 and CD68 revealed only very few scattered positive cells. There was no evidence for abnormal inclusions on staining for p62.
Genetic testing for FSHD was negative. RYR1 sequencing identified a heterozygous c.9713A>G; p.Glu3238Gly sequence variant. This variant has not previously been reported, however, it affects a highly conserved amino acid, and in silico analysis predicts that it may affect the function of the RyR1 protein. No additional genetic studies were performed in the family.
Family 4 - Patient 5
This 56 year old gentleman from Ghana presented with a longstanding history of muscle pain and recently evolved lower back pain which hindered him during his work as a janitor. He also reported an inability to run and had to hold on to the bannister to get up stairs.
In the family history he had had 5 sibs of whom four had died due to unknown causes; one sister was alive and supposedly healthy. His mother had died in her eighties but his father had died at a young age of unknown causes. There was no known family history of neurological or neuromuscular problems. His early motor developmental milestones had been normal. He had four healthy children.
On examination, he had a generally muscular appearance. Trendelenburg sign was positive and there was some scapular winging. Ankle jerks were absent.
CK was raised at 1407 IU/l (normal <171 IU/l). Acid maltase activity in leucocytes was normal. An EMG showed small MUAPs and fast recruitment of paraspinal muscles at the thoracic level.
Muscle biopsy showed muscle fibres with a smaller diameter. There some fibres with unevenness of oxidative enzyme and COX activity, with occasional fibres showing core-like lesions. There were no obvious ultrastructural abnormalities on EM.
A CT scan of the spine and the lower limb showed decreased attenuation in the paravertebral muscle on the lumbar and, to a lesser extent, the thoracic level. There was also decreased attenuation within the posterior thigh but CT imaging of the lower leg was normal.
Genetic testing for FSHD and DES gene mutations was negative. RYR1 sequencing revealed a heterozygous c.10354A>C; p.Llys3452Gln in exon 69. No further genetic studies were performed in his family.
Family 5 - Patient 6
This previously healthy 47 year old patient presented with progressive axial weakness and scapular winging. In the past medical history, he had never been able to run and had had difficulties with sports in school and was, for example, unable to climb up a rope. From his teens, he experienced fatigue and pain in his lower back muscles. Medical consultation at the time revealed a mild scoliosis but no other abnormalities. His lower back pain increased over the years and he developed a progressive lumbar lordosis. He mentioned that with increasing weakness of lower back muscles he tried to compensate by using his abdominals. In addition, going up stairs became more difficult. Furthermore, he suffered from myalgia and muscle cramps, predominantly affecting the muscles of his back, upper arms and legs. The pain was most intense during and after physical exercise.