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Cylindrical spirals in a familial neuromuscular disorder.

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Cylindrical Spirals in
a F d a l Neuromuscdar Disorder
Kevin E. Bove, MD, Susan T. Iannaccone, MD, Pamela K. H i l t o n , MS,
a n d Frederick Samaha, MD
Cylindrical spirals are u n i q u e membranous structures t h a t were detected i n skeletal muscle of a m o t h e r and one of
her two children; all three have percussion myotonia but n o evidence of weak or wasted skeletal muscles. Muscle
cramps, stiffness, posteffort muscle tightness, myotonic lid lag, a n d the cylinders appear or progress w i t h age, b u t
the disorder is asymptomatic i n t h e children a n d only mildly incapacitating i n the mother. The cylinders are 8 p
long and 1 p wide, are composed of spiraling double-laminate membrane resembling myelin, and are derived f r o m
abnormal subsarcolemmal tubulovesicular structures that are interpreted as pathological T-tubes.
Bove KE, Iannaccone ST, Hilton PK, et al: Cylindrical spirals in a familial neuromuscular disorder.
Ann Neurol 7 : 5 5 0 - 5 5 6 , 1980
Although distinctive intramuscular structures serve
t o identify certain congenital myopathies [ 14, 151,
disease-specific alterations a r e unusual i n this illdefined group of conditions. In a r e c e n t r e p o r t an
unusual structure, t e r m e d a cylindrical spiral, was observed in skeletal muscle of t w o unrelated m e n with
dissimilar histories and was i n t e r p r e t e d as n o t disease
specific [ 51. We have observed identical structures i n
a m o t h e r and one of h e r t w o children, all of w h o m
have percussion myotonia.
Case Reports
Patient 1
A 31-year-old woman presented with the chief complaint
of muscle cramps. She had been considered a clumsy child
who had frequent falls resulting in minor injuries. T h e
cramping had been particularly troublesome during the
year prior to presentation and interfered with her housework. She reported not being able to get out of bed because of severe muscle stiffness lasting one to three days
and occurring every four to six weeks. She also described a
“drawing” of muscles that generally developed after exertion. This could involve any skeletal muscle group but was
usually proximal. She had had a heart murmur as a child
that cleared spontaneously. She denied an unusual incidence of pulmonary infections. She finished high school
but had a history of depression and had used chlordiazepoxide (Librium) intermittently for ten years. She denied paresthesias, fasciculations, or dysphagia.
Physical examination revealed a healthy-appearing
woman who had no evidence of muscle stiffness o r weakness. Deep tendon reffexes were normal, as were findings
on sensory and cerebellar testing. No abnormalities ocFrom the Departments of Pathology, Pediatrics, and Neurology,
University of Cincinnati College of Medicine and the Children’s
Hospital Research Foundation, Cincinnati, OH.
curred on examination of the cranial nerves except for
eyelid myotonia. She had marked lid lag. There was also
percussion myotonia and myoedema of both upper extremities but no grip myotonia. There was no atrophy or
fasciculation. The thyroid was of normal size and without
Results of various laboratory tests were within normal
limits, as were serum and urinary amino acid chromatograms. An electrocardiogram showed nonspecific ST and
T-wave abnormalties. An electromyogram demonstrated
insertional irritability and bizarre high-frequency potentials
as well as classic myotonic discharges. An oral challenge with
4 gm of potassium chloride was followed by a rise in serum
potassium to 6.5 mEq per liter but no alteration in her
muscle strength. Ischemic exercise of the forearm produced normal elevation of serum lactate and pyruvate concentrations.
Patient 2
The 10-year-old son of Patient 1 (Fig 1A) was the product
of a normal full-term pregnancy and delivery. His growth
and development were considered normal. He had no
complaints of cramps o r stiffness. His performance in
school was satisfactory. Physical examination showed no
abnormalities except that he also demonstrated lid lag and
percussion myotonia. Muscle strength and bulk were not
impaired. Laboratory tests and an electrocardiogram and
electromyogram were also normal.
Patient 3
The 8-year-old daughter of Patient 1 (Fig 1B) had a normal
prenatal and infantile history. However, during the two
years prior to investigation she had become somewhat
clumsy. H e r mother reported that the patient tripped freReceived Sept 10, 1979, and in revised form Oct 30. Accepted for
publication Nov 4, 1979.
Address reprint requests to Dr Bove, Department of Pathology,
Children’s Hospital Medical Center, Cincinnati, OH 45225.
0~64-5134/80/060550-07$01.25@ 1979 by Kevin E. Bove
sections were stained using hematoxylin and eosin, Luxol
fast blue, Sudan black, and the Gomori trichrome procedures. Unstained sections were examined in polarized and
fluorescent light. The residual frozen tissue sample was
stored at -70°C for six months and then subjected to qualitative energy-dispersive x-ray (EDX) analysis.
Fig I . Son (A)and daughter (B) of Patient 1. Both have a
normal physique with slight lambar lordosis.
quently. The child had no complaints of fatigability,
cramps, or stiffness. The only abnormal findings on physical examination were myotonia and myoedema in response
to percussion of the forearm and marked lid lag. Laboratory
data were within normal limits, and no abnormalities were
found by electromyogram.
Muscle biopsy specimens were obtained from the quadriceps muscle of the mother (Patient 1) and biceps muscles
of her children (Patients 2 and 3) using isometric clamps.
One portion was fixed in 3% glutaraldehyde buffered in
sodium cacodylate, postfixed in osmium tetroxide, and embedded in epoxy resin (Epon). Semithin sections were
stained with methylene blue-azure 11. Ultrathin sections
were stained with 1% uranyl acetate and 1% lead hydroxide and were examined with a Zeiss Em92AS electron
microscope. Another sample of muscle was flash frozen in
liquid nitrogen. Cryostat sections were prepared and
stained using the following methods: hematoxylin and
eosin, modified trichrome, periodic acid-Schiff, Sudan
black, Baker acid hematin, Luxol fast blue, Sudan IV, Nile
blue sulfate, von Kossa, alizarin red, succinic acid dehydrogenase, phophoryiase, and myosin adenosine triphosphatase (ATPase) at p H 9.4 and 4.6. A third sample was
fixed in 10% formalin and embedded in paraffin. These
Light Microscopic Observations
PATIENT 1. Muscle fibers were uniform in size and
shape, averaging 60 to 70 p in diameter. Inflammation, fibrosis, and sarcoplasmic degeneration were
absent. Central nuclear migration and short chains of
nuclei were rarely observed. In cryostat sections
stained with the modified trichrome method, aggregates of granules and rods staining dark blue-purple
were observed in the cross sectioned profiles of 25 to
50% of the muscle fibers (Fig 2A). Located just beneath the sarcolemma, these bacilliform inclusions
often surrounded sarcolemmal nuclei, were rarely
seen deep within muscle fibers, and were neither
birefringent nor autofluorescent.
Discrimination of two major fiber types by the
myosin ATPase reactions performed at pH 9.4 and
4.6 was excellent. Negative-staining subsarcolemmal
aggregates such as those described were detected in
both type I and type I1 fibers but were more common
in type I1 fibers.
The battery of histochemical staining methods
applied to the inclusions was mostly unsuccessful.
Positive reactions were obtained in frozen sections
with hematoxyiin alone (pale blue), modified trichrome method without hematoxylin (purple), Sudan
black (weak gray), and Baker acid hematin (strong
black). Negative reactions were obtained with the
periodic acid-Schiff, Nile blue sulfate, alizarin red,
von Kossa, and Luxol fast blue methods. We were
unable to demonstrate the bodies in paraffin sections
by any of these methods. They were dark blue in
semithin Epon sections stained with methylene blueazure I1 (Fig 2B) and were weakly osmiophilic in unstained semithin sections.
2. Biceps muscle samples were examined
using the same methods as in Patient 1, and the results were qualitatively similar. Fewer fibers (approximately 10%) contained granular subsarcolemma1 aggregates than in the mother’s muscle sample.
PATIENT 3. Paraffin, frozen, and semithin Epon
sections of biceps muscle were normal by light microscopy.
Electron Microscopic Observations
No ultrastructural alterations were found in the muscle specimen from Patient 3 . In the mother and son,
Bove et al: Cylindrical Spirals in a Familial Neuromuscular Disorder
Patients 1 and 2, the subsarcolemmal bodies had a
uniform cylindrical configuration, measuring up to 8
p long and as much as 1 p in diameter. In both
mother and son the cylinders had 10 to 16 lamellae.
A long central channel containing glycogen granules
communicated at one or both ends of each cylinder
with the cytosol. In cross section, mature cylinder
walls were composed of dense double-unit membranes coiled in a tight, continuous spiral around the
central lumen (Fig 3). The outermost and innermost
layers were usually composed of a single-unit membrane. Near the cylinder ends, multiple points of
continuity between the dense lamellae and outer
membrane were consistently present (Fig 4).
An exhaustive search for the structural origin of
the cylindrical bodies was conducted by studying ultrathin sections of skeletal muscle from 15 epoxy
resin blocks each in Patients 1 and 2. In Patient 1,
proximity of the cylinders to subsarcolemmal mitochondria or T-tubes was occasionally found, but
there were no convincing examples of continuity.
The search for formative stages in the development of cylinders was fruitful only in muscle from
Patient 2. In this sample, occasional clusters of cylin-
Annals of Neurology
Vol 7 No 6 June 1980
F i g 2. (A) Normal-sized muscle fibers in cross section show peripherally located nuclei and subsarcolemmal and perinurlear
aggregates of blue-purple granules (arrows). (Frozen section,
modified trichrome stain; x 700 before 5 % reduction.) ( B )
Cylindrical configuration of inclusions i s demonstrated in
longitudinally sectioned muscle. Short nuclear chains are associated. (Semithin Epon section, methylene blue-azure 11:
X1,500 before J% reduction.)
ders had fewer lamellae or were surrounded by
myriad small vesicles and tortuous tubules, many
with saccular invaginations resembling altered Ttubules. In such regions the outer lamellae of the
developing cylinders were in continuity with these
abnormal tubulovesicular structures (Figs 5, 6), the
single membranes of which adhered and partially
fused, forming a dense double lamina 200 A thick.
Initial adherence appeared to occur between the
outer or cytosol sides of adjacent tubular profiles (Fig
6). The resultant bilaminate membranes appeared to
enter a continuous spiral, o r in some profiles a discontinuous spiral, terminating after ly2 to 2 turns in
a vesicular core enclosing glycogen-rich cytosol (Fig
F i g 3. I n cross section each cylinder is composed of a continuous
spiral of dense double membrane enveloped by a thin, delicate
outer membrane and exhibiting a central channel containing
glycogen. (U ranyl acetatellead hydroxide; x 2 5,000 before
25 % reduction.)
Fig 4. I n longitudinal section the central channel of each cyl
inder communicates with the cytosol. Cylinder walls are
composed of parallel lamellae that fuse with the outer
membrane near the blunt ends (arrow). (Uranyl acetatellead
hydroxide; X27,000 before 10% reduction.)
Bove e t al: Cylindrical Spirals in a Familial Neuromuscular Disorder
5). The nature of the process of cylinder formation
and the morphometry of the cylinders were not
further elucidated. Although the associated abnormal
tubules are similar in dimension to T-tubes, no abnormalities were observed in deeper-lying T-tubules
or lateral sacs. Myofibrils and mitochondria were
generally well preserved, as were a small number of
fortuitously included myelinated nerves.
Three patients-a
mother and her two childrenexhibited percussion myotonia, and two of them had
cylinders. Neurological examination of the father was
normal. The mother, who had the greatest number of
cylinders within muscle cells, also complained of
painful cramps and stiffness. H e r children were
asymptomatic, though one of them was considered to
be clumsy. None of the three had muscle weakness
or wasting.
Myotonia is by definition painless [17]. Painful
cramps are most often associated with denervation or
with a metabolic myopathy such as McCardle disease
[ 101. Our patients had no clinical, electrophysiological o r histochemical evidence of denervation. A defect in glycogen metabolism was ruled out by the
normal response to ischemic exercise [ 131, normal
phosphorylase histochemical profile, and lack of
glycogen accumulation in the muscle.
Electrical myotonia was demonstrated only in the
mother. No one in the family had a typical phenotype
554 Annals of Neurology Vol 7
No 6 June 1980
F i g 5 . Postulated genesis of cylinders from congeries of
tubulowesicular structures (arrows) is based upon proximity
and continuity. The resultant spirals are continuous (") or
discontinuous (""I, depending on whether the cylinders are
cross sectioned i n the middle or near the end. (Uranyl acetatel
lead hydroxide; ~ 2 5 , 0 0 0before 25 % reduction.)
for myotonic dystrophy; strength of facial and other
muscles as well as intelligence were normal. Although minimal myopathic features were found in
biopsy specimens, the serum creatine phosphokinase
was normal and there were no ringbinden, fibrosis, or
regenerating fibers [7]. Other syndromes frequently
associated with myotonia include myotonia congenita
and hypothyroid myopathy [ 121. Muscle pain, a complaint of Patient 1, is not a feature of myotonia congenita, and none of our patients had hypertrophied
muscles or evidence of abnormal thyroid function.
The occurrence of episodic weakness and exacerbation of complaints by cold in Patient 1 are suggestive of hyperkalemic periodic paralysis [ 161.
Serum potassium levels rose after an oral challenge
with potassium chloride, but there was no loss of
strength. Although this patient complained of pain
and stiffness on exposure to cold, her strength did
not change when she was subjected to cold stress.
Moreover, the typical vacuolar myopathy of periodic
paralysis was not seen on biopsy.
The cylinders resembled nemaline rods in size and
F i g 6. Higher magnification of cylinders resolves the double
membranes of each lamella, single-unit membrane structure of
the inner and outer envelope, and continuity with adjacent
tubules. The intratubular and bilaminar vesicles (”) may result from invagination. (Uranyl acetatellead hydroxide;
~ 5 6 , 0 0 0before 10% reduction.)
configuration, but rods are more easily stained by a
variety of procedures, are red in sections stained with
modified trichrome, and occur predominantly in
type I fibers. In electron micrographs, cylinders are
easily distinguished from my elin figures-disorganized spheroidal whorls of cytomembrane that are
nonspecific byproducts of organellar degeneration
found in a variety of normal and diseased cells, including muscle cells. Myelin figures of lysosomal
origin are particularly numerous in toxic myopathies
[ 3 ] , while nonlysosomal origin is postulated for the
membranous whorls seen in inclusion body myositis
[4]. The figures may appear to be derived from Ttubules [2] or mitochondria, though this finding has
no proved specificity. In our patients, myelin figures
were a minor feature topographically unrelated to the
In 1974 Giambarelli e t a1 [S] described “concentric
laminated bodies” (CLB) in muscle from five patients
with unrelated neuromuscular diseases. They reviewed the literature and found multiple clinical entities in which such structures were occasionally
found in muscle cells. CLB are typically found with
difficulty, and Giambarelli and co-workers, who could
not demonstrate origin from mitochondria or sarcolemma, suggested a derivation of CLB from myofilaments.
The CLB is similar in size to the cylindrical spiral
and has a core of glycogen, but, unlike the cylinder, it
has not been seen on light microscopy, is fibrillar
rather than membranous, is described as being composed of concentric rather than spiraling layers, has
ill-defined boundaries, and is characterized by the
presence of periodic densities on each layer. The
uncertain origin of the CLB contrasts with the demonstrable derivation of cylinders from sarcoplasmic
origin that is supported by the
affinity of the cylinders for osmium and their strong
staining reaction with the Baker acid hematin
method, indicating a high phospholipid content.
Because the cylinders are composed of laminated
membrane arranged in a “jellyroll” or spiral around a
central lumen, we propose that a homology may exist
with the structure of the myelin sheath. The major
periodicity of myelin is 110 to 160 A and results
from a specific arrangement of lipids and proteins in
Bove et al: Cylindrical Spirals in a Familial Neuromuscular Disorder
juxtaposed neurolemma [ 101. The dark 30 A lines in
myelin represent the fused outer cell membrane and
the less dense band, the fused inner membrane surfaces. The cylinder seems to arise from juxtaposition
of membranes that are in continuity with vesicles derived from abnormally tortuous and invaginated subsarcolemmai T-tubes. We postuiate that the narrow
intralamellar space within the cylinders results from
incomplete fusion of the outer or cytosol surfaces of
the opposed membranes and that the clear, broad
interlamellar space is continuous with tubulovesicular lumina. Because we were unable to demonstrate
convincing continuity of the abnormal tubules or
cylinders with the extracellular space, and because
extracellular peroxidase fails t o penetrate the cylinders [51, origin from the sarcoplasmic reticulum is an
alternative possibility. Weak affinity of cylinders for
hematoxylin would be consistent with high calcium
content, but more specific stains for calcium (von
Kossa and alizarin red methods) and preliminary
EDX analysis indicate no calcium accumulation in the
Despite similarities in structure, important histochemical differences exist between true myelin and
the cylinder membranes. Myelin is strongly sudanophilic in frozen sections and has high affinity
for Sudan black in paraffin sections; neither quality
was found in cylinders. The strong blue-purple coloring of the cylinders treated with the modified trichrome method in frozen sections of peripheral
nerves is unlike the red staining of myelin sheaths of
motor nerves in the same sections. Finally, myelin
is birefringent in polarized light and weakly
autofluorescent in ultraviolet light; neither was true
of the cylinders. Osmiophilia and strong reactivity
with the Baker acid hematin method were the only
common histochemical reactions.
In this family, the apparent formation of cylindrical
spirals from superfluous subsarcolemmal membranes
suggests that a dominantly inherited structural defect
exists in these membranes. Cramps, stiffness, percussion myotonia, lid lag, posteffort muscle tightening,
and clumsiness are functional manifestations that may
appear o r progress with age but are not incapacitating. The cylinders appear to accumulate with time.
Cardiac muscle is not clinically involved. The clinical
spectrum in our patients resembles that in the two
previously reported patients with cylindrical spirals,
one of whom had progressive muscle cramps with
556 Annals of N e u r o l o g y Vol 7
No 6 June 1980
pain, and the other a progressive familial gait disorder [51.
We are indebted to James Anthony, MD, who referred Patient 1
to us for evaluation of a possible muscle disorder. We are also
indebted to Ann Geddes, PhD, who provided energy-dispersive
& fi.e-ze-$xhi.p&$e.. ay!:.z&rs az
Mia,=.;- \ 7 & q
Laboratories of the Proctor and Gamble Company.
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