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Arecessive form of central core disease transiently presenting as multi-minicore disease is associated with a homozygous mutation in the ryanodine receptor type 1 gene.

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A Recessive Form of Central Core Disease,
Transiently Presenting as Multi-Minicore
Disease, Is Associated with a Homozygous
Mutation in the Ryanodine Receptor
Type 1 Gene
Ana Ferreiro, MD,1 Nicole Monnier, PhD,2 Norma B. Romero, MD, PhD,1 Jean-Paul Leroy, MD,3
Carsten Bönnemann, MD,4,5 Charles-Antoine Haenggeli, MD,6 Volker Straub, MD,7 Wolfgang D. Voss, MD,8
Yves Nivoche, MD,9 Heinz Jungbluth, MD,10 Arnaud Lemainque, PhD,11 Thomas Voit, MD, PhD,7
Joël Lunardi, PhD,2 Michel Fardeau, MD,1 and Pascale Guicheney, PhD1
Multi-minicore disease is an autosomal recessive congenital myopathy characterized by the presence of multiple, shortlength core lesions (minicores) in both muscle fiber types. These lesions being nonspecific and the clinical phenotype
being heterogeneous, multi-minicore disease boundaries remain unclear. To identify its genetic basis, we performed a
genome-wide screening in a consanguineous Algerian family in which three children presented in infancy with moderate
weakness predominant in axial muscles, pelvic girdle and hands, joint hyperlaxity (hand involvement phenotype), and
multiple minicores. We mapped the disease to chromosome 19q13 in this family and, subsequently, in three additional
families showing a similar phenotype, with a maximum LOD score of 5.19 for D19S570. This locus was excluded in 16
other multi-minicore disease families with predominantly axial weakness, scoliosis, and respiratory insufficiency (“classical” phenotype). In the Algerian family, we identified a novel homozygous missense mutation (P3527S) in the ryanodine receptor type 1 gene, a positional candidate gene responsible for the autosomal dominant congenital myopathy
central core disease. New muscle biopsies from the three patients at adulthood demonstrated typical central core disease
with rods; no cores were found in the healthy parents. This subgroup of families linked to 19q13 represents the first
variant of central core disease with genetically proven recessive inheritance and transient presentation as multi-minicore
disease.
Ann Neurol 2002;51:750 –759
Congenital myopathies are early-onset hereditary muscle disorders defined by particular architectural changes
of the muscle fibers, in the absence of necrotic or regenerative phenomena. Among these distinctive
changes, core lesions, consisting of localized areas of
mitochondria depletion and sarcomere disorganization,
characterize two different entities: central core disease
(CCD)1 and multi-minicore disease (MmD).2 Both
conditions typically present with neonatal hypotonia,
delayed motor development, and generalized muscle
weakness and amyotrophy, nonprogressive or slowly
progressive. However, they display significant clinical,
morphological, and inheritance pattern differences; as a
result, the nosological boundaries between both congenital myopathies generally are considered well defined.
The diagnosis of MmD is based on the presence in
most muscle fibers of multiple minicores, which are
poorly circumscribed, segmentary lesions spreading
only a few sarcomeres along the fiber longitudinal axis
From the 1INSERM U523, Institut de Myologie, Groupe Hospitalier Pitié-Salpêtrière, Paris; 2Laboratoire de Biochimie de l’ADN,
Hôpital La Tronche, Grenoble; 3Service d’Anatomie Pathologique,
CHU Brest, Brest, France; 4Children’s Hospital of Philadelphia,
Philadelphia, PA; 5Kinderklinik and Poliklinik, Georg August Universitat Göttingen, Göttingen, Germany; 6Département de Neuropédiatrie, Hôpital des Enfants, Geneva, Switzerland; 7Department
of Pediatrics, University of Essen, Essen; 8Kinderklinik auf der Bult,
Hannover, Germany; 9Département d’Anesthésie, Hôpital Robert
Debré, Paris, France; 10Department of Paediatrics and Neonatal
Medicine, Hammersmith Hospital, Imperial College School of
Medicine, University of London, London, United Kingdom; and
11
750
© 2002 Wiley-Liss, Inc.
Production Génotypage, Centre National de Génotypage, Evry,
France.
Received Dec 26, 2001, and in revised form Feb 21, 2002. Accepted for publication Feb 28, 2002.
Published online May 21, 2002, in Wiley InterScience
(www.interscience.wiley.com). DOI: 10.1002/ana.10231
Address correspondence to Dr Ferreiro, INSERM U523, Institut de
Myologie, Groupe Hospitalier Pitie-Salpetriere, 47 bd. de l’Hopital,
75651 Paris, France. E-mail: a.ferreiro@myologie.chups.jussieu.fr
and affecting both type 1 and type 2 fibers. From a
clinical point of view, MmD is a heterogeneous condition in which four different subgroups have been distinguished.3 The phenotype of the most prevailing,
classical MmD form is characterized by the axial predominance of muscle weakness; severe scoliosis and respiratory insufficiency are common. The moderate
form with hand involvement consists of generalized
muscle weakness predominating in the pelvic girdle
and including hand weakness, amyotrophy, and hyperlaxity; scoliosis and respiratory involvement are mild or
absent. The two other forms of the disease are characterized by a clinical picture similar to the classical one,
but including ophthalmoplegia,4 and by antenatal onset with arthrogryposis,5 respectively. It is now agreed
that MmD is an autosomal recessive condition.3 Because the morphological lesions defining MmD are
nonspecific,5 its boundaries remain undefined, and its
genetic basis remains unknown.
In contrast, CCD (MIM 117000) is characterized by
the presence of core lesions that have sharply defined
borders, involve exclusively type 1 fibers, and are considered to extend throughout the entire fiber length;
they are more often central and unique but can be predominantly eccentric6 or multiple within one fiber.
The clinical picture is marked by moderate muscle
weakness predominating in proximal lower limbs; respiratory insufficiency is rare. CCD is defined as an
autosomal dominant condition caused in at least half
of the families by heterozygous mutations of the ryanodine receptor type 1 gene (RYR1),6 –10 a large and
complex gene11 located on chromosome 19q13.1.12,13
To identify the genetic basis of MmD, we analyzed a
series of autosomal recessive MmD families.5 We describe here a homogeneous subgroup of four of these
families in which genetic analysis through a positional
candidate gene approach and subsequent phenotype reevaluation provided evidence that this condition actually represents a pathological variant of CCD with
autosomal recessive inheritance and transitory presentation as MmD.
Patients and Methods
Patients: Diagnostic Criteria
The diagnosis of MmD was established after analysis by light
and electron microscopy of a muscle biopsy from at least one
patient per family. Our inclusion criteria are summarized in
Table 1. Twenty informative families (eight consanguineous,
12 with two affected siblings each), including 34 patients
with an initial diagnosis of classical (17) or hand involvement (3) MmD, were analyzed. All parents were clinically
normal. No case presenting ophthalmoplegia, congenital arthrogryposis, or atypical clinical features was included in this
study.
Morphological Methods
Open muscle biopsies were performed after informed consent was obtained. Specimens for light microscopy analysis
and for ultrastructural studies were immediately frozen or
fixed and processed as previously described.5
In Vitro Contracture Testing
In vitro caffeine-halothane contracture testing was performed
on one patient (Patient 3) with a quadriceps muscle biopsy,
according to the protocol of the European Malignant Hyperthermia Group.14
Genomic DNA and RNA Isolation
Genomic DNA was extracted from blood samples after informed consent was obtained, with standard procedures. Total RNA was extracted from frozen muscle specimens with a
guanidium thiocyanate-phenol-chloroform method.15
Genotyping
Whole-genome screening was performed with 280 highly
polymorphic (CA)n microsatellite markers from the Généthon human linkage map,16 distributed at 10 to 15cM intervals.
Polymerase chain reactions were conducted with 30ng of
genomic DNA using fluorescent primers and TaqGold polymerase (0.5 units; Applied Biosystems, Foster City, CA) as
described.17 Fractionation and data collection were performed on 4% acrylamide gels with an ABI 377 DNA sequencer. All genotypes were scored with ABI GeneScan
Table 1. Inclusion and Exclusion Criteria
Inclusion Criteria
Early-onset global weakness
Autosomal recessive transmission
Serum creatin kinase level ⱕ3⫻ normal
Type 1 fiber predominance or uniformity
Multiple minicores
As the main morphological abnormality
In a significant proportion of type 1 and, when present, type 2 fibers
Both in light and electron microscopy
Short in longitudinal sections
Exclusion Criteria
Endocrinopathy or chronic intake of drugs
EMG evidence of peripheral nerve
abnormalities
Morphological evidence of
Necrosis or regeneration
Significant endomysial fibrosis
Glycogen or lipid content abnormalities
Inflammatory infiltrates
Grouping, atrophic angulated fibers
Diffuse desmin accumulation
EMG ⫽ electromyogram.
Ferreiro et al: Central Core Disease
751
Table 2. Pairwise Cumulated LOD Scores for Linkage between the Disease Phenotype and Markers on Chromosome
19q13 in the Four Families
LOD Scores at ␪ ⫽
Marker
D19S225
D19S208
D19S224
D19S220
D19S570
D19S897
D19S421
D19S422
D19S420
D19S217
D19S412
D19S553
0.0
0.01
0.05
0.1
0.2
0.3
0.4
⫺4.55
3.39
2.28
0.09
5.19
3.42
2.54
2.41
1.22
0.63
0.41
⫺10.43
0.15
3.30
2.80
1.08
5.04
3.32
2.46
3.06
1.76
1.78
1.96
⫺2.03
1.11
2.97
2.96
1.45
4.45
2.94
2.15
3.20
2.02
2.29
2.34
⫺0.29
1.23
2.54
2.69
1.39
3.72
2.47
1.77
2.87
1.87
2.20
2.20
0.23
0.95
1.70
1.90
1.01
2.33
1.59
1.08
1.96
1.32
1.59
1.57
0.42
0.54
0.92
1.07
0.59
1.17
0.84
0.55
1.06
0.74
0.87
0.86
0.31
0.19
0.30
0.37
0.23
0.35
0.29
0.20
0.34
0.25
0.26
0.26
0.13
LOD ⫽ logarithm of odds.
Analysis 2.1.1 and Genotyper 2.0 software (Applied Biosystems; Fig 1).
allele frequencies with the MLINK program (accessed from
http://www.hgmp.mrc.ac.uk).
Linkage Analysis
Ryanodine Receptor Type 1 Mutation Screening
Linkage analysis was performed with the LINKAGE 5.2
package (accessed from http://www.hgmp.mrc.ac.uk) under
the assumptions of autosomal recessive inheritance and an
equal recombination frequency for men and women. MmD
being a rare, early-onset disease, a disease-gene frequency of
0.0001, one liability class, and a penetrance of 0.95 were
assumed. Two-point LOD scores were calculated for equal
Because of the large size of RYR1, mutation screening was
performed when possible on complementary DNA (cDNA)
from a skeletal muscle biopsy. cDNA synthesis was conducted with specific primer mixes, and the resulting first
strands were amplified in overlapping fragments, ranging
from 400 to 700bp, with primers designed from the published RYR1 cDNA sequence. The amplified fragments,
Table 3. Clinical Features
Family I
Algerian
⫹ (Second degree)
(Patient no.)
Origin
Consanguinity
1
First signs
Facial weakness
Muscle weakness
distribution
Hand involvement
Joint contractures
Joint hyperlaxity
Scoliosis
Age at muscle
biopsy (yr)
Age at last examination (yr)
Evolution
Family II
Turkish
⫹ (Fourth degree)
(Patient no.)
2
3
Family III
German
⫺
(Patient no.)
Family IV
Turkish
⫺
(Patient no.)
4
5
6
7
8
NH
club feet
DMM
⫹/⫺
General, ⬎
distal
Poor motor
performance
⫹/⫺
Proximal,
axial and
hands
⫹
Poor motor
performance
⫺
Proximal,
axial and
hands
⫹⫹
NH
club feet
DMM
⫹⫹
Proximal,
axial and
hands
⫹⫹
NH
NH
NH
DMM
⫹
Proximal,
axial and
hands
⫹⫹
⫹⫹
Proximal,
axial and
hands
⫹⫹
⫹
Proximal,
axial and hands
Poor motor
performance
⫺
General, ⬎
distal
⫹⫹
⫹
⫹⫹⫹
Mild, only
in heels
Mild, only
in heels
Mild, only
in heels
⫺
⫺
⫺
⫺
⫹
⫹/⫺
2 and 18
⫹⫹⫹
⫺
4 and 21
⫹⫹
⫺
21
⫹⫹⫹
⫺
3 and 5
⫹
⫺
Not done
Hips, distal
arthrogryposis
⫹⫹
⫺
5
⫹⫹⫹
⫺
Not done
⫹⫹⫹
⫺
13
26
24
22
8
14
11
16
13
Stable
Stable
Stable
Stable
Stable
Slowly progressive?
Stable
Slowly progressive
Although the clinical pattern was homogeneous, intra- and interfamilial variation in severity of the disease was observed. No case presented
respiratory failure, despite a reduced vital capacity in three patients (1, 2, and 4). Cardiac function was always normal.
DMM ⫽ delayed motor milestones; NH ⫽ neonatal hypotonia.
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spanning the whole RYR1 cDNA, subsequently were sequenced with an ABI 3700 apparatus.
When muscle samples were unavailable, RYR1 mutation
screening was performed on genomic DNA and focused on
the 3⬘ end of the gene, containing most of the mutations
involved to date in CCD.6 Exons 94 to 106 were analyzed
by single-strand conformation analysis as previously described,6 except exon 102, which was analyzed by direct sequencing.
HhaI restriction analysis of exon 71 was used to detect the
P3527S mutation (Fig 2).
Results
Identification of a Potential Recessive Linkage to
Chromosome 19q13
We initially analyzed an informative consanguineous
Algerian family (Family I) in which three affected children presented with the moderate form with hand involvement of MmD. Genome-wide screening with 280
microsatellite markers showed that three markers
(D2S380, D19S412, and D19S907) were homozygous
in the three affected sisters and heterozygous in the two
healthy siblings. An analysis of 35 flanking microsatellite markers led to the exclusion of putative linkage to
the D2S380 region and confirmed that both parents
share a 27.5cM18 common haplotype located in
19q13.1–13.4 and inherited by all the affected siblings
in a homozygous way (Fig 1a).
We subsequently analyzed the markers in this region
in 19 other classical or hand involvement MmD families. In three of them, one consanguineous (Family II)
and two multiplex (Families III and IV), the disease
was potentially linked to chromosome 19q13.1 (see Fig
1b). Markers D19S570 and D19S897 gave the highest
cumulative LOD scores, 5.19 and 3.42, respectively at
␪ ⫽ 0.00 (see Table 2). These markers are located in a
3cM linked interval shared by the four families and
containing a candidate gene, RYR1, which encodes a
calcium release channel of the skeletal muscle sarcoplasmic reticulum.
The patients of the four linked families showed a
similar, relatively mild phenotype. In the remaining 16
MmD families, linkage to chromosome 19q13 was excluded (data not shown). They presented with a classical, globally more severe phenotype, marked by the
axial predominance of muscle weakness and frequently
including severe scoliosis and early respiratory insufficiency.
Identification of the Ryanodine Receptor
Type 1 Mutation
In Family I, RYR1 cDNA was entirely analyzed for Patient 3 by direct sequencing. We found a single homozygous amino acid change, P3527S, resulting from
a C to T transition at position 10579. This change
abolished an Hha restriction site in exon 71. The seg-
regation study showed that the consanguineous parents
were heterozygous carriers of the P3527S change, the
three affected sisters were homozygous, and the two
unaffected siblings were heterozygous (II4) and noncarrier (II5), respectively, for the mutation (see Fig 2).
The substitution was absent from 170 unrelated control chromosomes, including 70 chromosomes from a
North African population. Proline 3527 is strictly conserved in RyR1, RyR2, and RyR3 isoforms in human
and other vertebrate species (see Fig 2) and is localized
upstream of the calcium channel transmembrane domain.
In Families II, III, and IV, screening of exons 94 to
106, coding for the C-terminal domain of RyR1, was
completed on genomic DNA and did not show any
mutation. The presence of the P3527S mutation in
these three families was excluded by restriction analysis;
subsequent sequencing of exon 71 failed to disclose any
abnormality. Further analysis using cDNA from recently obtained muscle samples is in progress.
Clinical Features of Moderate Multi-Minicore
Disease with Hand Involvement
The eight patients from the four linked families shared
clinical features (see Table 3) characteristic of the
MmD phenotype that we called the moderate form
with hand involvement because of particular, although
nonexclusive, hand clinical abnormalities. This includes
Patient 4 (Family II), who initially had been considered to have a mild, classical form of MmD.5 A first
description of patients from Families I and II has been
reported previously (Cases 36 –38 and Case 16, respectively, in the previous report).5
All patients showed early, generalized, moderate
muscle weakness involving mainly neck and trunk flexors, limb girdles, and hands. Muscle power scored in
general between 4 and 5 according to the Medical Research Council scale. Pelvic girdle tended to be more
severely affected than shoulder girdle. Distal lower
limbs were normal or almost normal. In contrast, in
distal upper limbs, hand hypotonia and hyperlaxity
(Fig 3) were accompanied by uniform amyotrophy of
intrinsic hand muscles and moderate weakness (Medical Research Council scale score 4). Hyperlaxity was
also present, to a lesser degree, in all the remaining
limb joints; Patients 2 and 3 suffered repeated patellar
and knee dislocations. A variable degree of facial muscle weakness was present in most cases (see Fig 3); eye
movements were normal. Apart from mild or moderate
heel contractures observed in four patients, no other
joint retractions were present. Only Patient 1 showed
minimal, stable scoliosis. No patient presented signs of
respiratory failure, including the three patients (1, 2,
and 4) with moderate reduction of vital capacity (52–
70%).
Although the clinical pattern was homogenous,
Ferreiro et al: Central Core Disease
753
Fig 1. Pedigrees and haplotype analysis of
the four linked families at 19q13. Affected individuals are denoted by filled
symbols. For compactness, only the most
relevant markers are shown. Haplotypes/
markers that are homozygous in the affected individuals from consanguineous
parents or segregate with the disease phenotype in multiplex families are boxed.
Markers used in the first genome screening
are in boldface. All the affected individuals have the two at-risk haplotypes. Noticeably, five of the six unaffected siblings
from the four families have inherited one
at-risk haplotype.
marked intrafamilial and interfamilial variation in the
severity of the disease was observed (see Table 3). The
most severe cases (Patients 1 and 6) presented with
congenital club feet, neonatal hypotonia and abnormal
motor development, and have never been able to run.
Patient 1, now aged 25 years, can hardly walk with
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help and needs assistance for all everyday life activities.
She presents a nasal voice and subjective dysphagia; her
vital capacity is reduced to 52% of the expected values.
In contrast, Patients 5, 7, and 8 presented in childhood
with poor motor performance, have normal vital capacity, and lead active lives with only moderate difficulties
in climbing stairs or rising from a chair. Patients 2, 3,
and 4 show intermediate severity.
Muscle weakness remained stable in most cases.
Family I had reported subjective progression of the disease, but we could not substantiate any objective modification after 3 years of follow-up.
Serum creatine kinase levels were normal in all patients, as well as in the parents and unaffected siblings
from Family I. Radiological examinations of lower
limb muscles in four patients, with computed tomography scans (Patients 1–3) or magnetic resonance imaging (Patient 4), showed a constant pattern that is illustrated in Figure 3.
Heart function tests (electrocardiogram and echocardiography), performed in at least one patient per family, showed no abnormalities. Mental function was
normal. No episode of malignant hyperthermia or family history of anesthetic deaths was retrieved in any of
the four families.
Fig 2. Screening of the P3527S mutation in Family I by restriction analysis of exon 71 (a) and evolutionary conservation
of the proline residue (b). The 398bp amplified products were
digested with the enzyme HhaI and analyzed by electrophoresis
on an 8% polyacrylamide gel. Because of the presence of two
restriction sites, a normal allele causes three bands of 177,
136, and 85bp. The presence of the mutation abolishes one of
the two sites, resulting in two bands of 177 and 221bp. The
177bp constant fragment was used as an internal control of
digestion. MWM ⫽ molecular weight marker.
Morphological Findings and Reevaluation
In Family I, the diagnosis of MmD had been established on the basis of two muscle biopsies performed in
the two eldest sisters at 2 and 4 years of age, respectively (Fig 4). The biopsy from Patient 1 showed only
type 1 fiber uniformity with rare and small zones of
Z-disk streaming on electron microscopy examination.
The first biopsy from Case 2 disclosed both type 1 uniformity and multiple minute zones lacking oxidative
Fig 3. Clinical (A) and radiological (B) features. Patients 1 (a), 2 (b), 3 (c), and 8 (d) at 23, 21, 19, and 13 years of age, respectively. (A) Note the neck and shoulder amyotrophy, absence of retractions, flat feet (Patient 3), inward rotated patellae, and
facial involvement of variable severity. Hypotonia, amyotrophy, and hyperlaxity were present in hands. (B) Axial lower limb computed tomography scan from Patient 1. Gluteus maximus and quadriceps were preferentially involved; severe fatty replacement was
evident in the vastus lateralis, vastus intermedius, and vastus medialis and contrasted with a relatively spared rectus femoris.
Ferreiro et al: Central Core Disease
755
activity, corresponding at the ultrastructural level to
small regions of sarcomere disarray and absence of mitochondria.
In view of the genetic results, we performed additional muscle biopsies in the three patients at adult age
(Fig 5). Type 1 uniformity was corroborated in all
cases. The morphological pattern showed a drastic
modification in Patient 1, with extensive fiber loss and
adipous replacement noticeably unaccompanied by endomysial fibrosis; sharply defined, long core lesions were
present in the few spared fibers. The new sample from
Patient 2 showed less oxidative-negative lesions than the
first biopsy, but they were larger and more defined; only
in one fiber were some rods found. The muscle sample
from Patient 3 evidenced a morphological pattern typical of CCD, with sharply defined, subsarcolemmal core
lesions spanning most of the fiber length and abundant
rods, predominantly within the cores.
In addition, we performed a deltoid muscle biopsy
on their asymptomatic 50- and 52-year-old parents.
The mother’s muscle was normal on light microscopy;
electron microscopy disclosed, after extensive sampling,
only one isolated atrophic fiber with rods. The biopsy
from the father showed only abnormal variability in
fiber diameters and uneven oxidative staining without
core lesions, either with light or electron microscopy
analysis. Histometric analysis excluded type 1 fiber predominance from both samples.
An in vitro malignant hyperthermia contracture test
performed for Patient 3 demonstrated the absence of
malignant hyperthermia susceptibility.
Fig 4. Initial diagnostic muscle biopsies in Family 1: serial
transverse cryostat sections. (a, b) Patient 1 at 2 years of age,
left deltoid. Only mild fiber size variability and type 1 fiber
uniformity were observed. Myosin adenosine triphosphatase
(ATPase) at pH 9.4 ⫻40 (a) and NADH-tetrazolium reductase (NADH-TR) ⫻80 (b). (c, d) Patient 2 at 4 years of age,
right quadriceps. Multiple minute lesions lacking oxidative
activity were present in most fibers. NADH-TR ⫻40 (c), and
⫻80 (d).
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Biopsies from Patients 4, 6, and 8 performed at the
ages of 3, 5, and 13 years, respectively, showed type 1
fiber predominance and relative hypotrophy, as well as
multiple short cores involving both fiber types. A second muscle biopsy taken from Patient 4 at 5 years of
age showed more marked particular features that are
illustrated in Figure 6.
Discussion
Substantial efforts have been made in recent years to
achieve a better characterization of the two congenital
myopathies with cores, dominant CCD, and recessive
MmD.3 The assignment of the CCD locus to chromosome 19q1312 and the subsequent identification of
RYR1 heterozygous mutations in CCD patients6 –10
represented significant progress. Although the genetic
basis of MmD was unknown, the differences in phenotype and transmission pattern between MmD and
CCD generally have led them to be considered separate
entities. Some early reports emphasized the coexistence
of both typical cores and minicore lesions19,20 and suggested a variation of the morphological pattern of core
myopathies with age.19,21,22 However, no RYR1 abnormalities had been described in these mixed or in typical
MmD cases. Therefore, the potential nosological relationship between CCD and MmD remained undefined.
To identify the genetic basis of MmD, we analyzed a
series of recessive MmD families.5 Linkage studies, mutation screening, and subsequent phenotype reevaluation provided evidence that a homogeneous subgroup
of four of these families is affected by a pathological
variant of CCD with autosomal recessive inheritance
and transitory presentation as MmD.
The common clinical phenotype was marked by a
relatively mild muscle weakness, distinct joint hyperlaxity, hand involvement, and absence of significant
scoliosis or respiratory insufficiency. The predominant involvement of proximal lower limbs, the presence of feet deformities in two of eight patients, and
the relatively spared respiratory function are consistent with the classically described CCD features. In
contrast, joint hyperlaxity in CCD patients has been
seldom reported. However, in a series of 15 CCD
cases evaluated on orthopedic grounds,23 hyperlaxity
was a constant finding, which suggests it might have
been underestimated previously. The same could be
true concerning hand involvement, which remains the
most atypical finding in our families in comparison
with classic, dominant CCD cases.
From a morphological point of view, the most remarkable finding was the drastic modification of the
myopathological pattern documented in Patients 1 and
2 after 18 and 20 years of evolution, respectively.
While the deltoid and quadriceps biopsies performed
in early childhood showed only type 1 fiber uniformity
(Patient 1) or lesions consistent with MmD (Patient
Fig 5. Adult-age muscle biopsies in Family 1: serial transverse cryostat sections. (a–c) Patient 1 at 18 years of age; right deltoid.
There was severe fiber loss with adipous tissue infiltration, but no fibrosis was observed (a). Well-defined core lesions, often unique,
sometimes bordered by an oxidative-positive rim (a), were present in most fibers and occasionally coexisted with minicores (c).
NADH-tetrazolium reductase (NADH-TR) ⫻80 (a), succinate dehydrogenase (SDH) ⫻80 (b), and ⫻160 (c). (d–f) Patient 2 at
21 years of age, left deltoid. Type 1 fiber uniformity was accompanied by oxidative-negative lesions (asterisk) less frequent but
larger and more defined than in her first biopsy. Myosin ATPase at pH 9.4, ⫻40 (d), NADH-TR ⫻80 (e), and SDH ⫻80 (f).
(g–i) Patient 3 at 21 years of age, right quadriceps. Most fibers showed subsarcolemmal (eccentric) clear-cut cores with perilesional
rims (g, h). A significant proportion of cores contained rods (i), which were more rarely found isolated. The region framed in (h) is
enlarged in (i). Cytochrome C oxidase (COX) ⫻80 (g), NADH-TR ⫻80 (h), and Gomori’s modified trichrome ⫻160 (i).
2), those taken at adulthood disclosed more severe abnormalities highly characteristic of CCD. Noticeably,
the second biopsy from Patient 1, taken from the same
muscle territory as the first one (right and left deltoid,
respectively), showed not only a distinct core myopathy
but also severe fiber loss. This modification contrasts
with the absence of clinical progression of the disease
and supports the hypothesis that core lesions may not
represent a primary developmental abnormality but
could be secondary to a maintained abnormal contraction caused by any excitation-contraction coupling de-
fect. The finding of T-tubule and lateral vesicle arrays
within the cores in Patient 4 could also be explained by
this mechanism because analogous triadic junction abnormalities have been shown recently to be induced by eccentric exercise in healthy animals.24 No similar lesions
were, however, ever found in the classical MmD cases.5
Therefore, autosomal recessive CCD can, at an
early stage, transitorily present with minicore lesions.
This could explain the absence of typical central cores
in the muscle biopsies from the three remaining
linked families, which were taken during childhood.
Ferreiro et al: Central Core Disease
757
Fig 6. Morphological findings in Patient 4: second muscle
biopsy. Transverse cryostat sections, hematoxylin-eosin (H and
E) ⫻40 (a), NADH-tetrazolium reductase (NADH-TR) ⫻80
(b), electron micrograph longitudinal section (c). The predominant type 1 fibers were either hypotrophic or hypertrophic,
whereas all the type 2 fibers had large diameters; centrally
located nuclei were observed in greater than 50% of the fibers
(a). Short and poorly circumscribed core lesions were visible on
H and E–stained sections as basophilic areas (a); some of
them were central (b, asterisk) and coexisted with well-defined
cores (b, arrow). T-tubule and lateral vesicle arrays, forming
pentads or heptads, were found within the ultrastructural lesions (c, asterisks).
A second muscle biopsy at the adult age should,
therefore, be considered in any unclear case of core
myopathy and, by extension, could be helpful in ascertaining a diagnosis in any congenital myopathy.
Nevertheless, the exclusion of linkage to 19q13 in 16
of the 20 MmD families analyzed proves that a genetic heterogeneity is at the origin of MmD, which
should not be automatically considered a recessive or
early presentation of CCD.
The notion of autosomal recessive inheritance is difficult to assert in CCD; wide intrafamilial variation in
penetrance and severity has been repeatedly described,
and RYR1 neomutations are common. That two of the
four linked families in our study were consanguineous,
that all parents were asymptomatic, and that five of the
six unaffected siblings have inherited one at-risk haplotype support this mode of transmission that has been
proven for Family I. The minor abnormalities identified in the parents’ biopsies, none of which fulfilled the
diagnostic criteria of CCD, may represent the minimal
758
Annals of Neurology
Vol 51
No 6
June 2002
phenotypic expression of a carrier status of a recessive
RYR1 mutation.
The fact that the newly identified P3527S mutation
fails to produce a CCD phenotype in the heterozygous
state might be explained by its position in exon 71, as
there appears to be a correlation between the localization of RYR1 defects and their functional consequences. Heterozygous RYR1 mutations cause two related but different dominant conditions: CCD and
malignant hyperthermia, a pharmacogenetic disorder of
skeletal muscle Ca2⫹ regulation triggered in genetically
predisposed individuals by exposure to common anesthetic agents.25 All but one of the 23 RYR1 mutations
inducing a malignant hyperthermia phenotype reported
to date have been localized in the N-terminal coding
region,26 –28 thereby affecting the cytoplasmic domain
of the protein that possibly interacts with the dihydropyridine receptor.11 In contrast, most of the heterozygous mutations identified in clinically and morphologically typical CCD cases cluster in the last 15 exons,
encoding the C-terminal transmembrane domains of
RyR1 that contribute to the formation of the Ca2⫹
conducting pore.6 Because an analysis of the whole
gene usually is not performed, the frequency and functional consequences of mutations in other domains of
RYR1 are yet unknown. However, it can be hypothesized that the P3527S mutation localized in an intermediate exon of RYR1 does not affect either the channel function or the interaction with the
dihydropyridine receptor. Therefore, it can have minor
functional effects unless it affects both alleles and can
be unrelated to malignant hyperthermia.
Besides the fact that the P3527S RYR1 mutation was
not found in 170 control chromosomes, its causative
effect is supported by the nature of the amino acid
modification, the change of an evolutionary conserved
proline residue into a serine. In addition, its localization in a cytoplasmic domain upstream of the calcium
channel is consistent with a possible defect of the channel regulation. In silico modeling indicated that the
new serine may introduce a potential casein kinase
phosphorylation site, although there is no proof of the
in vivo activity of this enzyme in skeletal muscle. Several putative calmodulin-binding sites have been assigned to the intermediate domain of RyR1 in the
3010 to 322529 and 3553 to 366230 regions, and one
may speculate that the loss of a proline residue could
introduce a conformational change in the region interfering with phosphorylation of the protein. Finally, sequencing of the whole RYR1 cDNA excluded other abnormalities in this gene. Functional analysis of P3527S
effects on Ca2⫹ currents and their regulation will help
us to fully understand the functional consequences of
this new mutation.
This work demonstrates that classic morphological
lesions may undergo age-related modifications and
therefore represent necessary but undependable diagnostic criteria in the complex overlap of phenotypes
between MmD and CCD. It also represents the first
description of a genetic defect underlying the MmD
phenotype and establishes its genetic heterogeneity. We
hope that it contributes to clarifying the complex nosology of the congenital myopathies with cores.
Note Added in Proof
Corroborating results from a recent independent study
are currently in press: Jungbluth H, Müller CR,
Halliger-Keller B, et al. Autosomal-recessive inheritance
of RYR1 mutations in a congenital myopathy with
cores. Neurology (in press).
This work was supported by funds from the INSERM, the Association Française contre les Myopathies, and the Fondation ElecricitéSanté. H.J. is a Muscular Dystrophy Campaign Research Fellow
(RA1/587/1).
We thank all the families that participated in this study. We are
especially grateful to Dr A. Engel (Mayo Clinic, Rochester, MN)
for continuous encouragement. We thank Dr F. Muntoni (London), Dr B. Eymard (Paris), and Dr F. Hanefeld (Goettingen)
for constructive discussions and Dr R. Egensperger and Dr G.
Schreiber (Goettingen) for help with the pathological and clinical
analyses, respectively. We thank Mrs Pavek (Centre National de
Genotypage, Evry), Mrs Collin, and Mrs Rouche (INSERM
U523, Paris) for technical assistance.
Electronic Database Information: Online Mendelian Inheritance in
Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim
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Ferreiro et al: Central Core Disease
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