Molecular mechanisms and phenotypic variation in
RYR1-related congenital myopathies
Haiyan Zhou,1,?Heinz Jungbluth,1,2,?Caroline A. Sewry,1,3Lucy Feng,1Enrico Bertini,4Kate Bushby,5
Volker Straub,5Helen Roper,6Michael R. Rose,7Martin Brockington,1Maria Kinali,1Adnan Manzur,1
Stephanie Robb,1Richard Appleton,8Sonia Messina,4,9Adele D’Amico,4Ros Quinlivan,3Michael Swash,10
Clemens R. Mu « ller,1 1Susan Brown,1SusanTreves12and Francesco Muntoni1
1Dubowitz Neuromuscular Centre, Imperial College, Hammersmith Hospital, London W12 0NN,UK,2Department of
Paediatric Neurology, Evelina Children’s Hospital, St Thomas’ Hospital, London SE17EH,UK,3Centre for Inherited
Neuromuscular Disorders, Robert Jones & Agnes Hunt Orthopaedic Hospital,Oswestry,UK,4Unit of Molecular
Medicine,Ospedale Bambino Gesu ' , Rome, Italy,5Institute for Human Genetics, International Centre for Life,
University of Newcastle uponTyne, Newcastle uponTyne,UK,6Department of Paediatrics, Birmingham Heartlands
Hospital,UK,7Department of Neurology, King’s College Hospital, London,UK,8Alder Hey Children’s Hospital,
Liverpool,UK,9Department of Neurosciences, Psychiatry and Anaesthesiology,University of Messina, Messina, Italy,
10Department of Neurology,The Royal London Hospital, London,UK,1 1Institute fu « r Humangenetik,Universita « t
Wu « rzburg, Biozentrum am Hubland,Germany and12Departments of Anaesthesia and Research, Basel University
Hospital, 4031Basel, Switzerland
*These authors contributed equally to this work.
Correspondence to: Professor Francesco Muntoni, Dubowitz Neuromuscular Centre, Imperial College, Hammersmith
Hospital, Du Cane Road, London W12 0NN.
Dominant mutations in the skeletal muscle ryanodine receptor (RYR1) gene are well-recognized causes of both
malignant hyperthermia susceptibility (MHS) and central core disease (CCD). More recently, recessive RYR1
mutations have been described in few congenital myopathy patients with variable pathology, including multi-
minicores. Although a clinical overlap between patients with dominant and recessive RYR1mutations exists, in
most cases with recessive mutations the pattern of muscle weakness is remarkably different fromthat observed
in dominant CCD.
In order to characterize the spectrum of congenital myopathies associated with RYR1mutations, we have inves-
tigated a cohort of 44 patients from 28 families with clinical and/or histopathological features suggestive of RYR1
involvement. We have identified 25 RYR1 mutations, 9 of them novel, including 12 dominant and 13 recessive
mutations.With only one exception, dominant mutations were associated with a CCD phenotype, prominent
cores and predominantly occurred in the RYR1 C-terminal exons 101 and 102. In contrast, the 13 recessive RYR1
mutations were distributed evenly along the entire RYR1gene and were associated with a wide range of clinico-
Protein expression studies in nine cases suggested a correlation between specific mutations, RyR1protein levels
and resulting phenotype: in particular, whilst patients with dominant or recessive mutations associated with
typical CCD phenotypes appeared to have normal RyR1 expression, individuals with more generalized
weakness, multi-minicores and external ophthalmoplegia had a pronounced depletion of the RyR1 protein.
The phenomenon of protein depletion was observed in some patients compound heterozygous for recessive
mutations at the genomic level and silenced another allele in skeletal muscle, providing additional information
on the mechanism of disease in these patients.
Our data represent the most extensive study of RYR1-related myopathies and indicate complex genotype-
phenotype correlations associated with mutations differentially affecting assembly and function of the RyR1
calcium release channel.
Keywords: skeletal muscle ryanodine receptor gene (RYR1); central core disease (CCD); multi-minicore disease (MmD);
doi:10.1093/brain/awm096Brain (2007) Page1of13
? The Author (2007).Publishedby Oxford University Pressonbehalfofthe Guarantorsof Brain. Allrightsreserved.For Permissions, please email: firstname.lastname@example.org
Brain Advance Access published May 4, 2007
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Abbreviations: CCD¼central core disease; CNM¼centronuclear myopathy; MHS¼malignant hyperthermia
susceptibility; MmD¼multi-minicore disease; RYR1¼skeletalmuscle ryanodine receptor gene;SEPN1¼selenoprotein N gene
Received December 22, 2006. Revised March 23, 2007 . Accepted March 28, 2007
The congenital myopathies are a heterogeneous group of
inherited neuromuscular disorders characterized by the
predominance of a particular histopathological feature—
central cores, nemaline rods, multi-minicores and central
concerning the most common of these myopathies—
central core disease (CCD), nemaline myopathy (NM),
myopathy (CNM)—have demonstrated genetic, pheno-
typic and histopathological
entities, and marked phenotypic variability (Jungbluth
et al., 2003).
The skeletal muscle ryanodine receptor (RYR1) gene
encodes the principal sarcoplasmic reticulum (SR) Ca2þ-
release channel (RyR1) with a crucial role in excitation–
contraction (E–C) coupling. RYR1 mutations have been
associated with the malignant hyperthermia susceptibility
(MHS) trait, an abnormal pharmacogenetic response to
muscle relaxants and volatile anaesthetics, and various
congenital myopathy phenotypes including CCD and
Jungbluth et al., 2002). More recent studies suggest that
mutations is much wider, comprising central cores, multi-
ple cores, central nuclei, nemaline rods and marked type 1
predominance or uniformity without additional structural
changes (Monnier et al., 2000; Sewry et al., 2002).
Typical CCD is a dominantly inherited condition with a
consistent clinical phenotype characterized by hip girdle
weakness with frequent orthopaedic complications, such as
dislocation of the hips and scoliosis, but usually absence of
significant bulbar and respiratory involvement. The dis-
order is named after the prominent core areas devoid of
oxidative enzyme activity that may be central or peripheral
and run a substantial length along the longitudinal fibre
axis. In contrast, MmD is a recessively inherited condition
with diverse clinicalphenotypes:
subgroup of MmD patients is characterized by severe
spinal rigidity, early scoliosis and respiratory impairment,
and is associated with recessive mutations in the seleno-
protein N (SEPN1) gene (Ferreiro et al., 2002b). Other
variants of MmD are associated with recessive RYR1
mutations and clinical manifestations include a distribution
of weakness which may resemble CCD, or a predominantly
axial myopathy with external ophthalmoplegia, or pro-
nounced distal weakness and wasting (Jungbluth et al.,
2005). The pathological hallmark of MmD associated with
recessive SEPN1 mutations are multiple focal areas devoid
of oxidative enzyme activity (‘minicores’). While the
clinico-pathological features associated with classical CCD
overlap between different
and SEPN1-related MmD are quite distinct, it has been
suggested that in some families dominant RYR1-related
CCD and recessive RYR1-related MmD may represent a
clinico-pathologic continuum rather than separate entities
samples from typical cases of CCD do not show prominent
cores (Sewry et al., 2002) but it is not clear if this
may relate to age or sampling, as a significant feature
of CCD is differential involvement of muscles.
The molecular basis of the wide phenotypic spectrum
associated with dominant and recessive RYR1 mutations is
slowly emerging. Dominant RYR1 mutations affecting the
cytoplasmic N-terminal (MHS/CCD region 1, amino acids
35–614) and central (MHS/CCD region 2, amino acids
2163–2458) domains of the RyR1 protein give rise
predominantly to the MHS phenotype, whereas the classical
CCD phenotype is closely associated with dominant RYR1
C-terminal mutations (MHS/CCD region 3, amino acids
4550–4940). Whilst more than 100 dominant RYR1
mutations have been described to date (Treves et al.,
2005; Robinson et al., 2006), only nine RYR1 mutations
have been reported in association with MmD phenotypes
(Ferreiro et al., 2002a; Monnier et al., 2003; Jungbluth
et al., 2005; Zhou et al., 2006b; for a recent review see
Zorzato et al., 2007).
In order to provide further insight into the correlation
between RYR1 mutations and congenital myopathies with
cores, we have characterized a large cohort of patients’
with clinical, histopathological and/or muscle imaging
features suggestive of RYR1 involvement. Twenty-five
causative RYR1 mutations have been identified in 44
patients from 28 unrelated families, including nine novel
mutations. These data feature the largest cohort of recessive
RYR1 mutations reported and provide new insight into the
complex genotype–phenotype correlations associated with
mutations affecting the RyR1 protein.
Patients and methods
Patients were selected on the basis of clinical presentation, muscle
histopathological features and, when available, muscle imaging
findings. Inclusion criteria for the study were (i) clinical features
suggestive of RYR1 involvement, in particular central cores,
multiple cores or significant unevenness of stain with techniques
for oxidative enzyme or (iii) other histopathological features
suggestive of RYR1 involvement such as type 1 predominance or
uniformity, or increased central nuclei; cases with the latter
histopathological findings but no central or multiple cores on
muscle biopsy were only included if clinical findings and features
on muscleMRIwere suggestive
as previously reported (Jungbluth et al., 2004). Mutations in
Page 2 of13 Brain (2007)H. Zhou et al.
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genes involved in conditions with similar clinico-pathological
features, in particular the SEPN1 gene (Ferreiro et al., 2002b),
were excluded whenever appropriate.
Histopathological studies were performed on all patients where
skeletal muscle biopsies were available. Muscle biopsies were
obtained from the quadriceps. Cryostat sections were stained with
haematoxylin and eosin (H&E), periodic acid Schiff (PAS),
nicotinamide dehydrogenase tetrazolium reductase (NADH-TR),
succinate dehydrogenase (SDH) and cytochrome oxidase (COX),
according to standard procedures (Dubowitz and Sewry, 2007).
Fibre typing was assessed by immunolabelling of fast and slow
myosin heavy chain isoforms.
Muscle magnetic resonance imaging
Muscle MRI images were obtained from the thigh and lower legs;
all patients were fully cooperative and no sedation or general
anaesthesia was required for the MRI examination. Muscle MRI
was performed using conventional T1 weighted spin echos
(TR¼500ms, TE¼20ms) on a 1.0-Tesla HPQ system (Marconi
Medical Systems, Cleveland, OH). Non-contrast enhanced images
were obtained from pelvis and thighs and calves. The axial plane
was selected with respect to the long axis of the body. This
involved two sequential scans. We obtained 15 slices from each
site. Slices were 5mm thick and the gap between slices varied from
10 to 50mm depending on the site and on the size of the patient.
Scanning time averaged 20min for each patient.
Total RNA was extracted from patients’ frozen skeletal muscle
tissue, and complementary DNA was synthesized by using
SuperScript III first strand synthesis system kit (Invitrogen). The
entire coding sequence was amplified by 27 overlapping fragments
followed by the direct sequencing in both directions. Genomic
DNA was extracted from peripheral blood lymphocytes following
the manufacturer’s instructions (Nucleon). The C-terminal RYR1
mutational hot spot (Davis et al., 2003) including exon 95 to
exon103 were screened by PCR and direct sequencing by using
genomic DNA as template. For patients who had no mutations in
the C-terminal hot spot and from whom muscle cDNA was not
available, we also studied exons 1, 4, 12, 14, 33, 39, 44, 45, 46, 47,
48, 49, 50, 51, 52, 53, 57, 68, 73, 79, 90, 91 and 92 using genomic
DNA as a template. Once a sequence change was identified, a
panel of 4200 chromosomes from the normal control population
were screened to establish whether the variant detected was likely
to be a polymorphism or a pathogenic mutation.
Skeletal muscle proteins from nine patients were extracted in
sample buffer consisting of 75mM Tris–HCl, 1% SDS, plus a
cocktail of protease inhibitors (antipain, aprotinin and leupeptin).
Twenty micrograms of protein was resolved using a NuPAGE Pre-
cast gel (3–8% Tris–acetate, Invitrogen) and then transferred
electrophoretically to a nitrocellulose membrane (Hybond-ECL,
Amersham). The nitrocellulose membrane was blocked in 10%
semi-skimmed milk in Tris-buffered saline buffer and then probed
with mouse anti-RyR1 monoclonal antibody (Abcam) (1 : 2500)
and mouse anti-desmin monoclonal antibody (DAKO) (1 : 1000)
at room temperature for 1h. After washing, the membrane was
incubated with peroxidase-conjugated donkey anti-mouse IgG
(Jackson) (1 : 50 000) for 1h at room temperature and visualized
using chemiluminescence (ECLþ Plus, Amersham).
There was marked variability in distribution and severity of
weakness and associated clinical findings. The largest group
of patients demonstrated
associated with typical central core disease, comprising
proximal weakness affecting predominantly the hip girdle,
mild facial weakness, with sparing of extraocular muscles
and absence of significant respiratory or bulbar involve-
ment. Scoliosis and congenital dislocation of the hips were
frequent complications. The second largest group of
patients featured more generalized muscle weakness and
wasting and associated extraocular muscle involvement;
bulbar involvement was also pronounced in this group and
two patients required gastrostomy insertion. Respiratory
involvement was more pronounced compared to patients
with features of classical CCD but none required nocturnal
ventilation. The third group comprised three severely
affected neonates from three families. In all three groups,
CK was normal or only mildly elevated. A total of four
patients suffered malignant hyperthermia reactions. The
main clinical features are summarized in Table 1.
Muscle MR imaging
Muscle MR imaging of the thigh and the lower leg was
performed in 13 patients, and the characteristic MR images
are shown in Figure 1.
In nine patients with typical clinical features of CCD and
dominant RYR1 mutations, there was a consistent pattern
of selective muscle involvement characterized by relative
sparing of rectus femoris, gracilis and adductor longus
within the thigh (Figure 1A and B), and sparing of the
tibialis anterior and gastrocnemii within the lower leg but
increased signal within the vasti, adductor magnus and
soleus (Jungbluth et al., 2004). In four patients with more
generalized weakness and wasting, extraocular muscle
involvement and recessive RYR1 mutations, there was
diffuse involvement of the thigh and lower-leg muscles,
with some residual selectivity corresponding
observed in the first group (Figure 1C); the latter pattern
was also observed in one single case with features of
centronuclear myopathy (Figure 1D).
The main pathological features related to the size of the
areas devoid of oxidative enzyme activity (cores), alterations
in fibre type proportions and the presence of central and/or
internal nuclei. Most cases with dominant mutations had
large cores that were central or peripheral. They were
RYR1gene mutations in congenital myopathiesBrain (2007) Page 3 of13
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T able1 Summary of pathological and clinical features and mutations details of patients with RYR1mutations
Exon Nt change AA changeMWScOph FD RIMHC FTUIN
AD11 95 c.13994T4 Cp.L4665P De novo
c.13913G4A22 95 p.G4638DFamilial
Davis et al. (2003)
31 100 c.14474G4C p.R4825PP^^^ This study
41 101 c.14582G4A p.R4861HG, F^^^^^
Davis et al. (2003)
51 101c.14582G4A p.R4861HD^^^^^
61 101 c.14581C4T p.R4861CP^^^^^
Davis et al. (2003)
71 101c.14581C4T p.R4861CP
^ Davis et al. (2003)
81 101 c.14581C4T p.R4861CP^^^^
^ Davis et al. (2003)
91 101 c.14581C4T p.R4861CG
Davis et al. (2003)
102 101 c.14588_14606
Zorzato et al. (2003)
Davis et al. (2003)
123 102c.14741G4C p.R4914TP^^^^^
Davis et al. (2003)
131 102c.14740A4 Gp.R4914QP
^ Davis et al. (2003)
Page 4 of13
H. Zhou et al.
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142 102c.14667C4 Gp.C4883X#
151 47 c.7523G4A p.R2508HD
Wu et al. (2006)
161 90 c.12335C4T p.S4112LP
Jungbluth et al. (2007)
AR 1713 c.212C4A
^ Zhou et al. (2006b)
Jungbluth et al.
100c.14365-2 A4 T acceptor
p.V4849I 201 101c.14545G4A Homozygousx
Jungbluth et al.
Zhou et al. (2006b)
Jungbluth et al (2005)
mThe data presented here are from the proband of each family.dAt the RNA level the altered acceptor splice site was found to cause the deletion of first102 nucleotides of exon100.
xConsanguineous.#Predicted to create a stop codon although at the RNA level an alternative splice site was created. ?Initial biopsy resembled centronuclear myopathy; cores were
only identified on 2nd biopsy (Jungbluth et al., 2007).
Abbreviations: AD¼autosomal dominant; AR¼autosomal recessive; Nt¼nucleotide; AA¼amino acid; MW¼muscle weakness; G¼generalized weakness; P¼predominant
proximal weakness; F¼marked facial weakness; D¼predominant distal weakness; Sc¼scoliosis; Oph¼ophthalmoplegia; FD¼marked feeding difficulties; RI¼respiratory
impairment; MH¼malignant hyperthermia.
C¼cores; þ¼unevenness of stain; þþ¼multiple cores; þþþ¼central cores; FTU¼fibre type uniformity or type1predominance; IN¼increased internal or centrally located
nuclei. NA: Histopathological slides are not available to be viewed.
RYR1gene mutations in congenital myopathies
Page 5 of13
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usually single in cross section but occasionally more than
one per fibre was seen. In one family (case 14), clear cores
were not a feature on oxidative stains in either the mother
(who had two muscle biopsies 30 years apart) or in her
child; however, cores were apparent with electron micro-
scopy in the mother. In the majority of cases, irrespective of
the type of mutation, fibre typing was indistinct or uniform
with oxidative enzymes. Myosin immunolabelling showed
that most fibres expressed slow myosin, with a few co-
expressing fast myosin. In the recessive cases, oxidative
activity showed unevenness of stain or multiple small areas
devoid of activity (minicores). Internal nuclei, some of
which were central were increased in 19 cases. In one
severely affected case (case 16), the pathological features
resembled centronuclear myopathy with abundant central
nuclei and central NADH-TR activity; cores were not
apparent in the first biopsy taken at 1 year of age but were
apparent in a sample from a different muscle taken at age 9
years (Jungbluth et al., 2007). In one severely affected
neonate (case 1), connective tissue and fat were pro-
nounced. The pathological features observed in this cohort
of patients are summarized in Table 1.
Twenty-five different nucleotide variations were identified
in 44 patients from 28 unrelated families, including 22
exonic missense mutations, two splice-site mutations and
one genomic in frame deletion (Table 1). Sequence
comparison across different RyR isoforms and throughout
different species suggested that the changes identified were
significant as they affected evolutionary conserved domains
(Table 2). All novel variations were also excluded from
4200 control chromosomes by either specific restriction
enzyme digestion or direct sequencing.
Twelve mutations were
(Figure 2). Eight of them had arisen de novo, in keeping
Fig.1 Muscle MRI in RYR1-related congenital myopathies.T1-weighted MR images, transverse sections through the proximal thigh in an
1 1-year old boy (A) (case 6) and a12-year old girl (B) (case10) with central core disease (CCD), a 41-year old male with multi-minicore
disease (MmD) and external ophthalmoplegia (C) (case 25) and a 9-year old girl with centronuclear myopathy (CNM) and external
ophthalmoplegia (D) (case16). In patients with typical CCD due to heterozygous dominant mutations affecting C-terminal exons100^102
(A^B), there is a consistent pattern of selective muscle involvement characterized by marked increase in abnormal signal within vasti
(VL,VI), sartorius (S) and adductor magnus (AM) and relative sparing of rectus femoris (RF), adductor longus (AL), gracilis (G) and
semitendinosus (St). In patients with MmD and external ophthalmoplegia due to recessive RYR1mutations (C) and CNM and external
ophthalmoplegia due to a heterozygous dominant RYR1mutation (D), thereismore diffuseinvolvement (corresponding to more generalized
weakness and wasting on clinical examination) but persistent relative sparing of the rectus femoris (RF) compared to the vastus
intermedius (VI) and the gracilis (G) compared to the sartorius (S).
Page 6 of13Brain (2007) H. Zhou et al.
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with the sporadic occurrence of the condition, and four
variations were inherited from the affected parents, three of
them paternally transmitted and one of them maternally
Thirteen substitutions were recessive (Figure 2), consist-
ing of four compound heterozygous, three homozygous and
six monoallelically expressed mutations which exerted their
pathogenicity only in association with non-transcription of
another allele (see later).
The four compound heterozygous substitutions identified
in two unrelated patients were inherited from both clinically
asymptomatic parents. No muscle samples were available for
histological studies from the parents of these two patients.
Two of the three homozygous missense mutations were
identified in consanguineous families, except for p.R2435L,
which was found in a non-consanguineous family originating
from the same village in Italy.
In six families, we were able to demonstrate silencing of
one allele associated with inheritance of a missense
mutation on the other allele, as recently reported (Zhou
et al., 2006a). In these patients the observed sequence
change was clearly heterozygous in genomic DNA from
patients but transcription analysis using skeletal muscle
derived cDNA showed monoallelic expression, and the only
allele which was transcribed was the mutant one (Zhou
et al., 2006a). Segregation analysis studies in informative
families demonstrated that the mutant allele was inherited
from an asymptomatic father whilst the non-transcribed
allele was of maternal origin, suggesting the possibility
of epigenetic silencing of the RYR1 gene in these cases
(Zhou et al., 2006a).
Characterization ofnovel RYR1mutations
Nine of the RYR1 mutations were novel, including four
dominant and five recessive changes, while 16 have been
reported previously (Table 1).
Four novel dominant mutations comprised of p.S4112L
in exon 90; p.L4665P in exon 95; p.R4825P in exon100; and
an alternative splice-site mutation c.14667C4G in exon102.
The de novo p.S4112L mutation was identified in a patient
in whom the main histopathological features on initial
muscle biopsy were those of centronuclear myopathy
(Jungbluth et al., 2007). p.L4665P was identified in a
neonate with a severe CCD variant. This de novo mutation
resulted from the variation of two consecutive nucleotides
at residues c.13994T4C and c.13995C4T, a unique change
probably caused by two nucleotides inversion which has
never been reported before and not found in the parents or
in 4200 control chromosomes. The p.R4825P change
associated with typical CCD is a novel mutation; however,
substitution of the arginine residue at position p.R4825 by
another amino acid has been previously reported in another
CCD family (Monnier et al., 2001). The substitution
c.14667C4G was predicted at the genomic level to cause
a stop codon, yet RNA studies showed the creation of an
alternative splice site in exon 102 resulting in the deletion
of the first 21 base pairs of the exon (r.14647_14667del),
corresponding tothe in-frame
p.C4883_Y4889del. The deleted seven amino acids are
located in the pore region of the calcium ion release
channel of RyR1 protein. The affected mother carried the
same changeas theproband,
Five novel recessive mutations included one homozygous
recessive (p.R3772Q), and four heterozygous missense
mutations in combination with monoallelic expression in
muscle (p.M402T, p.R2939K, p.A4329D and p.T4709M).
The variants p.S71Y in exon 3, recently identified in a MH
pedigree (Galli et al., 2006), and p.N2283H in exon 42,
occurring in MHS domain 1 and 2, respectively, were
identified in a patient with a myopathy and central cores
and had been inherited from the asymptomatic parents.
The homozygous p.R3772Q mutation affects a well-
conserved arginine residue in two consanguineous families
where all affected members have multiple cores. The
mutations p.M402T, p.R2939K, p.A4329D and p.T4709M
were monoallelically transcribed in skeletal muscle. Whilst
substitutions p.R2939K, p.A4329D and p.T4709M all affect
highly conserved residues, the methionine residue at
position p.M402 affected by the p.M402T mutation is
conserved across species only in RyR1 but not in RyR2 and
T able 2 Amino acid sequence alignment of ryanodine receptor1 (RyR1) in mammalians and other human RyR isoforms,
RyR2 and RyR3
Amino acid position
71 109 402?2283 2423 2435 2508 2939 3448 3772 4112 4329 4638 4665 4709 4825 4849 4861 4893 4914
?The residue is conserved among mammalians in RyR1but not in human RyR2 and RyR3.
RYR1gene mutations in congenital myopathiesBrain (2007)Page 7 of13
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RyR3 isoforms (Table 2); however, two nearby residues
p.R401 and p.I403 have been implicated in MHS/CCD
(Quane et al., 1993; Galli et al., 2002; Monnier et al., 2005)
indicating the significance of this region in the pathogenesis
of those conditions.
Recurrent RYR1mutations and associated phenotype
p.R4861 is the residue most commonly mutated in core
myopathy patients. In our study, four CCD patients carried
a c.14581C4T (p.R4861C) transition and two patients
harboured a c.14582G4A (p.R4861H) change. Substitutions
at R4861 were also reported to be common in a cohort of
Japanese patients with central cores on muscle biopsy,
where they also had occurred de novo (Wu et al., 2006).
The p.R2508H substitution, reported at high frequency in
theJapanese population, wasalso found in one of our patients
with a MHS history, scoliosis and progressive muscle
weakness but no visible cores on muscle biopsy (case 15).
We identified a homozygous p.R2435L change in an
Italian CCD patient (case 18); this mutation has been
previously reported as a dominant mutation in an Italian
MH family (Barone et al., 1999), while another hetero-
zygous substitution of the same residue (p.R2435H) has
been reported in a large Canadian pedigree with MHS and
variable expression of clinical myopathic features (Shuaib
et al., 1987; Zhang et al., 1993). In our study, we found that
the mutation p.R2435L caused a typical congenital core
myopathy only when inherited at the homozygous state,
while the heterozygous carrier parents were clinically
The homozygous p.R3772Q mutation has been identified
in two unrelated consanguineous families from North
Fig. 2 Schematic representation of the skeletal muscle ryanodine receptor tetramer taken fromTreves et al., 2005, with permission
showing the position of the dominant and recessive RYR1mutations identified in the patients presented in this report. Dominant and
recessivemutations are shownwith circlesinred andgreen, respectively. All dominantmutations, exceptmutations p.R2508H andp.R4112L
which were associated with atypical myopathy with MH and CNM, are located in domain 3. Recessive mutations are distributed evenly
throughout the gene. Monoallelically expressed mutations are marked by an asterisk; two pairs of compound heterozygous mutations are
indicated by symbols i and #, respectively; homozygous recessive mutations were labelled with symbol x.
Page 8 of13Brain (2007)H. Zhou et al.
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Africa and Asia, respectively. One patient developed a MH
reaction during an orthopaedic operation for bilateral
clubfeet (case 22). Another highly consanguineous family
where both parents and all offspring (case 21) were
homozygous for the same change showed features of a
moderate congenital myopathy but had not developed any
Skeletal muscle RyR1protein expression
in patients with RYR1mutations
Investigation of the consequences of individual mutations
on the expression of the RyR1 protein clearly showed a
differential effect on the level of protein present in skeletal
muscle. In particular in three patients with dominant
RYR1 mutations (two with classical CCD phenotype
carrying p.G4638D and p.R4861H, one carrying p.S4112L
associated with a centronuclear myopathy phenotype), in
one patient with compound heterozygous MH changes
(p.S71Yþp.N2283H) and one patient with homozygous
MH/CCD change (p.R2435L), RyR1 protein levels were
similar to control (Figure 3). In contrast, in four patients
p.A4329D and p.T4709M) associated with monoallelic
transcription of the mutant allele, there was a dramatic
reduction of RyR1 protein levels as determined by western
blot analysis (Figure 3).
We report 12 dominant and 13 recessive RYR1 mutations
in a cohort of 44 congenital myopathy patients from
28 families, representing the largest series of recessive RYR1
mutations reported to date. Our findings suggest potential
molecular mechanisms implicated in the marked pheno-
typic variability associated with dominant and recessive
These were mainly found in the C-terminal and only in one
case in the central domain of the RYR1 gene. In more than
80% of cases dominant RYR1 mutations involved exons 100
to 102 of the RYR1 coding sequence. Our findings lend
support to the idea that the C-terminal is a mutational
hotspot for CCD and suggest that focusing on RYR1 exons
100 to 102 could be a highly efficient screening strategy in
patients with typical features. In contrast to other recent
mutational studies on CCD (Wu et al., 2006), we identified
only one single dominant mutation outside the C-terminal
hotspot (Exon 47, c.7523G4A, p.R2508H); this discrepancy
is likely to reflect different inclusion criteria between the
two studies, as our cohort only included patients with a
congenital myopathy phenotype, while the presence of
central or multiple cores on muscle biopsy without any
associated muscle weakness, a criterion used in the recent
study from Wu et al. (2006), was considered insufficient
Most CCD-related dominant RYR1 mutations resulted in
the substitution of a charged polar residue, typically
arginine, with an uncharged polar residue. When mapped
to the transmembrane topology of the RyR1 protein
(Du et al., 2002, 2004), most dominant CCD-related
RYR1 mutations localize to the M8/M10 loop surrounding
the pore helix region, demonstrating the unique role of this
part of the protein in the pathogenesis of CCD; another
two dominant CCD mutations localized in or close to M6,
implying the potential importance of this transmembrane
Fig. 3 The effect of RYR1mutations on protein expression.Western blot analysis of skeletal muscle biopsies from patients described in this
study. Patients carrying mutations p.R4861H (heterozygous), p.G4638D (heterozygous), p.S71Yþp.N2283H (compound heterozygous) and
p.R2435L (homozygous) with normal biallelic transcription showed normal levels of RyR1expression as compared to control muscle. In
contrast patients carrying mutations p.R109W, p.M402T, p.A4329D and p.T4709M with monoallelic transcription had dramatically
decreased levels of RyR1.The patient with CNM carrying mutation p.S4112L (heterozygous) showed normal level of expression of RyR1.
Desmin immunoreactivity was used as a muscle-specific loading control.
RYR1gene mutations in congenital myopathiesBrain (2007) Page 9 of13
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Most patients with dominant C-terminal mutations had
the classical CCD phenotype, characterized by variable
degrees of proximal weakness pronounced in the hip
girdle and absence of significant bulbar, respiratory or
namely congenital dislocation of the hips and scoliosis,
were common features. In this group of patients, muscle
MRI showed a highly consistent pattern of selective
muscle involvement; this was even more reproducible
Interestingly, some patients carrying the same amino
acid substitution showed marked variation in age of onset
and severity, suggesting either a non-stochastic distribu-
tion of mutant and wild-type RyR1 proteins or other
for proper channel function andstability
had typical central cores on muscle biopsy, in one
family harbouringa seven
(p. C4883_Y4889del), clear cores were not present at
the light microscopy level, although they could be seen on
muscle biopsy from the affected mother 30 years after
the first, non-diagnostic biopsy. The profound muscle
weakness present in this family confirms the lack of direct
correlation between muscle weakness and the propensity
to develop cores in RYR1-related myopathies.
Two cases harbouring C-terminal mutations presented
with unusual clinical and/or histopathological features: one
infant who had a unique nucleotide inversion (c.13994T4C,
c.13995C4T) in exon 95, resulting in a p.L4665P substitu-
tion, presented with an unusually severe neonatal pheno-
type and marked increase in connective tissue, leading to
an initial suspicion of congenital muscular dystrophy;
on the second
Fig. 4 Schematic diagram showing the distribution of mutations associated with congenital myopathies in the C-terminal region of RYR1.
(A) Closed circles signify mutations which are dominant and are associated with the CCD phenotype ?; closed squares signify mutations
which are recessive and associated with the MmD phenotype g and the closed triangle signifies the mutation associated with the
CNM phenotype m. (B) A 34-amino-acid deletion p.S4789_K4822del, affecting most of the transmembrane segments M7a and M7b,
did not cause any phenotype when expressed in the heterozygous state.The schematic diagram is based on the proposed model of Du’s
(Du et al., 2002).
Page10 of13Brain (2007)H. Zhou et al.
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this patient also had a severe MH reaction when the muscle
biopsy was taken. Another patient with a heterozygous
missense mutation in exon 90 (p.S4112L) had a phenotype
suggestive of centronuclear myopathy with more severe
muscle involvement and
(Jungbluth et al., 2007). In contrast to CCD-related
mutations, this change does not localize to the RyR1
channel pore but to a previously identified calmodulin-like
binding site (Xiong et al., 2006). In contrast to recessive
RYR1 mutations associated with a similar clinical pheno-
type including external ophthalmoplegia (see later), the
dominant p.S4112L substitution did not affect the amount
of protein present in the muscle, as indicated by western
blot analysis; this may indicate an alternative pathogenic
mechanism, for example disturbed protein–protein interac-
tions, with similar functional consequences despite normal
In contrast to dominant RYR1 mutations, recessive RYR1
mutations were evenly distributed throughout different
domains. Clinical manifestations associated with recessive
RYR1 mutations were variable, ranging from a typical CCD
phenotype to generalized muscle weakness and wasting with
associated external ophthalmoplegia, variable degrees of
bulbar involvement and respiratory impairment.
Recessive RYR1mutations previouslyassociated
A typical CCD phenotype was found in two families
carrying apparently recessive RYR1 missense mutations
affecting the N-terminal (MHS/CCD region 1, p.S71Y) and
central (MHS/CCD region 2, p.N2283H and p.R2435L)
domains of the protein; these mutations were previously
associated with dominantly inherited MHS phenotypes;
recently performed functional studies indicate that the
MHS phenotype was due to the p.N2283H mutation
(Zhou et al., 2006b). Interestingly, the fact that dominant
MHS-associated RYR1 mutations may give rise to a
congenital myopathy phenotype in the compound hetero-
zygous or homozygous state is novel and suggests a
combined deleterious effect on the tetrameric RyR1 protein.
The hypothesis of a cooperative effect is further supported
by the previous finding of a more severe IVCT response in
individuals homozygous for MHS-related RYR1 mutations
(Lynch et al., 1997), and the observation of a congenital
both presenting with genetically unresolved MH (Deufel
et al., 1992).
the offspringof parents
Recessive RYR1mutations with monoallelic expression
and protein depletion
Our data support and extend our recent observation that
different mutations affect the amount of mutant RyR1
proteins present in skeletal muscle (Zhou et al., 2006b).
Whilst the amount of expressed RyR1 protein was normal
in patients with a typical CCD phenotype associated with
both recessive and dominant RYR1 missense mutations, we
found a marked reduction of RyR protein in muscle
biopsies from patients with more severe clinical phenotypes
including generalized muscle weakness and wasting and
external ophthalmoplegia. The more diffuse involvement in
these patients was also apparent on muscle MRI which
showed only remnantsof the
characteristic of CCD patients. In these patients a hetero-
zygous RYR1 missense mutation was expressed on the
background of a second non-transcribed allele, confirming
RYR1 epigenetic silencing as an important aetiological
mechanism in RYR1-related congenital myopathies (Zhou
et al., 2006a).
While some of the recessive RYR1 missense mutations
identified were expected to result in severe disruption of the
RyR1 protein structure, these did not cause a clinical
phenotype in the heterozygous state, suggesting either that
the domains concerned are not crucial to the formation of
a functional RyR1 channel, or that only a small amount of
functional RyR1 isrequired
phenotype. For example, the heterozygous carrier state of
the intron99 splice-site mutation (c.14365-2A4T) leading to
a 34-amino-acid deletion p.S4789_K4822del did not cause
any clinical phenotype in the patient’s asymptomatic father,
despite affecting most of the transmembrane segments M7a
and M7b (Figure 4B); this corresponds to observations in
one consanguineous family reported previously where
homozygosity for an intronic RYR1 splice-site mutation
caused marked protein depletion and a severe phenotype
with external ophthalmoplegia in the proband but no
symptoms in the carrier parents (Monnier et al., 2003).
to maintaina normal
Functional effects of RYR1mutations
Functional consequences of individual RYR1 mutations at
the cellular level are currently only partially understood,
and five ofthe mutations
p.S71Yþp.N2283H) have been previously studied in vitro
(Zorzato et al., 2003; Ducreux et al., 2006; Zhou et al.,
2006b). Whilst it is widely accepted that dominant RYR1
mutations causing MH increase the sensitivity of the RyR1
protein to activation and dominant CCD-mutations are
associated with a reduction of stimulated calcium release,
the scarce data available on recessive RYR1 mutations and
those associated with allele silencing suggest the possibility
of altogether different pathogenic mechanisms. In contrast
mutation implicated in a typical CCD phenotype and
associated with reduction of sarcoplasmic reticulum cal-
cium stores (‘leaky’ channel) commonly seen in this
phenotype, the homozygous recessive p.V4849I substitution
in stimulated or
RYR1gene mutations in congenital myopathies Brain (2007) Page11of13
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spontaneous calcium release. The recombinant channels
with compound heterozygous p.S71Yþp.N2283H substitu-
tions lost activity upon isolation, whilst the p.R109W
substitution expressed in the homozygous state leads to
complete loss of calcium conductance (Zhou et al., 2006b).
These findings in conjunction with the observation of
reduced RyR1 protein levels suggest that some recessive
RYR1 mutations may affect protein assembly or protein
stability in a manner not detectable in commonly applied
in vitro models, and that the quantitative lack of functional
RyR1 protein causes phenotypes more severe than those
associated with simple RyR1 malfunction. Future functional
studies on the emerging spectrum of RYR1 mutations will
further clarify interactions and function of this complex
protein assembly, while protein studies might provide an
additional tool to investigate patients with recessive RYR1
The initial part of this work was supported by a grant from
the Muscular Dystrophy Campaign to FM, HJ and CAS.
The authors also wish to thank the National Specialist
Commissioning Advisory Group (NSCAG) support to the
Hammersmith Hospital Neuromuscular Centre for rare
congenital myopathies and muscular dystrophies.
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