HUMAN M UTATION
M UTATION IN BRIEF
HUMAN MUTATION Mutation in Brief 31: E1544-E1550 (2010) Online
Received 31 Decem ber 2009; accepted revised m anuscript 12 April 2010.
© 2010 WILEY-LISS, INC.
Recessive Mutations in RYR1 Are a Common Cause
of Congenital Fiber Type Disproportion
Nigel F. Clarke1,2, Leigh B. Waddell1,2, Sandra T. Cooper1,2, Margaret Perry2,3, Robert L.L. Sm ith4,
Andrew J. Kornberg5, Francesco Muntoni6, Suzanne Lillis7, Volker Straub8, Kate Bushby8, Michela Guglieri8, Mary
D. King9, Michael A. Farrell10, Isabelle Marty11, Joel Lunardi11, Nicole Monnier11, and Kathryn N. North1,2
1Institute for Neuroscience and Muscle Research, Children’s Hospital at Westm ead, Sydney, Australia; 2Discipline of
Paediatrics and Child Health, University of Sydney, Sydney, Australia; 3Anaesthetic Departm ent, The Children’s Hospital at
Westm ead, Sydney, Australia; 4John Hunter Children’s Hospital and University Discipline of Paediatrics and Child Health,
Newcastle, Australia; 5Departm ent of Neurology, Royal Children’s Hospital, Melbourne, Australia; 6Dubowitz Neurom uscular
Centre, Institute of Child Health, London, UK; 7Diagnostic Genetics Laboratory, Guy’s Hospital, London, UK; 8Institute of
Hum an Genetics, Newcastle University, International Centre for Life, Central Parkway, Newcastle upon Tyne, UK; 9Neurology
Departm ent, Children’s University Hospital Tem ple St, Dublin, Ireland; 10Departm ent of Neuropathology, Beaum ont Hospital,
Dublin, Ireland. 11Biochim ie et Génétique Moléculaire, CHU Grenoble / INSERM U836, Grenoble, France
* Correspondence to Dr. Nigel Clarke, Children’s Hospital at Westm ead, Locked Bag 4001, WESTMEAD, NSW 2145,
Australia. Telephone: +61 2 98451453. Fax: +61 2 9845 3389. E-m ail: NigelC@chw.edu.au.
Com m unicated by Ravi Savarirayan
ABSTRACT: The main histological abnormality in congenital fiber type disproportion (CFTD) is
hypotrophy of type 1 (slow twitch) fibers compared to type 2 (fast twitch) fibers. To investigate
whether mutations in RYR1 are a cause of CFTD we sequenced RYR1 in seven CFTD families in
whom the other known causes of CFTD had been excluded. We identified compound
heterozygous changes in the RYR1 gene in four families (five patients), consistent with autosomal
recessive inheritance. Three out of five patients had ophthalmoplegia, which may be the most
specific clinical indication of mutations in RYR1. Type 1 fibers were at least 50% smaller, on
average, than type 2 fibers in all biopsies. Recessive mutations in RYR1 are a relatively common
cause of CFTD and can be associated with extreme fiber size disproportion. ©2010 Wiley-Liss, Inc.
KEY WORDS: RYR1, congenital myopathy, congenital fiber type disproportion, multi-minicore disease
Congenital fiber type disproportion is a form of congenital myopathy in which consistent type 1 fiber
hypotrophy relative to type 2 fibers is the main histological abnormality [Clarke and North, 2003]. Of the four
genetic causes that have been reported to date, TPM3 (MIM# 191030) appears a common cause compared to
ACTA1 (MIM# 102610) , TPM2 (MIM# 190990) and SEPN1 (MIM# 606210) [Laing et al., 2004; Clarke et al.,
Recessive Mutations in RYR1 Cause CFTD E1545
2006; Clarke et al., 2008; Brandis et al., 2008; Lawlor et al., 2009]. Despite recent advances, a genetic cause is
not found in at least 50% of CFTD patients.
The gene that encodes the ryanodine receptor type 1 (RYR1; MIM# 180901) was first identified as the
principle cause of autosomal dominant central core disease and malignant hyperthermia (MH) [reviewed in
Robinson et al., 2006]. In recent years, recessive mutations in RYR1 have been found in many patients with
multi-minicore disease who had relative type 1 fiber hypotrophy as a secondary histological abnormality
[Jungbluth et al., 2002; Monnier et al., 2008]. For this reason we considered RYR1 a good candidate for CFTD.
PATIENTS AND METHODS
We investigated RYR1 in seven families. Six unrelated patients were enrolled in a study on CFTD [Clarke et
al., 2008] who met the following criteria; type 1 fibers smaller than type 2 fibers by at least 25% as the main
histological abnormality, clinical features consistent with congenital myopathy, normal sequencing of ACTA1
and TPM3, and availability of either frozen muscle or cultured fibroblasts. This study was approved by the
human ethics committees of the Children’s Hospital at Westmead, Sydney, Australia (ID:2000/068). A seventh
family (Patients 4 and 5) was ascertained during routine diagnostic studies in the UK.
In six probands (including Patients 1-3 reported here) we sequenced the coding regions of the RYR1 gene from
cDNA (GenBank NM_000540.2) using methods previously described [Monnier et al., 2001]. Patient mRNA was
extracted from either archived frozen muscle samples (five patients) or from cultured myotubes derived from a
fibroblast cell line transduced with the myoD virus (Patient 3) [Cooper et al., 2007] and cDNA was generated
using reverse transcriptase superscript III (Invitrogen, CA) according to manufacturer’s instructions. We
amplified the RYR1 coding sequence using overlapping primer pairs and sequenced the fragments on an ABI
3100 DNA Analyzer (Applera, Courtaboeuf, France). Sequence variants and adjacent intron/exon boundaries
were confirmed by sequencing genomic DNA (Ensembl ENSG00000196218).
In the seventh proband (Patient 4), the entire coding regions of the RYR1 gene and intron/exon boundaries
were sequenced from genomic DNA and abnormalities were confirmed in genomic DNA from Patient 5.
Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation
initiation codon in the reference sequence.
Frozen muscle samples were available for protein studies in Patients 1 and 2. We performed
immunohistochemistry (IHC) and Western blotting using patient frozen muscle for the ryanodine receptor type 1
(RyR1) to investigate protein localisation and levels using the 34C antibody (University of Iowa Hydridoma
Bank) and methods previously described [Cooper et al., 2003; Monnier et al., 2008].
Patient 1 was born at term with severe generalised hypotonia and weakness. He sat at age 1 year, stood with
support at age 2 years, drooled and had poor weight gain, constipation and recurrent respiratory infections. At age
2 years there was generalised muscle wasting, severe generalised weakness and hypotonia, quiet voice, ptosis,
ophthalmoplegia and moderate facial weakness. Serum CK and nerve conduction studies (NCS) were normal.
Electromyography (EMG) showed myopathic abnormalities. Genetic testing was normal for TPM2 and myotonic
E1546 Clarke et al.
dystrophy (DM1). He required nocturnal continuous positive airways pressure from age 2 years, gastrostomy
feeding from 2½ years and died at age 3 years from respiratory failure.
Patient 2 had severe hypotonia at birth and required resuscitation due to poor respiratory effort. He had
reduced subcutaneous fat with excess skin folds, marked axial hypotonia, a frog-leg posture, poor anti-gravity
movements of the limbs, mild ptosis, mild ophthalmoplegia and a weak cry. He was intubated at 26 days of life
for respiratory failure and died following extubation at age 1 month. Brain MRI and serum CK were normal.
EMG was myopathic. Genetic testing for DM1 and myotubular myopathy was normal.
Patient 3 was born at term and had generalised hypotonia and marked truncal weakness at birth. He initially
fed poorly, was delayed in walking and never ran. He required surgery for undescended testes, strabismus,
scoliosis (12 years) and Achilles contractures (14 years). At age 29 years Patient 3 was thin with generalised
muscle wasting (height 1.77 m, weight 36 kg; Figure 1, A-C) and walked only a few meters. He had a long thin
face, bilateral ptosis, ophthalmoplegia, convergent strabismus, mild facial weakness, a narrow high-arched palate
and reduced mouth opening. There were mild hip flexion contractures. Weakness was most pronounced in
proximal limb muscles. CK levels were normal. Forced vital capacity was 50% of predicted at age 12 years.
SEPN1 gene analysis showed heterozygosity for a known recessive mutation (p.G943A) but a second mutation
was not identified on sequencing genomic or cDNA and the phenotype was considered atypical for SEPN1-
Patients 4 and 5 are siblings. Patient 4 (female) had normal early motor milestones and first presented at
age 2 years with difficulty running. There was a slow decline in motor abilities. At age 18 years she had
difficulty climbing stairs or walking more than 15 minutes. She used a Gowers’ manoeuvre to rise and had
moderate generalised weakness, mild facial weakness (Figure 1D) and ptosis. CK levels, lung function tests and
genetic testing of SEPN1 and DOK7 (MIM# 610285) were normal. EMG showed myopathic changes. Muscle
MRI at age 12 years showed muscle signal abnormalities in a pattern typical for RYR1-related myopathies
(Figure 1E), which prompted RYR1 gene testing. Patient 5 was noted to have mild proximal lower limb weakness
at birth. He walked at 14 months and had a slow loss of muscle strength during childhood and reduced exercise
stamina. On examination at age 12 years he had a myopathic face, generalised muscle hypotrophy, mild proximal
limb weakness, bilateral mild scapular winging and a mild scoliosis. Neither sibling had ophthalmoplegia or
All patients were of European descent and had normal genetic analysis of ACTA1, TPM3 and SMN1 (MIM#
600354). Sequencing of TPM2 and SEPN1 was not performed unless specifically mentioned.
We found compound heterozygous sequence changes in RYR1 in four families; Patient 1: c.6104A>T
(p.His2035Leu) and c.738T>G (p.Tyr246X), Patient 2: c.10204T>G (p.Cys3402Gly) and c.13480G>T
(p.Glu4494X), Patient 3: c.9978C>A (p.Asn3326Lys) and c.9000+1G>T, and Patients 4 and 5: c.1205T>C
(p.Met402Thr) and c.5333C>A (p.Ser1778X). None of the novel sequence variants were present in 100
chromosomes from healthy individuals of European descent.
On cDNA analysis, levels of transcripts containing two nonsense mutations (c.738T>G change in Patient 1,
c.13480G>T in Patient 2) appeared low based on the sequencing trace files, to the degree that these mutations
were difficult to distinguish from non-specific background peaks, likely due to nonsense-mediated mRNA decay
(NMD). The c.9000+1G>T change in Patient 3, which abolishes the consensus splice donor site for intron 59,
was associated with skipping of exon 59 alone or exons 59 and 60, which are both out-of-frame transcripts that
are predicted to encode markedly truncated, non-functional proteins. Both abnormal transcripts were present at
similar levels as the normally spliced sequence, based on PCR amplification of patient cDNA (non-quantitative).
Three out four missense variants we identified (p.His2035Leu, p.Cys3402Gly, p.Asn3326Lys) are novel variants
localized in cytoplasmic protein domains of unknown function. All are non-conservative substitutions. Two
affect residues well-conserved among species and RyR isoforms (His2035, Asn3326) while Cys3402 is
conserved only in mammals and in the RyR3 isoform. The c.1205T>C (p.Met402Thr) variant we found in
Patients 4 and 5 has been previously reported in a patient with recessive core myopathy [Zhou et al., 2006] and is
located in a domain of the protein associated with malignant hyperthermia.
Recessive Mutations in RYR1 Cause CFTD E1547
Figure 1. Clinical photographs and muscle MRI images. A-C: Patient 3 at age 29 years, D: Patient 4 at age 18 years. Patient 3
has a long myopathic face, ptosis, convergent strabismus and hip contractures. In comparison, Patient 4 has only mild facial
weakness during maximal eye closure. E: MRI scan from Patient 4 performed at age 12 years of the pelvis (i), upper thigh (ii)
and calf (iii). Abnormal high signal (pale) is seen in the gluteus maximus (GM), vastus lateralis (VL), adductor magnus (AM)
and soleus (S) muscles while the rectus femoris (RF), semi-tendinosis (St), gracilis (G), tibialis anterior (TA) and
gastrocnemius (GN) muscles are spared.
Interestingly, all patients had one ‘null’ RYR1 allele (predicted to encode no full-length RyR1 protein)
and an allele with a heterozygous missense change. Parental DNA was available from both parents in Patients 1
and 2, and from the mother of Patient 3. All parents were heterozygous for one of the sequence changes,
consistent with autosomal recessive inheritance. The mother of Patient 3 had isolated unilateral ptosis but
otherwise all parents were clinically unaffected.
In all biopsies, a consistent difference in fiber size was the most striking histological abnormality (Figure 2),
in keeping with a diagnosis of CFTD. Type 1 fibers were between 50 and 84% smaller than type 2 fibers (Table
1). No cores or rods were seen on standard histological stains. Two quadriceps muscle biopsies were available for
Patient 1, taken at ages six months and three years, and the fiber size disproportion was more extreme in the
second biopsy (Figure 2B) due to a decrease in mean type 1 fiber diameter and further hypertrophy of type 2
fibers. No further abnormalities were seen on electron microscopy (EM). Internalized nuclei were present in
between 1.5-10% of fibers in the patients’ biopsies. Widespread myofibrillar disarray, was seen on EM of Patient
2’s biopsy, particularly in small fibers. The only abnormality on EM in Patient 3 was minor irregularity of the Z-
bands and EM was not available for Patient 4.
Table 1. Muscle fiber measurements
Diam = mean diameter (μm), SD = standard deviation, quad = quadriceps, m = months, yr = years, %FSD = Percentage
fiber size disproportion = 100 x (Mean type 2 diameter - mean type 1 diameter)/mean type 2 diameter.
Patient Muscle Age at biopsy Type 1 fibers
% Diam. (SD)
Type 2A fibers
% Diam. (SD)
E1548 Clarke et al.
Figure 2. ATPase stains and Western blot analysis of
patient muscle biopsies. A-D: ATPase (pH 4.3) stains of
muscle biopsies from Patient 1 taken at ages 6 months
(A) and 3 years (B), Patient 2 (C) and Patient 3 (D).
Type 1 fibers stain dark, type 2 fiber stain pale and
intermediate staining fibers represent 2C fibers (hydrid
type 1/2A). The size bar indicates 50 μm in each image.
In Patient 1 the discrepancy in fiber sizes is more
marked and more consistent in the second biopsy (B)
compared to the first (A). E: Western blots of the
ryanodine receptor type 1 (RyR1) in Patient 1 (P1), his
father (FP1) with age matched controls (C) and in Patient
2 (P2). RyR1 levels are reduced in patients and in the
father of Patient 1 who while asymptomatic, is
heterozygous for a RYR1 nonsense mutation. Multiple
bands are often seen due to rapid protein degradation
and do not indicate pathology. Myosin (MyHC) is
shown to indicate protein loading.
RyR1 Protein Studies
Western blotting showed reduced RyR1 levels in muscle from Patients 1 and 2, and the father of Patient 1
(who was heterozygous for p.Tyr246X) (Figure 2E). The pattern of RyR1 expression appeared normal by IHC in
muscle from Patient 1 and control (data not shown).