Download Central core disease due to recessive mutations in RYR1 gene: Is it

SHORT REPORT
ABSTRACT: Central core disease (CCD) is an autosomal-dominant congenital myopathy, with muscle weakness and malignant hyperthermia (MH)
susceptibility. We identified two of nine Brazilian CCD families carrying two
mutations in the RYR1 gene. The heterozygous parents were clinically
asymptomatic, and patients were mildly affected, differing from the few
autosomal-recessive cases described previously. Recessive inheritance in
CCD may therefore be more common than previously appreciated, which
has important implications for genetic counseling and MH prevention in
affected families.
Muscle Nerve 35: 670 – 674, 2007
CENTRAL CORE DISEASE DUE TO RECESSIVE
MUTATIONS IN RYR1 GENE: IS IT MORE
COMMON THAN DESCRIBED?
PATRÍCIA M. KOSSUGUE, MS,1 JÚLIA F. PAIM, MD, MS,2 MONICA M. NAVARRO, MD,2
HELGA C. SILVA, MD, PhD,3 RITA C. M. PAVANELLO, MD,1 JULIANA GURGEL-GIANNETTI, MD, PhD,4
MAYANA ZATZ, PhD,1 and MARIZ VAINZOF, PhD1
1
Department of Genetics, Human Genome Research Center, IB-USP, R. do Matão,
106, São Paulo, SP—CEP 05508-900, Brazil
2
Pathology Department, Genetics Department, and Neurophysiology Clinic,
Sarah Network of Rehabilitation Hospitals, Belo Horizonte, Minas Gerais, Brazil
3
Department of Pathology—FMUSP and Department of Anesthesiology,
UNIFESP, São Paulo, Brazil
4
UFMG, Belo Horizonte, Minas Gerais, Brazil
Accepted 30 October 2006
Central
core disease (CCD) is a rare congenital
myopathy, with high inter- and intrafamilial phenotype variability ranging from asymptomatic to severely affected individuals. Clinical signals include
hypotonia, delayed motor milestones, proximal muscle weakness, and skeletal anomalies such as hip
dislocation, scoliosis, and foot deformities.1,21
The diagnosis of CCD is confirmed by the presence within muscle fibers of cores, which are lesions
of the sarcomeres with sarcomeric disorganization,
lack of mitochondria, and lack of oxidative activity.17
These cores, mainly in type I muscle fibers, may be
single or multiple, central or peripheral, and occasionally associated with rods.13,20
Abbreviations: ATPase, adenosine triphosphatase; CCD, central core disease; CK, creatine kinase; DNA, deoxyribonucleic acid; EMG, electromyography; HE, hematoxylin and eosin; MH, malignant hyperthermia; NADH, nicotinamide adenine dinucleotide; PCR, polymerase chain reaction; RYR1,
ryanodine receptor 1; SDH, succinate dehydrogenase; SSCP, single-strand
conformation polymorphism
Key words: central core disease; congenital myopathy; malignant hyperthermia; ryanodine receptor; RYR1
Correspondence to: M. Vainzof; e-mail: [email protected]
© 2007 Wiley Periodicals, Inc.
Published online 16 January 2007 in Wiley InterScience (www.interscience.
wiley.com). DOI 10.1002/mus.20715
670
Short Reports
The main gene associated with this disorder is
the ryanodine receptor (RYR1) gene at chromosome
19q13,15 which is also linked to malignant hyperthermia (MH). Both MH and CCD are disorders of Ca2⫹
release channels, and patients with CCD are at risk
for MH because there are mutations that cause both
phenotypes.9
The RYR1 gene is a very large gene, with 106
exons. However, RYR1 mutations are essentially clustered in three regions: regions 1 and 2 reside in the
myoplasmatic foot domain of the protein, and region 3 is located in the transmembrane/luminal
region of the highly conserved C-terminal domain.
Recently, the majority of the novel mutations linked
to CCD was found to be concentrated in region 3,
particularly between exons 95 and 103.3
The human RYR1 gene encodes a 5037 aminoacid protein, the functional skeletal-muscle calcium
release channel, which is composed of four RYR1
subunits of 565 kDa each and a number of accessory
proteins that are thought to regulate and stabilize
the calcium channel.2,6,11,22,23 This protein is located
in the junctional terminal cisternae of the sarcoplasmatic reticulum membrane.
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Table 1. Family data.
Family
1
2
3
4
5
6
7
8
9
Familial/sporadic
Inheritance
Exon
Mutation
Reference no.
Familial
Familial (2 affected)
Sporadic
Familial (8 affected)
Sporadic
Sporadic
Familial (8 affected)
Familial (3 affected)
Sporadic
AR
AR
New*
AD
?
New*
AD
AD
?
101
94 and 101
101
102
102
101
—
—
—
V4849I
R4558Q/A4846V
R4861H
G4897V
R4914T
R4861C
—
—
—
8
Novel/novel
14
Novel
3
3
—
—
—
*Not detected in the parents.
AD, autosomal dominant; AR, autosomal recessive.
CCD is usually transmitted through autosomaldominant inheritance with variable penetrance but a
few reported cases have autosomal-recessive inheritance.5,8,10,18 The pattern of inheritance is important
for genetic counseling and prevention of malignant
hyperthermia.
Here, we screened the hot spot C-terminal region
of the RYR1 gene (encompassing exons 94 to 106)
for mutation, and identified two of nine Brazilian
CCD families with autosomal-recessive mutations,
suggesting that this pattern of inheritance is more
frequent than expected.
PATIENTS AND METHODS
Thirteen exons from the C-terminal region of the
RYR1 gene were screened for mutations in 15 patients, from 9 unrelated families. A total of 200 chromosomes, from 100 control individuals, matched for
ethnic background, age, and sex were used to confirm the frequency of the novel mutations.
Genomic deoxyribonucleic acid (DNA) was extracted from peripheral blood lymphocytes.12 The
C-terminal region (exons 94 to 106) was analyzed
using polymerase chain reaction (PCR) amplification with described primers.14 The mutation screening was performed by single-strand conformation
polymorphism (SSCP) analysis16 through electrophoresis on MDE gel (FMC Bioproducts, Rockland,
ME). Bands with altered migration were sequenced
using a sequencing kit on a MegaBACE 1000 automated DNA sequencer (Amershan Biosciences, Piscataway, NJ).
Muscle biopsy was taken from the biceps muscle
in the first family and from the quadriceps muscle in
the second family, for diagnostic purpose, cryoprotected, and snap-frozen in liquid nitrogen. Routine
histological and histochemical analyses included
staining for hematoxylin and eosin (HE), modified
Gomori trichrome, succinate dehydrogenase (SDH),
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nicotinamide adenine dinucleotide (NADH), acid
phosphatases, and adenosine triphosphatase (ATPase) at pH 9.4 and 4.3.4
Informed consent was obtained from each subject.
RESULTS
Seven different mutations were identified in six of
the nine families (Table 1).
Three mutations were new ones (families 2 and
4), whereas the other four have been described previously (families 1, 3, 5, and 6). In two sporadic cases
(families 3 and 6), the mutation was not present in
the parents, and were therefore new mutations. In
the other two sporadic cases (families 5 and 9), the
parents were not available for analysis.
In two families (1 and 2), mutations were identified in the two alleles, confirming an autosomalrecessive disease (Fig. 1).
In a 9-yearold affected girl, we identified the homozygous
V4849I mutation in exon 101 (a previously described
mutation). The patient had muscle weakness and
difficulties in running, jumping, and climbing stairs.
Her serum creatine kinase (CK) was normal, and
electromyography (EMG) showed myopathic features. Histological analysis of the muscle biopsy
showed discrete myopathic alterations, a total type I
predominance, and a pattern of several diffuse large
and mini-cores within muscle fibers (Fig. 2A). The
parents were clinically asymptomatic, consanguineous, and carriers of the same mutation in a heterozygous state. No previous history of neuromuscular
diseases was present in this large family with many
probable heterozygote relatives (Fig. 2A).
Autosomal-Recessive Families: Family 1.
2. Two different and novel mutations
(R4558Q and A4846V) were identified in two afFamily
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FIGURE 1. Molecular analysis of the RYR1 gene in the patients from family 1 (A) and family 2 (B). 䡩, female; ▫, male; 〫, unidentified
member of the family. Filled symbols are affected members.
fected sibs: a 46-year-old woman and a 42-year-old
man. The R4558Q mutation (c.13673 G⬎A) in exon
94 was inherited from the sibs’ mother and the
A4846V mutation (c.14537 C⬎T) in exon 101 from
the father, both clinically unaffected (Fig. 1). These
mutations were confirmed by automatic sequencing
FIGURE 2. Histological findings in the patients from family 1 (A) and family 2 (B). In the patient from family 1, hematoxylin and eosin (HE)
shows variability in fiber size and some degeneration; nicotinamide adenine dinucleotide (NADH) reaction illustrates the pattern of the
cores, and a total predominance of type I fibers is observed on the adenosine triphosphatase (ATPase) 9.4 reaction. In patient II-2 from
family 2, HE shows large hypertrophied fibers, splitting and several internal nuclei, and one or two large and well-defined cores can be
observed both in NADH and succinate dehydrogenase (SDH) reactions.
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and were not found in 200 chromosomes from normal controls. The two affected sibs reported symptoms and weakness since childhood. The oldest sister
had foot deformities, surgically repaired during
childhood, and her younger brother has also facial
weakness and muscular hypotrophy. Complementary studies in the affected male disclosed normal
serum CK levels. The EMG showed myopathic features, with small-amplitude, short-duration, polyphasic motor unit potentials. There was a rapid recruitment of motor units, and long-duration, highamplitude motor unit potentials were present in
both tibialis anterior and right gastrocnemius muscles. Motor and sensory conduction velocities were
normal. Muscle biopsy showed fiber-size variation,
with the presence of some very large fibers, and very
discrete perimyseal fibrosis. A large number of internally located nuclei were observed. A type I fiber
predominance was present and in the oxidative reaction, one or two large cores were identified in
almost all muscle fibers (Fig. 2B).
DISCUSSION
Since the finding of the association of the RYR1 gene
with CCD, several groups have been searching for
mutations in this gene in large cohorts of families
with affected patients. However, in the last 15 years,
only isolated mutations were identified in scattered
families. Recently, with the identification of a hot
spot of mutations in the C-terminal region of the
RYR1 gene, several new and recurrent mutations
have been identified in many families.3 Mutations in
this region were found in six of our nine families
with CCD, confirming this hot spot for mutation also
in Brazilian patients.
CCD has been classified mainly as an autosomaldominant disease, and three among our nine families showed a typical AD pattern of inheritance. The
recessive form of CCD is rare, with only four reported cases.5,8,18 However, we identified mutations
with this pattern of inheritance in at least two of our
nine analyzed families. It is possible that several sporadic cases will now be identified as having mutations
in the two alleles, showing that an AR pattern is in
fact not rare. Additionally, more studies of this region of the RYR1 gene may result in the identification of mutations in the second allele in cases that
were believed to have dominant inheritance. This
could explain the reduced penetrance and variable
clinical phenotype found in some families in which
individuals with one affected allele have only a susceptibility to MH whereas others, with two affected
alleles, have a severe CCD phenotype. In accordance
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with this hypothesis, Romero et al.18 described CCD
patients with a severe clinical course who were compound heterozygotes for the mutations G215E and
R614C in the RYR1 gene. Each mutation had already
been described as pathogenic, causing MH when
present in only one of the alleles.7,19 Furthermore,
some mutations that are now characterized as polymorphisms could contribute to the phenotype when
associated with other mutations.
The V4849I mutation, identified in one of our
patients, has previously been described as pathogenic in a consanguineous family with no history of
neuromuscular disorders.8 This mutation affects a
highly conserved and functionally important domain
of the RYR1 protein.8 The biopsy analysis in the
patient described by Jungbluth et al.8 showed the
presence of both mini-cores and single central cores,
and also a few rod-like structures. Although no rods
were identified in our patient with the same mutation, the pattern of the cores seems to be similar,
suggesting an association between the type of mutation and its consequence in muscle. Additionally, the
clinical picture of our patient is also similar to that in
the described family, i.e., moderate proximal weakness since childhood and predominantly axial and
proximal weakness with mild thoracolumbar scoliosis in early adulthood.8
The R4558Q and A4846V mutations, identified
in compound heterozygotes, are both novel mutations and were not found in 200 normal control
chromosomes. The alignment of the human RYR1
amino acid sequence to those of mouse, pig,
chicken, and fish, and also human RYR2 isoform,
demonstrates that the regions affected by these two
novel mutations are evolutionary conserved, adding
further evidence that they are pathogenic.
The parents of these patients are carriers of each
of these mutations in heterozygous states, but do not
have any clinical weakness. This suggests that the
R4558Q and A4846V mutations are pathogenic only
when found together.
The mild clinical course observed in the three
patients from our two AR families is surprising, since
a severe clinical course has been associated with the
AR forms of CCD. There are only two AR families
with compound heterozygotes reported in the literature18 and the patients described showed a severe
phenotype, with akinesia and hydramnion during
pregnancy. Our patients had clinical signs from
childhood and slow progression; both are in their
40s and are still ambulant.
In conclusion, although the classic pattern of
inheritance in CCD is autosomal dominant, our data
suggests that the autosomal-recessive form may be
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more common than expected. It is possible that
other mutations, previously found in asymptomatic
individuals and considered as nonpathogenic, are
responsible for muscle weakness if present in both
alleles, in homozygous or compound heterozygous
states. Considering that the parents in our two AR
families are all clinically asymptomatic heterozygous
carriers for one of the mutations, and since a significant clinical variability is observed in this disease, it
is recommended that exposure to volatile anesthetics should be avoided in such subjects because of the
possible high risk for malignant hyperthermia.
The collaboration of the following is gratefully acknowledged:
Viviane P. Muniz, Dr. Lydia U. Yamamoto, Dr. Ivo Pavanello, Dr.
Ana Cristina Cotta, Cleides Campos Oliveira, Telma Gouveia,
Luciana L. Q. Fogaça, Bruno L. Lima, Marta Cánovas, and Giselle
Izzo. This work was supported by FAPESP-CEPID and CNPq.
REFERENCES
1. Akiyama C, Nonaka I. A follow-up study of congenital nonprogressive myopathies. Brain Dev 1966;18:404 – 408.
2. Brillantes AB, Ondrias K, Scott A, Kobrinsky E, Ondriasova E,
Moschella MC, et al. Stabilization of calcium release channel
(ryanodine receptor) function by FK506-binding protein. Cell
1994;77:513–523.
3. Davis MR, Haan E, Jungbluth H, Sewry C, North K, Muntoni
F, et al. Principal mutation hotspot for central core disease
and related myopathies in the C-terminal transmembrane
region of the RYR1 gene. Neuromuscul Disord 2003;13:151–
157.
4. Dubowitz V. Muscle biopsy: a practical approach, 2nd ed.
London: Bailliere Tindall, 1985.
5. Ferreiro A, Monnier N, Romero NB, Leroy JP, Bonnemann C,
Haenggeli CA, et al. 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. Ann Neurol 2002;51:750 –759.
6. Franzini-Armstrong C, Protasi F. Ryanodine receptors of
stried muscles: complex channel capable of multiple interactions. Physiol Rev 1997;77:699 –729.
7. Gillard EF, Otsu K, Fujii J, Khanna VK, de Leon S, Derdemezi
J, et al. A substitution of cysteine for arginine 614 in the
ryanodine receptor is potentially causative of human malignant hyperthermia. Genomics 1991;11:751–755.
8. Jungbluth H, Muller CR, Halliger-Keller B, Brockington M,
Brown SC, Feng L, et al. Autosomal recessive inheritance of
RYR1 mutations in a congenital myopathy with cores. Neurology 2002;59:284 –287.
9. Loke J, MacLennan DH. Malignant hyperthermia and central
core disease: disorders of Ca2⫹ release channels. Am J Med
1998;104:470 – 486.
674
Short Reports
10. Manzur AY, Sewry CA, Ziprin J, Dubowitz V, Muntoni F. A
severe clinical and pathological variant of central core disease
with possible autosomal recessive inheritance. Neuromuscul
Disord 1998;467:467– 473.
11. Marks AR, Marx SO, Reiken S. Regulation of ryanodine receptors via macromolecular complexes. A novel role for
leucine/isoleucine zippers. Trends Cardiovasc Med 2002;12:
166 –170.
12. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic
Acids Res 1988;16:1215.
13. Monnier N, Romero NB, Lerale J, Nivoche Y, Qi D, MacLennan DH, et al. An autosomal dominant congenital myopathy
with cores and rods is associated with a neomutation in the
RYR1 gene encoding the skeletal muscle ryanodine receptor.
Hum Mol Genet 2000;9:2599 –2608.
14. Monnier N, Romero NB, Lerale J, Landrieu P, Nivoche Y,
Fardeau M, et al. Familial and sporadic forms of central core
disease are associated with mutations in the C-terminal domain of the skeletal muscle ryanodine receptor. Hum Mol
Genet 2001;2581:2581–2592.
15. Mulley JC, Kozman HM, Phillips HA, Gedeon AK, McCure JA,
Iles DE, et al. Refined genetic localization for central core
disease. Am J Hum Genet 1993;52:398 – 405.
16. Orita M, Iwahana H, Kanazawa H, Hayashi K, Sekiya T. Detection of polymorphisms of human DNA by gel electrophoresis as a single-strand conformation polymorphisms.
Proc Natl Acad Sci USA 1989;86:2766 –2770.
17. Quane KA, Healy JM, Keating KE, Manning BM, Couch FJ,
Palmucci LM, et al. Mutations in the ryanodine receptor gene
in central core disease and malignant hyperthermia. Nature
Genet 1993;5:51–55.
18. Romero NB, Monnier N, Viollet L, Cortey A, Chevallay M,
Leroy JP, et al. Dominant and recessive central core disease
associated with RYR1 mutations and fetal akinesia. Brain
2003;126:1–9.
19. Sambuughin N, Holley H, Muldoon S, Brandom BW, de
Bantel AM, Tobin JR, et al. Screening of the entire ryanodine
receptor type 1 coding region for sequence variants associated with malignant hyperthermia susceptibility in the North
American population. Anesthesiology 2005;102:515–521.
20. Scacheri PC, Hoffman EP, Fratkin JD, Semino-Mora C, Senchak A, Davis MR, et al. A novel ryanodine receptor gene
mutation causing both cores and rods in congenital myopathy. Neurology 2000;55:1689 –1696.
21. Shy GM, Magee KR. A new congenital non-progressive myopathy. Brain 1956;79:610 – 621.
22. Takeshima H, Nishimura S, Matsumoto T, Ishida H, Kangawa
K, Minamino N, et al. Primary structure and expression from
complementary DNA of skeletal muscle ryanodine receptor.
Nature 1989;439 – 445.
23. Zorzato F, Fujii J, Otsu K, Phillips M, Green NM, Lai FA, et al.
Molecular cloning of cDNA encoding human and rabbit
forms of the Ca2⫹ release channel (ryanodine receptor) of
skeletal muscle sarcoplasmatic reticulum. J Biol Chem 1990;
265:2224 –2256.
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