Brain Advance Access originally published online on April 18, 2006
Brain 2006 129(6):1470-1480; doi:10.1093/brain/awl077
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Central core disease is due to RYR1 mutations in more than 90% of patients
1 Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP) Kodaira, Tokyo 2 Department of Anesthesiology, Tokyo Rinkai Hospital Tokyo 3 Department of Anesthesiology, Saitama Medical School Saitama, Japan
Correspondence to: Ichizo Nishino, MD, PhD, Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), 4-1-1 Ogawahigashi-cho, Kodaira, Tokyo 187-8502, Japan E-mail: nishino{at}ncnp.go.jp
 |
Summary |
|---|
 Top Summary IntroductionMethodsResultsDiscussionReferences |
|---|
Ryanodine receptor 1 (RYR1) gene mutations are associated with central core disease (CCD), multiminicore disease (MmD) and malignant hyperthermia (MH), and have been reported to be responsible for 47–67% of patients with CCD and rare cases with MmD. However, to date, the true frequency and distribution of the mutations along the RYR1 gene have not been determined yet, since mutation screening has been limited to three ‘hot spots’, with particular attention to the C-terminal region. In this study, 27 unrelated Japanese CCD patients were included. Clinical histories and muscle biopsies were carefully reviewed. We sequenced all the 106 exons encoding RYR1 with their flanking exon–intron boundaries, and identified 20 novel and 3 previously reported heterozygous missense mutations in 25 of the 27 CCD patients (93%), which is a much higher mutation detection rate than that perceived previously. Among them, six were located outside the known ‘hot spots’. Sixteen of 27 (59%) CCD patients had mutations in the C-terminal ‘hot spot’. Three CCD patients had a probable autosomal recessive disease with two heterozygous mutations. Patients with C-terminal mutations had earlier onset and rather consistent muscle pathology characterized by the presence of distinct cores in almost all type 1 fibres, interstitial fibrosis and type 2 fibre deficiency. In contrast, patients with mutations outside the C-terminal region had milder clinical phenotype and harbour more atypical cores in their muscle fibres. We also sequenced two genes encoding RYR1-associated proteins as candidate causative genes for CCD: the 12 kD FK506-binding protein (FKBP12) and the
1 subunit of L-type voltage-dependent calcium channel or dihydropyridine receptor (CACNA1S). However, no mutation was found, suggesting that these genes may not, or only rarely, be responsible for CCD. Our results indicate that CCD may be caused by RYR1 mutations in the majority of patients.
Key Words: central core disease; genotype–phenotype correlation; muscular dystrophy; myopathy; ryanodine receptor 1 mutations
Abbreviations:
CACNA1S, 1 subunit of L-type voltage-dependent calcium channel; CCD, central core disease; CICR, calcium-induced calcium release; EC, excitation–contraction; FKBP12, FK506-binding protein, 12 kD; mGT, modified Gomori–Trichrome; MmD, multiminicore disease; MH, malignant hyperthermia; RYR1, ryanodine receptor 1; SERCA, sarco-endoplasmic reticulum Ca2+ ATPase; SR, sarcoplasmic reticulum
Received October 28, 2005. Revised March 1, 2006. Accepted March 8, 2006.
|
Introduction |
|---|
|
TopSummaryIntroductionMethodsResultsDiscussionReferences |
|---|
Central core disease (CCD) was the first described congenital myopathy in humans (Shy and Magee, 1956
), usually inherited in an autosomal dominant pattern, except for few reports on autosomal recessive cases (Manzur et al., 1998; Jungbluth et al., 2002). The clinical features are quite variable (Quinlivan et al., 2003), ranging from lack of visible weakness or abnormality to lack of independent ambulation. Most of the patients, in classical descriptions, have a slowly or non-progressive proximal muscle weakness and hypotonia during infancy that can persist throughout adolescence/adulthood, and have associated delayed motor development and reduced muscle bulk. In addition, musculoskeletal alterations including congenital hip dislocation, kyphoscoliosis and joint contractures are also common findings in CCD patients (Lorenzon et al., 2000; Quinlivan et al., 2003). The diagnosis is commonly made from muscle biopsy by the presence of cores in type 1 fibres, which are typically well demarcated and located centrally in the fibres. A few cores, however, can be located in the subsarcolemmal regions of individual type 1 muscle fibres. Longitudinal sections show that the core runs the whole length of the fibre. Recent reports have documented that the phenotypic presentation varies considerably from no visible disability to lack of independent ambulation. While the overall incidence of CCD is rare, the absence of symptoms in a significant number of patients may suggest that the actual incidence of CCD may be considerably higher than that perceived currently.
CCD has been linked to the gene encoding the skeletal muscle ryanodine receptor, RYR1, and is considered to be an allelic disease of malignant hyperthermia (MH) susceptibility. This is a pharmacogenetic disorder with autosomal dominant inheritance in which susceptible individuals develop generalized muscle contracture followed by a hypermetabolic state due to massive calcium release from the sarcoplasmic reticulum (SR), when they are exposed to inhaled general anaesthetics or to the depolarizing muscle relaxant succinylcholine. CCD patients have higher probability to be susceptible to MH. The RYR1 mutations linked to MH and CCD are clustered in three relatively restricted regions of the protein or ‘hot spots’: N-terminal (residues p.M1-R614), central (p.R2163–p.R2458) and C-terminal (p.R4136-p.P4973). They are also called domains 1–3, respectively (Treves et al., 2005). The first two domains are located in the soluble cytoplasmic regions of the protein. Most of the CCD mutations are clustered in domain 3, which is located in the C-terminus and comprises the transmembrane/luminal and pore-forming region of the channel (McCarthy et al., 2000; Monnier et al., 2001; Davis et al., 2003). Hence, mutations in this region of the protein may directly alter the permeation/selectivity/gating properties of the channel (Balshaw et al., 1999).
The RYR1 is one of the largest described genes in humans, spanning >159 kb in size on chromosome 19.q13.1. A 15 117-nucleotide-long open reading frame encoded in 106 exons (two of which are alternatively spliced) produces a 563 kD protein. In addition, it forms a homotetrameric structure that functions as an SR calcium-release channel regulating Ca2+ content in skeletal muscle during excitation–contraction (EC) coupling. Because of the size of the RYR1 gene, efficient routine screening for mutations has been difficult. Most of the RYR1 mutation screenings in CCD patients have been limited to the above-mentioned three ‘hot spots’ or even to the C-terminal region alone, and 47–67% patients were found carrying RYR1 mutations (Monnier et al., 2001; Davis et al., 2003; Shepherd et al., 2004), which suggested that CCD is a genetically heterogeneous disease. Here, we screened all the exons and flanking exon–intron boundaries of RYR1 in order to determine the frequency and distribution of mutations, and describe the genotype–phenotype correlation in Japanese patients with CCD.
Although no mutation in other genes has been associated with CCD, many studies have shown that mutants of the RYR1-associated proteins FKBP12 and CACNA1S cause EC uncoupling in vitro just as some RYR1 mutants do (Avila et al. 2003b; Lyfenko et al., 2004; Weiss et al., 2004), raising a possibility that FKBP12 and/or CACNA1S mutations may also be responsible for CCD. In addition, the mutations in the RYR1-binding domain of CACNA1S are thought to account for 1% of MH patients (Stewart et al., 2001). We therefore sequenced the entire open reading frame of FKBP12 and the part of CACNA1S that encodes the RYR1-binding region.
|
Methods |
|---|
|
TopSummaryIntroductionMethodsResultsDiscussionReferences |
|---|
Subjects
Unrelated Japanese CCD patients were selected for the study from the National Center of Neurology and Psychiatry (NCNP) database from 1982 to 2004. CCD diagnosis was established on the basis of characteristic muscle pathology findings of cores almost exclusively in type 1 fibres. We excluded multiminicore disease (MmD) cases, which had multiple cores in >70% of type 1 fibres in our series. Available blood samples from the patients' relatives were also included in the analysis. In addition, DNA samples from 150 subjects without any known muscle disease were studied. Informed consent was obtained from the patients or their parents, as well as from control subjects.
The patients' clinical features were assessed by careful review of their medical records. Pathological features of all patients were independently evaluated by four authors (S.W., M.C.V.M., I.N. and I.N.). All patients have undergone a battery of histochemical stains, including haematoxylin and eosin, modified Gomori–trichrome (mGT), NADH-tetrazolium reductase (NADH-TR) and myosin ATPase.
In order to evaluate the genotype–phenotype correlation in CCD patients, the patients were divided into four groups, respectively, provided that they had one heterozygous C-terminal mutation, one heterozygous non-C-terminal mutation, two heterozygous mutations or no mutation.
Data were entered in Statistics Software for Social Sciences (SPSS version 11.0). Demographic characteristics were analysed by computation of the frequency, the mean ± standard deviation (SD), or the mean ± standard error of means (SEM), whichever was appropriate. The data then were subjected to a univariate analysis (Fisher's exact test). For comparing age and type 2 fibre deficiency, Mann–Whitney test was employed.
Mutational analysis
Genomic DNA was extracted from either muscle biopsy samples or peripheral blood lymphocytes according to standard protocols (Sambrook et al., 2001). PCR primers were designed to amplify all 106 exons of RYR1, all five exons of FKBP12 and the seven exons of CACNA1S (exon 14–17 and 25–27) that encode the RYR1-interacting region (Peng et al., 1998). Amplified fragments were directly sequenced using BigDye Terminator® v3.1 Cycle Sequencing kits on ABI3100 automated Genetic Analyzer (Applied BiosystemsTM, USA). DNA sequences were analysed with the SeqScape program and compared with the reference genomic sequence of: RYR1 (Genbank J05200), FKBP12 (Genbank M92423) and CACNA1S (Genbank L33798).
|
Results |
|---|
|
TopSummaryIntroductionMethodsResultsDiscussionReferences |
|---|
Patients
A total of 27 unrelated Japanese CCD patients were selected for the study, consisting of 7 males and 20 females (Table 1) ranging in age from 2 to 63 years [29 ± 19 years (mean ± SD)] at the time of muscle biopsy. DNA from the parents of two patients (Patients 23 and 25) were also studied.
|
Patients 1–8 underwent muscle biopsy because they had pertinent family history of MH and were considered to be MH susceptible. In Japan, the diagnosis of MH susceptibility is achieved by detecting enhancement in the rate of calcium-induced calcium release (CICR) from the sarcoplasmic reticulum in chemically skinned muscle fibres (Ibarra et al., 2005
), reflecting the underlying pathomechanism of this disorder, that is, a lower activation threshold of the SR calcium-release channel. Cores were incidentally found in the muscle biopsies of Patients 1–8; thus a working diagnosis of CCD was made despite the absence of any muscle symptoms in most of these patients. CICR rate was enhanced in all eight patients. There was no description about the MH susceptibility status on the medical records of other subjects (Patients 9–27). Nemaline bodies were identified in Patients 11, 12 and 23 on mGT stain; hence they were labelled as having core/rod disease (CRD).
Mutations
A total of 23 different missense mutations affecting 19 residues in RYR1 were identified, consisting of 3 previously reported and 20 newly identified missense mutations in 93% of CCD patients (Tables 1 and 2). Two patients did not have any mutation in RYR1. All mutations were heterozygous single nucleotide changes, except for the substitution of two consecutive nucleotides (c.14761_14762TT>AC) in Patient 21, which was predicted to result in a single amino acid change (p.F4921T). There were two common heterozygous RYR1 mutations in this cohort: c.14581C>T (p.R4861C) and c.7522C>G (p.R2508C), identified in 4 out of 25 (16%) and in 3 out of 25 (12%) patients, respectively. No mutations were found in either FKBP12 or CACNA1S.
|
Twenty-two of 25 (88%) had only one heterozygous mutation: 14 (56%) with a heterozygous C-terminal mutation and 8 (32%) with a heterozygous non-C-terminal mutation. The remaining three (12%) patients had two heterozygous mutations (Patients 23, 24 and 25). Only the parents of Patients 23 and 25 were screened for the respective pair of mutations, as samples were not available from the parents of Patient 24. In Patient 23, the mutation p.D60N was identified in exon 3 of her maternal allele while p.L3606P was found in exon 73 of her paternal allele. In Patient 25, p.E512K in exon 14 was inherited from her mother while p.R4893P in exon 102 was acquired from her father. No muscle samples were available for histological studies from the parents of these two patients. RYR1 mutations were found in patients with CRD (Patients 11, 12 and 23) (Table 1, Fig. 1). None of the newly identified missense mutations were found in 300 control chromosomes. Eighteen of 19 amino acids predicted to be changed were highly conserved through RYR1 evolution and most of them were also conserved across the RYR species, RYR1, RYR2 and RYR3 (Table 2).
|
The mutations found in this cohort of CCD patients are shown in Fig. 2 at their respective location in the RYR1, along with those reported previously. In this study, 6 mutations were located outside the mutational ‘hot spots’, 4 in N-terminal ‘hot spot’ and 13 in the C-terminal region. These mutations were carried by 8 out of 25 (32%), 4 out of 25 (16%) and 16 out of 25 (59%) patients, respectively. We did not find any mutation in the central ‘hot spot’ or domain 2 in our cohort.
|
Genotype–phenotype correlation
Among eight patients with a heterozygous non-C-terminal mutation, two showed mild limb muscle weakness while the others were asymptomatic. The exact age of onset was difficult to ascertain because of the paucity of symptoms. Significant clinical findings during pregnancy and birth were not reported in this group. On muscle histochemistry, type 2 fibre deficiency was seen in only one patient, as he had <1% of type 2 fibres, while these were >12% in others [37 ± 4% (mean ± SD)]. Minimal endomysial fibrosis was found in three patients (37%), while the others had no increase in the interstitial fibrous tissue.
In contrast, limb muscle weakness was present in all patients with C-terminal mutations, and significant symptoms were manifested during the perinatal period (Table 1). There was statistically significant association between the presence of mutation in the C-terminal domain and clinical phenotypic characteristic in the following categories: limb muscle weakness, poor foetal movement during pregnancy, presence of joint dislocation and delayed motor milestone (Fig. 3A). Endomysial fibrosis and type 2 fibre deficiency were observed in most, if not all, patients with C-terminal mutations (Fig. 3B).
|
In terms of core structure, most patients had single cores (95% of fibres), but multiple cores were also seen especially in patients with heterozygous non-C-terminal mutations. In mutations outside domain 3, most cores were located in the periphery or subsarcolemmal areas (71%). In patients with C-terminal mutations, cores were noted to be characteristic: these were ovoid in shape and with clearly demarcated borders, and were predominantly located in the centre of the fibres [60 ± 7% (mean ± SEM)]; they also occurred singly (98 ± 4%). In addition, most of the cores (72 ± 5%) in this group appeared to be ‘rimmed’ (Fig. 4B), which may connote high enzymatic activities around the cores on NADH-TR stain. More cores in type 1 fibres were also noted (90 ± 4%), but in general the percentage of the fibres with cores varied from 3 to 100%. No correlation was seen between clinical severity and the percentage of cores in fibres.
|
Some fibres show ‘atypical’ cores, characterized by indistinct borders and whose shapes were inexplicitly ovoid (Fig. 4E). These were seen more in non-C-terminal mutations but no statistical significance was observed as compared with C-terminal mutations (Fig. 3B). Like in typical core structures, these were also noted to be either in the centre of the fibre or in the subsarcolemmal areas.
In Patients 1–8, from whom muscle biopsy was performed for CICR and cores were incidentally identified, all had non-C-terminal heterozygous mutations.
The clinical features of patients with two heterozygous mutations were more similar to those of the group with one heterozygous C-terminal mutation. In terms of muscle pathology, type 2 fibre deficiency was likewise seen (Table 2), similar to mutations involving the C-terminal region. This idea may also be buttressed by the fact that among a total of three patients with CRD, two carried a single C-terminal mutation, while the other had a compound heterozygous mutation. In terms of core pathology, however, they resemble that of non-C-terminal mutations: the cores were seen in 14 and 49% of type 1 fibres in Patients 24 and 25, respectively; and the characteristic ‘rimming’ of cores is less appreciated (Fig. 1).
In two patients in whom no mutation was found, the cores had peculiar features distinguishable from that seen in patients with identifiable mutations. Particularly, in Patient 26, the cores, albeit located centrally, occupied almost the whole diameter of the fibre, and, notably, the subsarcolemmal area around the cores had increased oxidative activity (Fig. 5). The cores in Patient 27 were initially considered to be regular cores but a closer look has shown that almost all the cores had indistinct borders; moreover, only <20% of type 1 fibres have cores (Fig. 5). In such cases, the term ‘core-like’ may be more appropriate to describe these structures.
|
|
Discussion |
|---|
|
TopSummaryIntroductionMethodsResultsDiscussionReferences |
|---|
RYR1 mutations in CCD
At least 44 reported RYR1 mutations have been associated with CCD, including 39 missense mutations and 5 deletions (Quane et al., 1993
; Zhang et al., 1993; Fletcher et al., 1995; Manning et al., 1998; Lynch et al., 1999; Monnier et al., 2000, 2001; Avila and Dirksen, 2001; Tilgen et al., 2001; Ferreiro et al., 2002; Jungbluth et al., 2002; Robinson et al., 2002; Sewry et al., 2002; Avila et al., 2003a; Quinlivan et al., 2003; Zorzato et al., 2003; Shepherd et al., 2004). All these mutations are clustered in the three ‘hot spots’, except for two (Fig. 2). RYR1 mutations have been reported to be responsible for 47–67% of patients suffering from CCD, implying that the disease is genetically heterogeneous (Vainzof et al., 2000; Monnier et al., 2001). However, neither the true frequency nor the distribution of CCD-causative mutations has been accurately determined to date, since mutation screening has been limited to the three ‘hot spots’ of the RYR1, or even only to the C-terminal region. According to in vitro studies, two RYR1-binding proteins, FKBP12 and CACNA1S, directly participate in or modulate EC coupling, while EC uncoupling is thought to be the linchpin in the pathogenesis of CCD (Avila et al., 2001; Lyfenko et al., 2004). In addition, mutations have been identified in RYR1-binding region of CACNA1S in 1% of patients with MH. We therefore regarded FKBP12 and CACNA1S genes as rational candidates for CCD.
We screened 27 Japanese patients with CCD, diagnosed on the basis of histological findings, without putting much emphasis on their clinical presentation as inclusion criteria, and found that RYR1 mutations occur in 93% (25 out of 27) of CCD patients, which is a much higher rate than that thought previously. The most common CCD mutation in our cohort was c.14581C>T (p.R4861C). Interestingly, the c.14582G>A (p.R4861H) mutation that affects the same amino acid is the most common CCD mutation in European countries, while it was identified in only one patient from our series. Almost all reported patients bearing c.14582G>A had a positive family history, but all patients carrying mutation c.14581C>T were sporadic in this study and also in the literature (Monnier et al., 2001; Tilgen et al., 2001; Davis et al., 2003), suggesting that the 14 581 nucleotide might be susceptible to change.
To our knowledge, this is the first endeavour to screen the entire coding region of RYR1 in a cohort of CCD patients. By screening the C-terminal region alone, we would have found RYR1 mutations only in 59% (16 out of 27) of CCD patients, which is consistent with detection rates in previous reports, missing the two compound heterozygous mutations. Even extending the mutation screening to the three ‘hot spots’, we would have found RYR1 mutations in only 67% (18 out of 27), also missing one compound heterozygous mutation. It thus becomes necessary to screen the entire RYR1 coding region in CCD patients, however impractical it may be owing to the size of the gene. Interestingly, most of mutations outside the ‘hot spots’ were localized in exons 47 and 48, which neighbour the central region ‘hot spot’ (exons 39–46). Hence, mutation detection rate in CCD patients could be increased up to 89% by including exons 47 and 48, which may be a more practical alternative than sequencing the entire RYR1 gene. In addition, FKBP12 and CACNA1S may not be causative genes for CCD or an extremely rare, if they are, since there was no mutation in FKBP12 or in the RYR1-binding region of CACNA1S genes.
We did not find any mutation in two patients in our cohort, suggesting that CCD may still be genetically heterogeneous even though there still remains the possibility that mutations may exist in unexamined regions, such as promoter region and introns, or that we may have overlooked a mutation. Notwithstanding this likelihood of other probabilities, the pathological characteristics of these patients are clearly different from the rest. If these patients were excluded from the analysis solely on the basis of the ‘uncharacteristic’ core-like structures, that is, if the inclusion criteria used were more stringent, the detection rate of mutations involving the RYR1 gene will considerably and significantly increase to 100%.
Probable autosomal recessive CCD
CCD was once thought to be inherited solely via an autosomal dominant mechanism, but actually rare instances of recessive inheritance have also been identified (Manzur et al., 1998; Ferreiro et al., 2002; Romero et al., 2003).
In this study, sequencing of the entire RYR1 coding regions led to the identification of three patients (Patients 23, 24 and 25) with two heterozygous mutations. Patients 24 and 25 needed respiratory mechanical assistance after delivery, while their parents were completely healthy and without any skeletal abnormalities. In both patients, each of the two heterozygous mutations was, respectively, found in each of the parents, confirming that these patients had a compound heterozygous mutation. In Patient 25, p.R4893P affected the third residue of a very well conserved GVRAGGGIGD luminal motif (amino acids p.G4891-D4900) that has been proposed to be a pore-forming fragment responsible for the electrophysiological characteristics of the channel (Zhao et al., 1999).
The parents of Patients 23 and 25 were asymptomatic, while they had heterozygous mutation, suggesting the recessive nature of these mutations. Since CCD patients could be clinically asymptomatic, however, we may not be able to completely exclude the possibility that their parents could have CCD. Nevertheless, judging from the clinical features and the mutation data, autosomal recessive mode of inheritance is most likely in these particular patients. Patient 24 also carried two heterozygous mutations (p.S427L in exon13, p.G4899E in exon 102), but further analysis was inevitably limited by the unavailability of samples from her parents, hampering full evaluation if this was indeed an autosomal recessive case. The p.G4899E mutation also affected the very well conserved GVRAGGGIGD luminal motif (G4899 underlined). Interestingly, this mutation has been reported in two papers as a causative heterozygous mutation (Monnier et al., 2001; Romero et al., 2003); however, since only the ‘hot spots’ were screened in these studies, the presence of another mutation is still possible and hence it might be impetuous to conclude that p.G4899E mutation is a causative dominant mutation.
Genotype–phenotype correlation
In general, CCD has a wide spectrum of phenotypic expression, ranging from the apparent absence of symptoms to the presence of perinatal complications and generalized muscle weakness. Mutations in the C-terminal region seem to be associated with certain clinical and pathological features: hypotonia during infancy, delayed motor development and limb muscle weakness; type 2 fibre deficiency and interstitial fibrosis; and characteristic cores with clearly demarcated borders, which are observed in almost all type 1 muscle fibres. In addition, ‘rimming’ on the borders of these cores is observed in much higher frequency; similarly, this phenomenon was also noted in the biopsy specimens of three families determined to have mutation in domain 3 (Sewry et al., 2002). These unique features therefore delineate C-terminal mutations from other groups, at least in terms of muscle pathology.
In contrast, most of CCD patients harbouring at least one mutation outside the C-terminal region had only mild musculoskeletal abnormalities such as joint contractures and scoliosis. This phenomenon may be explained by the leaky-channel model and EC uncoupling model. Some non-C-terminal mutations in RYR1 promote the leak of Ca2+ ions from the SR that may or may not be compensated by the activity of the sarco-endoplasmic reticulum Ca2+ ATPase (SERCA), resulting in an elevation of resting cytosolic Ca2+ and a depletion of SR Ca2+ stores. On the other hand, C-terminal mutations, especially those in the pore region of RYR1, may directly affect the channel gating properties, resulting in an abolition of orthograde activation by the voltage-gated L-type Ca2+ channel or, in other words, EC uncoupling. However, there is no compensatory mechanism to increase Ca2+ release as SERCA pumps do in the leaky model (Tong et al., 1999; Avila et al., 2001, 2003b; Lyfenko et al., 2004). This may explain why C-terminal mutations are associated with clinically evident muscle weakness.
Interestingly, all patients with enhanced CICR had a non-C-terminal heterozygous mutation. Although we did not measure CICR in patients with C-terminal mutations, our results may suggest that majority of CCD patients with MH susceptibility might have a non-C-terminal mutation, considering the facts that no patient with C-terminal mutation had a family history of MH susceptibility, and that one C-terminal mutation, p.I4898T, has been associated with normal halothane and caffeine sensitivities by an in vitro study (Lynch et al., 1999). In a recent study made by Monnier et al. (2005), where individuals from IVCT-confirmed MH-susceptible families have been screened for RYR1 mutations and CCD, some MH-normal patients had cores in type 1 fibres albeit the absence of mutation. It would thus be interesting to screen the whole RYR1 in these patients, as only selected exons were included for genetic screening. In our study, however, we definitely cannot assume that patients with C-terminal mutations may have normal CICR test, as further evaluations are necessary. Furthermore, in only such patients, multiple cores were seen in addition to central cores. Thus, non-C-terminal heterozygous mutations may be more associated with multiple cores and MH susceptibility, although the number of patients is too small to enable us to draw a conclusion. Previous reports have asserted that exclusive MH susceptible patients have mutations in the C-terminal region, but there is only limited study on the histopathological evaluation of these patients, revealing the presence of cores; it is also important to stress that these cores are rather not characteristic of CCD (Ibarra et al., 2006).
We have shown that the muscle pathology on the patients may differ among those with C-terminal mutations and those with mutations outside this area. Cores, moreover, were not only located in the centre of the muscle fibres but also in subsarcolemmal or peripheral areas, and atypical cores can also be seen in such cases, indicating that the possibility of RYR1 mutation cannot be excluded in patients with such atypical cores. Here, atypical cores of CCD were still different from the cores of MmD, for most of the cores were clearly demarcated and only some fibres appeared to have more than one core; nevertheless, histological, clinical, genetic features and mode of inheritance overlap between CCD and MmD, making the boundary of these two diseases blurred (Lyfenko et al., 2004; Mathews et al., 2004). Patients 24 and 25, who possessed double mutations, had pathology akin to characteristics of both C- and non-C-terminal mutations: type 2 fibre deficiency is seen, and cores are rather atypical and are only seen in moderate number on type 1 fibres. This may, in part, be explained by the fact that these patients carry mutations in and outside the C-terminal region.
In the past, we may have been biased in interpreting the phenotype–genotype correlation between CCD and RYR1 mutations, since mutation screening had been limited to the ‘hot spots’. Quane et al. (1993) claimed that various phenotypes could be observed even among patients with the same mutation. However, their patients may have had a compound heterozygous mutation and one of the mutations may have been overlooked. In fact, if we had just screened the RYR1 C-terminal region of patients with two heterozygous mutations (Patients 24 and 25), we would have found only one mutation, giving an impression of remarkable phenotypic difference between the patients and their healthy parents, regardless of the presence of the same ‘heterozygous’ mutation.
In summary, our comprehensive mutation screening revealed a surprisingly higher detection rate of RYR1 mutations in CCD patients than that revealed in previous reports, which accounts for >90% of the cases. Our results also suggest the possibility that many RYR1 mutations may have been overlooked by the regularly used screening methods that cover only three ‘hot spots’. At least, exons 47 and 48 should be included into the hot spot screening if a comprehensive mutation analysis of RYR1 is not feasible. In terms of muscle pathology, patients with mutations involving the C-terminal region have characteristic cores that can be easily distinguished from those with mutations outside this domain, and also from those patients without mutation in the RYR1. Needless to say, the pathomechanism of how these cores develop still remains to be elucidated.
|
Acknowledgements |
|---|
The authors thank the patients, their family and physicians for their cooperation. This study is supported partly by the ‘Research on Health Sciences focusing on Drug Innovation’ from the Japanese Health Sciences Foundation; partly by the ‘Research on Psychiatric and Neurological Diseases and Mental Health’ of ‘Health and Labour Sciences Research Grants’ and the ‘Research Grant (17A-10) for Nervous and Mental Disorders’, from the Ministry of Health, Labour and Welfare; partly by the ‘Grant-in-Aid for Scientific Research’ from the Japan Society for the Promotion of Science; and partly by the ‘Program for Promotion of Fundamental Studies in Health Sciences’ of the National Institute of Biomedical Innovation (NIBIO).
|
References |
|---|
|
TopSummaryIntroductionMethodsResultsDiscussionReferences |
|---|
Avila G and Dirksen RT. (2001) Functional effects of central core disease mutations in the cytoplasmic region of the skeletal muscle ryanodine receptor. J Gen Physiol 118:277–90.
Avila G, O'Brien J, Dirksen RT. (2001) Excitation-contraction uncoupling by a human central core disease mutation in the ryanodine receptor. Proc Natl Acad Sci USA 98:4215–20.
Avila G, O'Connell KM, Dirksen RT. (2003a) The pore region of the skeletal muscle ryanodine receptor is a primary locus for excitation-contraction uncoupling in central core disease. J Gen Physiol 121:277–86.
Avila G, Lee E, Perez CF, Allen PD, Dirksen RT. (2003b) FKBP12 binding to RYR1 modulates excitation-contraction coupling in mouse skeletal myotubes. J Biol Chem 278:22600–8.
Balshaw D, Gao L, Meissner G. (1999) Luminal loop of the ryanodine receptor: a pore-forming segment? Proc Natl Acad Sci USA 96:3345–7.
Davis MR, Haan E, Jungbluth H, Sewry C, North K, Muntoni F, et al. (2003) Principal mutation hotspot for central core disease and related myopathies in the C-terminal transmembrane region of the RYR1 gene. Neuromuscul Disord 13:151–7.[CrossRef][ISI][Medline]
Ferreiro A, Monnier N, Romero NB, Leroy JP, Bonnemann C, Haenggeli CA, et al. (2002) 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 51:750–9.[CrossRef][ISI][Medline]
Fletcher JE, Tripolitis L, Hubert M, Vita GM, Levitt RC, Rosenberg H. (1995) Genotype and phenotype relationships for mutations in the ryanodine receptor in patients referred for diagnosis of malignant hyperthermia. Br J Anaesth 75:307–10.
Ibarra MCA, Ichihara Y, Hikita M, Yoshida K, Junji S, Maehara Y, et al. (2005) Effect of bupivacaine enantiomers on Ca2+ release from sarcoplasmic reticulum in skeletal muscle. Eur J Pharmacol 512:77–83.[Medline]
Ibarra MCA, Wu S, Murayama K, Minami N, Ichihara Y, Kikuchi H, et al. (2006) Malignant hyperthermia in Japan: mutation screening of the entire ryanodine receptor type 1 gene coding region by direct sequencing. Anesthesiology In press.
Jungbluth H, Muller CR, Halliger-Keller B, Brockington M, Brown SC, Feng L, et al. (2002) Autosomal recessive inheritance of RYR1 mutations in a congenital myopathy with cores. Neurology 59:284–7.
Leong P and MacLennan DH. (1998) The cytoplasmic loops between domains II and III and domains III and IV in the skeletal muscle dihydropyridine receptor bind to a contiguous site in the skeletal muscle ryanodine receptor. J Biol Chem 273:29958–64.
Lorenzon NM and Beam KG. (2000) Calcium channelopathies. Kidney Int 57:794–802.[CrossRef][Medline]
Lyfenko AD, Goonasekera SA, Dirksen RT. (2004) Dynamic alterations in myoplasmic Ca2+ in malignant hyperthermia and central core disease. Biochem Biophys Res Commun 322:1256–66.[CrossRef][ISI][Medline]
Lynch PJ, Tong J, Lehane M, Mallet A, Giblin L, Heffron JJ, et al. (1999) A mutation in the transmembrane/luminal domain of the ryanodine receptor is associated with abnormal Ca2+ release channel function and severe central core disease. Proc Natl Acad Sci USA 96:4164–9.
Magee KR and Shy GM. (1956) A new congenital non-progressive myopathy. Brain 79:610–21.
Manning BM, Quane KA, Ording H, Urwyler A, Tegazzin V, Lehane M, et al. (1998) Identification of novel mutations in the ryanodine-receptor gene (RYR1) in malignant hyperthermia: genotype–phenotype correlation. Am J Hum Genet 62:599–609.[CrossRef][ISI][Medline]
Manzur AY, Sewry CA, Ziprin J, Dubowitz V, Muntoni F. (1998) A severe clinical and pathological variant of central core disease with possible autosomal recessive inheritance. Neuromuscul Disord 8:467–73.[CrossRef][ISI][Medline]
Mathews KD and Moore SA. (2004) Multiminicore myopathy, central core disease, malignant hyperthermia susceptibility, and RYR1 mutations: one disease with many faces? Arch Neurol 61:27–9.
McCarthy TV, Quane KA, Lynch PJ. (2000) Ryanodine receptor mutations in malignant hyperthermia and central core disease. Hum Mutat 15:410–7.[CrossRef][ISI][Medline]
Monnier N, Romero NB, Lerale J, Nivoche Y, Qi D, MacLennan DH, et al. (2000) 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 9:2599–608.
Monnier N, Romero NB, Lerale J, Landrieu P, Nivoche Y, Fardeau M, et al. (2001) 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 10:2581–92.
Monnier N, Kozak-Ribbens G, Krivosic-Horber R, Nivoche Y, Qi D, Kraev N, et al. (2005) Correlations between genotype and pharmacological, histological, functional, and clinical phenotypes in malignant hyperthermia susceptibility. Hum Mutat 26:413–25.[CrossRef][ISI][Medline]
Quane KA, Healy JM, Keating KE, Manning BM, Couch FJ, Palmucci LM, et al. (1993) Mutations in the ryanodine receptor gene in central core disease and malignant hyperthermia. Nat Genet 5:51–5.[CrossRef][ISI][Medline]
Quinlivan RM, Muller CR, Davis M, Laing NG, Evans GA, Dwyer J, et al. (2003) Central core disease: clinical, pathological, and genetic features. Arch Dis Child 88:1051–5.
Robinson RL, Brooks C, Brown SL, Ellis FR, Halsall PJ, Quinnell RJ, et al. (2002) RYR1 mutations causing central core disease are associated with more severe malignant hyperthermia in vitro contracture test phenotypes. Hum Mutat 20:88–97.[CrossRef][ISI][Medline]
Romero NB, Monnier N, Viollet L, Cortey A, Chevallay M, Leroy JP, et al. (2003) Dominant and recessive central core disease associated with RYR1 mutations and fetal akinesia. Brain 126:2341–9.
Sambrook J and Russell DW. (2001) Molecular cloning: a laboratory manual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
Sewry CA, Muller C, Davis M, Dwyer JS, Dove J, Evans G, et al. (2002) The spectrum of pathology in central core disease. Neuromuscul Disord 12:930–8.[CrossRef][ISI][Medline]
Shepherd S, Ellis F, Halsall J, Hopkins P, Robinson R. (2004) RYR1 mutations in UK central core disease patients: more than just the C-terminal transmembrane region of the RYR1 gene. J Med Genet 41:e33.
Shuaib A, Paasuke RT, Brownell KW. (1987) Central core disease. Clinical features in 13 patients. Medicine (Baltimore) 66:389–96.[Medline]
Stewart SL, Hogan K, Rosenberg H, Fletcher JE. (2001) Identification of the Arg1086His mutation in the alpha subunit of the voltage-dependent calcium channel (CACNA1S) in a North American family with malignant hyperthermia. Clin Genet 59:178–84.[CrossRef][ISI][Medline]
Tilgen N, Zorzato F, Halliger-Keller B, Muntoni F, Sewry C, Palmucci LM, et al. (2001) Identification of four novel mutations in the C-terminal membrane spanning domain of the ryanodine receptor1association with central core disease and alteration of calcium homeostasis. Hum Mol Genet 10:2879–87.
Tong J, McCarthy TV, MacLennan DH. (1999) Measurement of resting cytosolic Ca2+ concentrations and Ca2+ store size in HEK-293 cells transfected with malignant hyperthermia or central core disease mutant Ca2+ release channels. J Biol Chem 274:693–702.
Treves S, Anderson AA, Ducreux S, Divet A, Bleunven C, Grasso C, et al. (2005) Ryanodine receptor 1 mutations, dysregulation of calcium homeostasis and neuromuscular disorders. Neuromuscul Disord 15:577–87.[CrossRef][ISI][Medline]
Vainzof M, Muniz VP, Tsanaclis AM, Silva HC, Rusticci MS. (2000) Does the A3333G mutation in the CACNL1A3 gene, detected in malignant hyperthermia, also occur in central core disease? Genet Test 4:383–6.[Medline]
Weiss RG, O'Connell KM, Flucher BE, Allen PD, Grabnerm M. (2004) Dirksen RT. Function analysis of the R1086H malignant hyperthermia mutation in the DHPR reveals an unexpected influence of the III-IV loop on skeletal muscle EC coupling. Am J Physiol Cell Physiol 287:1094–102.
Zhang Y, Chen HS, Khanna VK, De Leon S, Phillips MS, Schappert K, et al. (1993) A mutation in the human ryanodine receptor gene associated with central core disease. Nat Genet 5:46–50.[CrossRef][ISI][Medline]
Zhao M, Li P, Li X, Zhang L, Winkfein RJ, Chen SR. (1999) Molecular identification of the ryanodine receptor pore-forming segment. J Biol Chem 274:25971–4.
Zorzato F, Yamaguchi N, Xu L, Meissner G, Muller CR, Pouliquin P, et al. (2003) Clinical and functional effects of a deletion in a COOH-terminal lumenal loop of the skeletal muscle ryanodine receptor. Hum Mol Genet 12:379–88.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  L. Morrison Unraveling RYR1 mutations and muscle biopsies Neurology, January 8, 2008; 70(2): 99 - 100. [Full Text] [PDF]
|
|
|
|
|
|
I. Sato, S. Wu, M. C. A. Ibarra, Y. K. Hayashi, H. Fujita, M. Tojo, S. J. Oh, I. Nonaka, S. Noguchi, and I. Nishino Congenital neuromuscular disease with uniform type 1 fiber and RYR1 mutation Neurology, January 8, 2008; 70(2): 114 - 122. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Zhou, H. Jungbluth, C. A. Sewry, L. Feng, E. Bertini, K. Bushby, V. Straub, H. Roper, M. R. Rose, M. Brockington, et al. Molecular mechanisms and phenotypic variation in RYR1-related congenital myopathies Brain, August 1, 2007; 130(8): 2024 - 2036. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||