Human Molecular Genetics, 2000, Vol. 9, No. 18 2599-2608
© 2000 Oxford University Press
An autosomal dominant congenital myopathy with cores and rods is associated with a neomutation in the RYR1 gene encoding the skeletal muscle ryanodine receptor
1Laboratoire de Biochimie de l’ADN, CHU Grenoble, Grenoble, France, 2INSERM U523 and Institut de Myologie, Hôpital de la Salpétrière, Paris, France, 3Département d’Anesthésie, Hôpital Robert Debré, Paris, France, 4Banting and Best Department of Medical Research, University of Toronto, Toronto, Canada and 5Laboratoire BECP/DBMS, CEA 2943 UJF–CEA, 17 rue des Martyrs, 38054 Grenoble Cedex 09, France
Received 26 June 2000; Revised and Accepted 13 September 2000.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Central core disease (CCD) and nemaline myopathy (NM) are congenital myopathies for which differential diagnosis is often based on the presence either of cores or rods. Missense mutations in the skeletal muscle ryanodine receptor gene (RYR1) have been identified in some families with CCD. Mutations in the
-tropomyosin and -actin genes have been associated with most dominant forms of NM. Analysis of the RYR1 cDNA in a French family identified a novel Y4796C mutation that lies in the C-terminal channel-forming domain of the RyR1 protein. This mutation was linked not only to a severe and penetrant form of CCD, but also to the presence of rods in the muscle fibres and to the malignant hyperthermia susceptibility (MHS) phenotype. The Y4796C mutation was introduced into a rabbit RYR1 cDNA and expressed in HEK-293 cells. Expression of the mutant RYR1 cDNA produced channels with increased caffeine sensitivity and a significantly reduced maximal level of Ca2+ release. Single-cell Ca2+ analysis showed that the resting cytoplasmic level was increased by 60% in cells expressing the mutant channel. These data support the view that the rate of Ca2+ leakage is increased in the mutant channel. The resulting chronic elevation in myoplasmic concentration is likely to be responsible for the severe expression of the disease. Haplotyping analysis indicated that the mutation arose as a neomutation in the proband. This first report of a neomutation in the RYR1 gene has strong implications for genetic linkage studies of MHS or CCD, two diseases characterized by a genetic heterogeneity. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Central core disease (CCD) and nemaline myopathy (NM) are congenital myopathies which are often difficult to distinguish at a clinical level, so the diagnosis is based on typical histological features. CCD is an autosomal dominant congenital myopathy first described by Shy and Magee (1). The disease is non-progressive or slowly progressive and presents with diffuse muscular weakness and hypotonia during infancy, delayed motor development and reduced muscle bulk. Muscle weakness of the lower extremities is frequently the leading manifestation. CCD exhibits great variability both clinically and histologically and the severity of symptoms may vary from normal to severe (2). Histological examination of CCD muscles shows the presence of central areas (cores) mainly in type 1 muscle fibres. These cores are clearly delineated from the normally structured zones of the muscle fibres by a sharp border and are characterized by lack of oxidative activity, sarcomeric disorganization, Z line streaming and proliferation of sarcotubular membranes.
CCD patients are at risk for malignant hyperthermia susceptibility (MHS), an autosomal dominant pharmacogenetic disorder of skeletal muscle Ca2+ regulation (3). Genetic studies have shown that both MHS (4) and CCD (5,6) can be caused by mutations of the ryanodine receptor gene (RYR1) located on chromosome 19q13.1 (7,8). The RYR1 gene encodes a skeletal muscle calcium release channel located in the junctional terminal cisternae of the sarcoplasmic reticulum membrane. The N-terminal part of the protein forms a large myoplasmic domain that faces and interacts with the dihydropyridine receptor, a voltage-dependent, dihydropyridine-sensitive calcium channel located in the plasmalemma and transverse tubules. All 23 MHS mutations in RYR1 that have been reported to date are localized in the cytoplasmic domain (9,10). Among them, seven mutations have been reported to be responsible for both MHS and CCD.
Because expression of the CCD phenotype is variable, the CCD diagnosis was based on histological signs as well as on clinical expression of the disease. The MHS and CCD mutants could not be distinguished on the basis of their sensitivity to caffeine and halothane when expressed in a heterologous system (11), but CCD mutant channels appeared to be more permeable than MHS mutant channels, raising resting Ca2+ concentrations and depleting Ca2+ stores (12). Recently, a mutation in the C-terminal end of the RyR1 protein that forms the transmembrane channel domain was reported to be responsible for a highly penetrant, clinically severe form of CCD in a large Mexican pedigree (13). MHS was not clearly associated with the CCD phenotype in this family. This CCD mutant channel also appeared to be highly permeable, supporting the view that this and other CCD mutations might be distinguished from MHS mutations by their high permeability and raising the possibility that MHS in this family might have been aborted by a diminished Ca2+ store.
Nemaline myopathy is a congenital myopathy (14). The major symptoms are slowly progressive weakness, delayed motor milestones and skeletal deformities. NM shows a large spectrum of clinical severity, ranging from almost asymptomatic adult patients with mild proximal weakness to newborns presenting with severe neonatal hypotonia, deformities and respiratory failure, leading to death during the first days of life (15). The main abnormal histological feature of NM is the presence of numerous rods that either form clusters at the periphery of the muscle fibres or disseminate throughout the fibres. -tropomyosin (TPM3) and skeletal -actin (ACTA1) genes have been demonstrated to be responsible for dominant forms of NM, whereas most of the autosomal recessive severe forms of NM are associated with nebuline gene mutations (16). The TPM3 gene, which encodes a sarcoplasmic isoform of -tropomyosin expressed in slow-twitch or type I muscle fibres, has been localized to chromosome 1q21–q23. Mutations in the TPM3 gene have been associated with both dominant and recessive nemaline myopathies (17,18). The ACTA1 gene, mapped to chromosome 1q42, encodes the skeletal muscle -actin protein. The spectrum of clinical phenotypes of dominant forms of NM associated with mutations in the ACTA1 gene range from mild to very severe lethal forms of the disease (19).
In an early study (20), characteristic rods and cores were described in the same patient, but no indication was given at that time of the MHS status of the family and no molecular studies could be carried out. A few other patients presenting with the same association of cores and rods were briefly reported more recently (21). Here we present evidence for autosomal dominant congenital myopathy with rods, cores and MHS in a family with four generations of the disease. Linkage analysis showed a clear segregation of the disease with the RYR1 gene and sequencing of the RYR1 cDNA from the affected proband identified a neomutation in the C-terminal, channel-forming domain of the protein. The hypothesis that central core disease and dominant nemaline myopathy might co-segregate as two different and independent dominant diseases in the family was also tested by analysis of linkage to the NEM1 locus and by sequencing of both TPM3 and ACTA1 genes.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Linkage of the clinical phenotype in the family with the RYR1 locus
Since MHS and CCD phenotypes have been associated with mutations in the RYR1 gene (5,6,13), we investigated the 19q13 locus using an intragenic dinucleotide repeat in RYR1 (22) and the flanking centromeric D19S191, D19S220 and telomeric D19S422, D19S417, D19S223 markers for linkage studies. No recombinant event was observed in this region in the pedigree and a combination of these markers in grandparental chromosomes allowed the definition of four haplotypes in the proband’s generation (Fig. 1). One of the chromosome 19q haplotypes (Fig. 1) clearly cosegregated with the disease. In full agreement with the haplotyping analysis, combined pairwise LOD scores with informative markers adjacent to or internal to the RYR1 gene yielded significant positive values for all informative markers (Table 1). The LOD score value of +2.94 at
= 0.0, obtained with the haplotype associated with the disease, almost reached the significant threshold of +3 and was equivalent to the maximal theoretical value that could be expected in this pedigree. These results strongly implicate an alteration in the RYR1 gene in the pathological phenotype observed in generations II, III and IV of the pedigree.
|
|
In contrast, negative LOD score values were obtained with the D1S442 and D1S2624 markers flanking the TPM3 gene when the NEM1 candidate locus was investigated with polymorphic markers of chromosome 1 (Table 1). However, a recombinant event occurred between the D1S305 and D1S2624 markers in the chromosome 1q21–23 region of the proband inherited from her mother (individual I-2). This recombined chromosome 1q region was transmitted to all of the affected offspring (Fig. 1). Linkage analysis restricted to the proband and her lineage yielded a LOD score value of 0.89 when analysing the segregation of this recombined haplotype with the pathological phenotype.
Since mutations in the TPM3 gene that lies in this recombined chromosomal region have been associated with NM in previous studies, the coding sequence of the TPM3 gene was entirely sequenced. No mutations were found and RNA analysis using RT–PCR did not reveal any alteration in cDNA production nor in size modifications (data not shown). The ACTA1 gene encoding -actin is located on chromosome 1q42 and has also been associated with dominant forms of NM (19). To exclude a possible causative role of this gene in the ‘NM-like’ phenotype due to a mutation that may have occurred in patient II-8, the coding sequence of the ACTA1 gene was entirely sequenced. No mutations were identified and no modifications in size or cDNA production were observed.
Y4796C mutation in the RYR1 gene is associated with the pathological phenotype
The cDNA of the RYR1 gene was obtained from a skeletal muscle biopsy of the proband (patient II-8). Direct sequencing of the overlapping PCR products obtained from the RYR1 cDNA yielded eight sequence changes. Five of these sequence changes, two of them heterozygous, did not alter the amino acid sequence and were considered to be polymorphic (Table 2). Two sequence changes leading to amino acid substitution at positions 1832 and 2550 were present as homozygous changes. As presented in Table 2, both amino acid substitutions, A1832G and V2550L, are the same as those found in rabbit, pig or chicken RyR1 sequences. These mutations were not found in 100 unrelated chromosomes. Therefore, it is unlikely that these conservative amino acid changes represent pathogenic mutations or frequent variants.
|
The eighth sequence change, the substitution G
A at position 14 387 in the cDNA lies in the coding sequence in exon 100. This nucleotide substitution results in the Y4796C mutation in the RyR1 protein. In this case, cysteine replaces a tyrosyl residue that is strictly conserved in RyR1, RyR2 and RyR3 isoforms in human and in other species such as rabbit and chicken. As shown in Figure 2, the Y4796C mutation was heterozygous in all affected patients (II-8, III-1, III-2, IV-1 and IV-2). The mutation was located on the same 19q chromosome previously associated with the pathological phenotypes through haplotype analysis. The mutation was absent from all nine non-affected members of the family including the father of the proband and patient I-1. Since the proband, II-8, inherited the 19q chromosome that is associated with the disease in the family from her father, who does not carry the mutation, the Y4796C mutation must have arisen as a neomutation in the proband, II-8. This mutation was absent in 100 unrelated chromosomes in the general population.
|
Functional characterization of the Y4796C mutation
The functional significance of RYR1 mutations associated with either MHS or MHS/CCD has been evaluated previously through Ca2+ photometry and imaging using HEK-293 cells transfected with wild-type or mutant RYR1 cDNAs (11–13) or with skeletal muscle sarcoplasmic reticulum obtained from MHS patients (27). It was clearly demonstrated that pathological mutations affecting the RyR1 protein result in alteration of Ca2+ homeostasis in transfected HEK-293 cells. To evaluate the effect of the Y4796C mutation on the Ca2+ release channel function of RyR1, the 14387A
G mutation was introduced into the corresponding position in rabbit cDNA and expressed in HEK-293 cells. Ca2+ photometry and Ca2+ imaging assay were used to investigate the effect of the mutation on the functionality of RyR1 through measurements of intracellular Ca2+ concentrations (11). As shown in Table 3, the behaviour of the mutant channel differed from that of wild-type RyR1. The channel was significantly more sensitive to gating by caffeine, the EC50 for caffeine-induced Ca2+ release being 1.56 ± 0.08 mM in wild-type and 0.98 ± 0.15 mM in the mutant RyR1. These results are in line with the diagnosis of MHS for patient II-8, as described above. Resting Ca2+ concentrations were significantly elevated in the HEK-293 cells expressing the mutant channel, rising from 81.0 ± 2.0 nM in cells transfected with wild-type RyR1 to 129.4 ± 4.3 nM in cells transfected with the Y4796C mutant RyR1 cDNA. These results suggest that Ca2+ flux from the Ca2+ store is enhanced and exceeds Ca2+ return to the store through the action of the Ca2+ pump. Thus, Ca2+ homeostasis in these cells is only achieved at a higher resting Ca2+ concentration. The view that the mutant channels are more leaky, leading to a depletion of the Ca2+ store, is supported by the fact that the amplitude of Ca2+ release from the Ca2+ store is diminished significantly in the cells expressing the Y4796C mutant channel (426.4 ± 14 nM for wild-type versus 356.0 ± 12.4 nM for the Y4796C mutant) and by the complementary observation that the maximal changes in 340:380 ratio are significantly reduced in the Y4796C transfected cells. These observations all suggest that the rate of Ca2+ leakage is increased in the Y4796C mutant channel.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
This report presents genetic and biochemical evidence for a mutation in the skeletal muscle ryanodine receptor that is tightly associated with congenital myopathy with rods and cores in a large French pedigree. The rod–core congenital myopathy phenotype was diagnosed on the basis of both clinical symptoms and histological features. One of the patients (II-8) was tested for the MHS trait using the European in vitro contracture testing (IVCT) protocol and was found to be strongly positive. Moreover, the mutation, when tested by expression in heterologous cell culture (Table 3) was shown to have the same high sensitivity to caffeine that has been associated with all of the MHS/CCD mutations tested to date (11). These observations suggest that the same mutation that gives rise to cores in skeletal muscle is also responsible for MHS sensitivity in this family.
In earlier studies, MHS and CCD phenotypes have been associated, either independently or in combination, with the RYR1 locus on chromosome 19q whereas the NM has been associated with the NEM1 locus on chromosome 1q. Therefore, it was important to establish whether linkage between the rod–core myopathy and either RYR1 or NEM1 could be observed in this family. Haplotype analysis, using microsatellite markers, led us to conclude that the rod–core phenotype is associated with a single mutation in the RYR1 gene. This conclusion is based on several observations: (i) a LOD score of 2.94 with no recombinants was obtained with the 2:1:1 (D19S220:D19S417:D19S223) haplotype, clearly associating the rod–core myopathy with the RYR1 locus in generations II, III and IV; (ii) the chromosome carrying the linked haplotype, minus the causal mutation, originated in the unaffected father of the patient (individual I-1 in Fig. 1), associating the appearance of the mutation with the appearance of the rod-core myopathy in the proband (patient II-8 in Fig. 1); and (iii) biochemical analysis of the RyR1 mutant protein showed that it had high caffeine sensitivity for channel opening and was highly permeable.
Mutations in the -tropomyosin gene TPM3 and in the skeletal muscle -actin gene ACTA1, both located on chromosome 1q, have been associated with the dominant form of NM. A recombinant event in the NEM1 locus region that occurred in the proband (patient II-8 in Fig. 1) and segregated with the rod–core myopathy was evidenced. However, no mutation could be found in either of the two candidate genes, TPM3 and ACTA1. Although we cannot exclude the possibility that the recombinant event observed in chromosome 1q or another mutation might have altered a still not identified gene that could participate in the clinical expression of the disease, our data are in full agreement with the hypothesis that the Y4796C mutation in the RYR1 gene, which segregated fully with the myopathy in the family, is the cause of the pathological phenotype. Consequently, clusters of rods should be considered only as ‘additional’ histological features, in line with their frequent and well-known non-specificity (28).
Haplotype and mutation analysis revealed that the proband inherited the chromosome 19q region bearing the mutation from her father (patient I-1 in Fig. 1) and that the mutation was not present in her father’s DNA. This is, to our knowledge, the first report of a neomutation in the RYR1 gene associated with a pathological phenotype. As MHS and CCD are genetically heterogeneous, this is of importance for studies aimed at the identification of the genetic defect responsible for the disease. In our case, linkage analysis based on polymorphic markers and including the normal clinical status of the proband’s father would have excluded the RYR1 locus on chromosome 19q as being associated with the myopathic trait in this family.
The causative role of the Y4796C mutation in this specific pathologic phenotype is supported by several pieces of evidence: (i) the mutation affected a well-conserved tyrosine at position 4796; (ii) it segregated with the disease phenotype in the family and was not present in non-affected individuals; (iii) the mutation was not found in a screening of 100 non-related chromosomes in the general population; and (iv) functional analysis showed that the presence of the mutation resulted in impaired cytoplasmic Ca2+ homeostasis.
MHS and CCD are considered as allelic diseases and >20 mutations (29) of the RYR1 gene have been associated with these two pathological phenotypes. Except for the I4898T mutation associated with CCD (13), all of the previously described mutations lie between amino acids 35 and 614 (MHS/CCD region 1) or between amino acids 2163 and 2458 (MHS/CCD region 2) in the myoplasmic region of the protein (9). The I4898T mutation is located in the C-terminal transmembrane/luminal region of the RyR1 protein that forms the Ca2+ channel (30). The mutation lies in a region of RyR1 that may interact with the luminal domain of triadin, a protein proposed to form a ternary complex with RyR1 and calsequestrin (31). Functional analysis of the Ca2+ release channel function of the mutant protein has shown that the I4898T mutation results in a leaky channel responsible for the severe and penetrant CCD phenotype observed in the patients harbouring the mutation (13).
The Y4796C mutation also mapped to the C-terminal part of the protein that is likely to constitute the channel-forming segment of RyR1 (30,32,33). Du and MacLennan (34) showed that this region is involved in channel function and regulation. More specifically, the Y4796C mutation lies in a region that has been proposed to correspond to the myoplasmic loop of RyR1, localized between transmembrane helices M2 and M3 in a model based on the presence of four transmembrane domains (35). Alternatively, in a model based on the presence of 10–12 transmembrane passages (24), the mutation would lie in a transmembrane helix. More recently, a functional interaction between the skeletal muscle ryanodine receptor and the cytoplasmic domain of triadin has been shown to play a critical role in the control of Ca2+ release channel behaviour (36). One may, therefore, hypothesize that Y4796 is located in an RyR1 domain that interacts with the cytoplasm domain of triadin.
Single-cell analysis of HEK-293 cells co-transfected with wild-type and Y4796C mutant RYR1 cDNA showed a significantly increased resting cytoplasm Ca2+ concentration. Although these results have been obtained in a non-muscle system, one may hypothesize that the RyR1 channel can also be leaky in the skeletal muscle of affected patients. The resulting chronic elevation in myoplasmic Ca2+ concentration would be responsible for the clinical expression of the disease. This is in agreement with a previous report showing that overexpression of an R163C mutated RyR1 in muscle cells does not result in a significant modification of the resting Ca2+ concentration (37). Unlike the patients in our family, patients harbouring the R163C mutation have MHS but they do not present clinical symptoms in absence of triggering agents. The data presented in this report and other preliminary data that we have obtained on a panel of families presenting a clear clinical and histological CCD strongly support the fact that mutations of the C-terminal part of RyR1 that forms the Ca2+ pore in the sarcoplasmic reticulum membrane are likely to be associated with clinically expressed phenotypes such as congenital myopathy with cores and/or rods.
It has been observed that clinical expression of CCD or NM or the amplitude of the response to halothane in MHS patients may vary from one affected patient to another in the same family. In our pedigree, the grandsons of the proband clearly expressed the disease more severely and more precociously than their father and their grandmother suggesting that the disease worsened throughout the different generations. A possible explanation that may be considered for additional investigation in this pedigree would be the presence of additional alterations in the mechanisms involved in the regulation of Ca2+ homeostasis related to the different genetic backgrounds of the patients.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Family report
The pedigree of a French family with four generations, with no known history of consanguinity is presented in Figure 1. The proband (patient II-8) is a 50-year-old woman who had developed non-progressive muscle weakness during infancy. Her motor milestones were delayed and she started walking at age 2.5 years. During infancy, she presented with hypotonia, walking difficulties and weakness when running or climbing stairs. In her twenties she developed Achilles’ tendon contractures, mild difficulty in standing up and discrete lordosis. She is, however, still completely autonomous in her daily life. She has no cardiac abnormalities and creatine kinase (CK) serum level is normal (50 IU/l). Both of her parents were considered to be healthy without any reported muscular symptoms. Her father lived to the age of 93 years. At the age of 44 years, a quadriceps muscle biopsy was taken when the patient underwent in vitro contracture testing for MHS. Histological examination and ultrastructural analysis led to the diagnosis of congenital myopathy with cores and rods.
The proband has two sons (patients III-1 and III-2), both of whom presented delayed motor development and a clinical history similar to that of their mother. Patient III-1, who is 31 years old, started walking at age 2.5 years. He had no muscle biopsy, but his clinical phenotype was similar to that of his younger brother. Patient III-2, who is 30 years old, started walking at the age of 2.8 years. Walking difficulties and mild proximal weakness were noted during infancy. CK serum level was normal (74 IU/l). At the age of 18, he presented with mild weakness and discrete Achille’s tendon contractures. At the age of 27, a discrete lordosis was noted. Histological and ultrastructural analysis of a deltoid muscle biopsy, obtained at the age of 30 years, demonstrated the presence of cores and rods. At this time, muscle testing indicated a moderate worsening of muscle strength especially in the limb girdle. He is the father of two affected boys who present with a more severe and earlier symptomatology (patients IV-1 and IV-2). Both boys were born at full term but at birth they presented with severe hypotonia and hip luxations.
Patient IV-1, presently 3 years old, is still not walking. He presents with mild hypotonia, hip dislocation, strabismus and ptosis of the left eye. A muscle biopsy was obtained from the quadriceps muscle. Histological and ultrastructural analysis showed the presence of cores and rods in muscle fibres, associated with a moderate increase in connective tissue. Although noticeable, rods were less abundant than in his grandmother’s fibres. Patient IV-2, presently 10 months old, is unable to sit upright without help. He presents with a severe and generalized hypotonia and he has important cyphosis and contractures. Hydramnios and weak fetal movements were noted during the pregnancy of the mother of patient IV-2.
Morphological studies
Parts of each muscle biopsy were frozen immediately in isopentane cooled in liquid nitrogen and stored at –80°C until processed for histochemical studies. A small specimen was frozen immediately in liquid nitrogen for RNA extraction and another specimen was fixed for ultrastructural analysis. Histochemical studies were carried out on 10 µm cryostat transverse sections from muscle biopsy specimens. Histoenzymological protocols and ultrastructural studies were carried out as described previously (38,39).
As presented in Figure 3, histochemical studies of cryostat sections performed on muscle biopsies obtained from patients II-8, III-2 and IV-1 showed two different types of abnormality. The first was the presence of centrally or eccentrically located cores which appeared as rounded, precisely delimited areas near the centres of muscle sections stained for the oxidative enzymes, NADH-tetrazolium reductase and succinate dehydrogenase (Fig. 3b, c and d). Central cores are usually considered to be histological landmarks of CCD. Multiple rods located in clearly different areas of the cores constituted the second abnormality. Rods were distinctly observed with cycles of trichrome Gomori stain in the muscle fibres of the proband (Fig. 3). Electron microscopy analysis confirmed the characteristic transversal and longitudinal situation of rods. Although the presence of rods is of lesser significance from a pathological point of view, rods are usually typical of NM. Similar rods were observed, although in smaller numbers and only in very atrophic muscle fibres in biopsies from patients III-2 and IV-1.
|
IVCT
IVCT was performed in patient II-8 using a quadriceps muscle biopsy. Muscle bundles were tested according to the caffeine–halothane contracture test of the European Malignant Hyperthermia Group (40). According to this protocol, a patient is diagnosed as susceptible to malignant hyperthermia (MHS) if the 2 mN threshold value for muscle tension is attained at concentrations of <2 mM caffeine and <2% halothane. Values presented in Table 4 clearly indicate that the patient is MHS. IVCT testing carried out according to the North American protocol (41) led to the same MHS diagnosis. IVCT conducted in the presence of ryanodine as a triggering agent also showed an abnormal response compared with normal patients: the 10 mN muscle tension was reached after 13 min in the presence of 2 µM ryanodine.
|
Genotyping and linkage analysis
Genomic DNA was extracted from whole blood using a guanidine method (42). DNA from family members was typed using the following microsatellite markers: D19S220, D19S422, D19S417, D19S223 and RYR1, an intragenic dinucleotide repeat, for analysis of linkage to the RYR1 locus on chromosome 19q13 and D1442, D1S305, D1S624 for analysis of linkage to the NEM1 locus on chromosome 1q. Primer sequences and allele sizes have been reported previously (22,43). For each primer pair, 0.4 pmol forward primer was 5' end-labelled with 32P and added to a 25 µl PCR mix containing 50 ng of genomic DNA, 200 µM dNTPs, 1 µM forward and reverse primers and 0.25 U of Taq polymerase (Quantum Appligène, Strasbourg, France). Amplification was performed for 25–30 running cycles consisting of 30 s at 94°C, 30 s at annealing temperature and 1 min at 72°C. After denaturation with formamide, amplified products were separated on a 6% polyacrylamide/8 M urea DNA sequencing gel prior to autoradiography. Consecutive allele numbers were assigned and the results were documented using Cyrillic 2.1 software (Cherwell Scientific Publishing, Oxford, UK). Two-point linkage analysis was performed using the 5.2 Linkage Package with the following parameters: the CCD allele frequency was estimated at 1:100 000; the penetrance of the index case was taken as 1.0. The penetrance of the disease heterozygote was set at 1.0 when based on histochemical diagnosis and 0.98 when based on clinical diagnosis. The phenocopy rate was set at 0 and 0.02 under the same conditions.
Sequencing
Total RNA was extracted from a frozen muscle specimen from patient II-8 using an acid guanidinium thiocyanate–phenol–chloroform method (44). First strand cDNA was synthesized from 1 µg of total RNA in a final volume of 20 µl using Long Expand Reverse Transcriptase (Roche, Meylan, France) at 48°C for 1 h according to the manufacturer’s instructions. Due to its size and nucleotide composition, the RYR1 first strand cD-NA was synthesized using three mixes of specific primers. Mix 1 corresponded to the following set of primers: 5'-CACGAAGTCGGTGTGTGAGA-3' (nucleotides 2658–2639), 5'-TGTTCTGTGCCCGCAAAGAGg-3' (nucleotides 7823–7804), 5'-TCATTTCGTTCTCATCCGCTTC-3' (nucleotides 12 451–12 530). Mix 2 contained three primers: 5'-CCTCCTTCGCAGTCCCTTCT-3' (nucleotides 4168–4249), 5'-GGTTCTCCACCATCTTCTCGATGT-3' (nucleotides 9430–9507), 5'-GGAGATGGGCAGCAAAGA-3' (nucleotides 14 530–14 613) and mix 3: 5'-ACCCTCTTCCTCCTCGTCCT-3' (nucleotides 5754–5835), 5'-TGTGGGACTTGGACCAGTAGA-3' (nucleotides 10 450–10 530), oligo(dT). The resulting first strands were then amplified in 500–700 bp overlapping fragments using a second set of primers. The TPM3 and ACTA1 cDNAs, respectively, were amplified in 2 and 4 overlapping fragments, starting from a first strand cDNA obtained using oligo(dT) as primer. Hot-start PCR was performed in a final volume of 50 µl using 0.5 U of Taq Plus Precision Polymerase (Stratagene, La Jolla, CA) and 1.5 mM MgCl2 or 0.5 U of Taq polymerase (Promega, Madison, WI) and 1 mM MgCl2 in the presence of 5% DMSO or glycerol, depending on the amplified products. RYR1, TPM3 and ACTA1 cDNAs were entirely sequenced. In order to eliminate any sequencing errors resulting from polymerase activity, independently amplified products of the fragments were sequenced, when necessary using an ABI 377 apparatus and the PCR primers as sequencing primers.
Mutation analysis
The 14387AG mutation that resulted in a Y4796C substitution changed the normal sequence GTATAT to GTGTAT. This allowed genomic DNA samples to be screened for the Y4796C mutation using a mutated primer. PCR amplification was performed with the following primers: For-Y4796C (5'-TCCAAGAGTGCTCCTCGTGT-3') and Rev-Y4796C (5'-CCAAGAGGGACATCACCGTA-3'). As indicated (bold) a mutation leading to the substitution GA was introduced in the sequence of the Rev-Y4796C primer. Amplification of genomic DNA using this modified primer generated an AccI restriction site only with the normal allele whereas the amplified fragment corresponding to the mutant allele remained uncut. PCR was performed in a 25 µl final volume using 0.25 U of Taq polymerase from Quantum Appligène under the following conditions: denaturation at 95°C for 30 s, annealing temperature at 58°C for 30 s and extension temperature at 72°C for 1 min for 30 cycles. Amplified products were then digested with AccI. The 105 bp mutant allele was uncut whereas digestion of the normal allele yielded two fragments of 86 and 19 bp. Digested fragments were analysed on an 8% acrylamide gel.
Construction and functional analysis of MHS/CCD mutants
Enzymes for DNA manipulation were from Roche, New England Biolabs (Beverly, MA), Promega and Amersham Pharmacia Biotechnology (Little Chalfont, UK). Fura-2 acetoxymethyl ester (Fura-2 AM) was from Molecular Probes (Eugene, OR). Caffeine and protease inhibitors were from Sigma. The methods used in construction and mutagenesis of full-length rabbit skeletal muscle ryanodine receptor (RYR1) cDNA cassettes were described previously (11,31). Fragments containing mutations were confirmed by sequence analysis and cloned into the pcDNA-RYR1 vector. Transfection of the cDNAs into HEK-393 cells was performed as described previously using the calcium phosphate precipitation method (30). Total proteins collected in detergent solution from one 35 mm plate were separated by 6% SDS–PAGE and RyR1 protein was detected using monoclonal antibody 34C (45).
Caffeine-induced changes in Fura-2 fluorescence were measured in a Photon Technologies (PTI) microfluorimetry system (Lawrenceville, NJ) as described previously (11,12). Briefly, cells on a glass coverslip were loaded with 2 mM Fura-2 AM in a physiological medium 48 h after transfection. About 50 cells in a selected region were excited alternately at 340 and 380 nm and fluorescence emission at 510 nm was measured. The 340:380 nm ratio was calculated using PTI Felix software and dose–response curves were normalized to the maximal release response observed at 10 mM caffeine. Acquired digital images (400 ms/frame) were analysed with PTI Image Master 2.0 software. The 340:380 nm ratios from single cells were converted to Ca2+ concentrations as described previously (46), using [Ca2+] = Kd x [(R – Rmin)/(Rmax – R)] x (Sf2/Sb2). Data analysis was performed using Origin software (Microcal Software, Northampton, MA). All data are given as means ± SE. An unpaired Student’s t-test was used for statistical comparison of mean values between samples. Statistical significance was set at P < 0.05.
|
ACKNOWLEDGEMENTS |
|---|
We thank all family members for their participation in this study, Joëlle Lerale for technical assistance and Dr Marc Fiszman for helpful comments. This work was supported in part by grants to J.L. from Association Française contre les Myopathies, Direction Régionale de la Recherche Clinique du CHU Grenoble and Fondation Daniel Ducoin and by grants to D.H.M. from the Medical Research Council of Canada, the Muscular Dystrophy Association (USA) and the Canadian Genetic Diseases Network of Centers of Excellence.
|
FOOTNOTES |
|---|
+ These authors contributed equally to this work
§ To whom correspondence should be addressed at: Laboratoire BECP/DBMS, CEA 2943 UJF–CEA, 17 rue des Martyrs, 38054 Grenoble Cedex 09, France. Tel: +33 4 76 76 55 73; Fax: 33 4 76 76 58 37; Email: jlunardi@chu-grenoble.fr
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
1 Shy, G.M. and Magee, K.R. (1956) A new congenital non-progressive myopathy. Brain, 79, 610–621.
2 Shuaib, A., Paasuke, R.T. and Brownell, A.K.W. (1987) Central core disease: clinical features in 13 patients. Medicine, 66, 389–396.[Medline]
3 Frank, J.P, Harati, Y., Butler, I.J., Nelson, T.E. and Scott, C.I. (1980) Central core disease and malignant hyperthermia syndrome. Ann. Neurol., 7, 11–17.[ISI][Medline]
4 Gillard, E.F., Otsu, K., Fujii, J., Khanna, V.K., DeLeon, S., Derdemezi J., Britt, B.A., Duff, C.L., Worton, R.G. and MacLennan, D.H. (1991) A substitution of cysteine for arginine 614 in the ryanodine receptor is potentially causative of human malignant hyperthermia. Genomics, 11, 751–755.[ISI][Medline]
5 Quane, K.A., Healy, J.M.S., Keating, K.E., Manning, B.M., Couch, F.J., Palmucci, L.M., Doriguzzi, C., Fagerlund, T.H., Berg, K., Ording, H. et al. (1993) Mutations in the ryanodine receptor gene in central core disease and malignant hyperthermia. Nature Genet., 5, 51–55.[ISI][Medline]
6 Zhang, Y., Chen, H.S., Khanna, V.K., De Leon, S., Phillips, M., Schappert, K., Britt, B.A., Brownell, K.W. and MacLennan, D.H. (1993). A mutation in the human ryanodine receptor gene associated with central core disease. Nature Genet., 5, 46–51.[ISI][Medline]
7 MacLennan, D.H., Duff, C., Zorzato, F., Fujii, J., Phillips, M., Korneluk, R.G., Frodis, W., Britt, A. and Worton, R.G. (1990) Ryanodine receptor gene is a candidate for predisposition to malignant hyperthermia. Nature, 343, 559–561.[Medline]
8 McCarthy, T.V., Healy, J.M.S., Heffron, J.J.A., Lehane, M., Deufel, T., Lehmann-Horn, F., Farrall, M. and Johnson, K. (1990) Localization of the malignant hyperthermia locus to human chromosome 19q12–13.2. Nature, 343, 562–564.[Medline]
9 Loke, J. and MacLennan, D.H. (1998) Malignant hyperthermia and central core disease: disorders of Ca2+ release channels. Am. J. Med., 104, 470–486.[ISI][Medline]
10 Leeb, T. and Brenig, B. (1998) Ryanodine receptors and their role in genetic diseases. Int. J. Mol. Med., 2, 293–300.[ISI][Medline]
11 Tong, J., Oyamada, H., Demaurex, N., Grinstein, S., McCarthy, T.V and MacLennan, D.H. (1997) Caffeine and halothane sensitivity of intracellular Ca2+ release is altered by calcium release channel (ryanodine receptor) mutations associated with malignant hyperthermia and/or central core disease. J. Biol. Chem., 272, 26332–26339.
12 Tong, J., McCarthy, T.V. and MacLennan, D.H. (1999) Measurements of resting cytosolic Ca2+ concentrations and Ca2+ store in HEK-293 cells transfected with malignant hyperthermia or central core disease mutant Ca2+ release channels. J. Biol. Chem., 274, 693–702.
13 Lynch, P.J., Tong, J., Lehane, M., Mallet, A., Giblin, L., Heffron, J.J.A., Vaughan, P., Zafra, G., MacLennan, D.H. and McCarthy, T.V. (1999) A mutation in the transmembrane/luminal domain of the ryanodine recptor is associated with abnormal Ca2+ releases channel function and severe central core disease. Proc. Natl Acad. Sci. USA, 96, 4164–4169.
14 Shy, G.M., Engel, W.K., Somers, J.E. and Wanko, T.(1963) Nemaline myopathy: a new congenital myopathy. Brain, 86, 793–810.
15 Wallgren-Pettersson, C. (1998) Genetics of the nemaline myopathies and the myotubular myopathies. Neuromusc. Disord., 8, 401–404.[ISI][Medline]
16 Pelin, K., Hilpela, P., Donner, K., Sewry, C., Akkary, P.A., Wilton, S.D., Wattanasirichaigoon, D., Bang, M.L., Centner, T., Hanafdeld, F. et al. (1999) Mutations in the nebulin gene associated with autosomal recessive nemaline myopathy. Proc. Natl Acad. Sci. USA, 96, 2305–2310.
17 Laing, N.G., Wilton, S.D., Akkari, P.A., Dorosz, S., Boundy, K., Kneebone, C., Blumberg, P., White, S., Watkins, H., Love, D.R. and Haan, E. (1995) A mutation in the tropomyosin gene TPM3 associated with autosomal dominant nemaline myopathy. Nature Genet., 9, 75–79.[ISI][Medline]
18 Tan, P., Briner, J., Boltshauser, E., Davis, M.R., Wilton, S.D., Lallgren-Pettersson, C. and Laing, N.G. (1999) Homozygosity for a nonsense mutation in the alpha-tropomyosin slow gene TPM3 in a patient with severe infantile nemaline myopathy. Neuromusc. Disord., 9, 573–579.[ISI][Medline]
19 Nowak, K.J., Wattanasirichaigoon, D., Goebel, H.H., Wilce, M., Pelin, K., Donner, K., Jacob, R.L., Hubner, C., Oexle, K., Anderson, J.R. et al. (1999) Mutations in the skeletal muscle -actin gene in patients with actin and nemaline myopathy. Nature Genet., 23, 208–212.[ISI][Medline]
20 Afifi, A.K., Smith, J.W. and Zellweger, H. (1965) Congenital non-progressive myopathy: central core disease and nemaline myopathy in one family. Neurology, 15, 371–381.
21 Fardeau, M. and Tomé, F.M.S. (1994) Congenital myopathies. In Engel, A.G. and Franzini-Amstrong, B. (eds), Myology, 2nd edn. McGraw-Hill, New York, NY, pp. 1487–1582.
22 Couch, F., Hogan, K., McCarthy, T. and Gregg, R. (1991) Dinucleotide repeat polymorphism at the RYR1 locus (19q13.1). Nucleic Acids Res., 19, 5094.
23 Phillips, M.S., Fujii, J., Khanna, V.K., DeLeon, S., Yokobata, K., de Jong, P.J. and MacLennan, D.H. (1996) The structural organization of the human skeletal muscle ryanodine receptor (RYR1) gene. Genomics, 34, 24–41.[ISI][Medline]
24 Zorzato, F., Fujii, J., Otsu, K., Phillips, M., Green, N.M., Lai, F.A., Meissner, G. and MacLennan, D.H. (1990) Molecular cloning of cDNA encoding human and rabbit forms of the Ca2+ release channel (ryanodine receptor) of skeletal muscle sarcoplasmic reticulum. J. Biol. Chem., 265, 2244–2256.
25 Harbitz, I., Kristensen, T., Bosnes, M., Kran, S. and Davies, W. (1992) DNA sequence of the skeletal muscle calcium release channel cDNA and verification of the Arg615–Cys615 mutation, associated with porcine malignant hyperthermia, in Norwegian landrace pigs. Animal Genet., 23, 395–402.[ISI][Medline]
26 Ottini, L., Marziali, G., Conti, A., Charlesworth, A. and Sorrentino, V. (1996) Alpha and beta isoforms of ryanodine receptor from chicken skeletal muscle are the homologues of mammalian RyR1 and RyR3. Biochem. J., 315, 207–216.
27 Richter, M., Schleithoff, L., Deufel, T., Lehmann-Horn, F. and Herrmann-Frank, A. (1997) Functional characterization of a distinct ryanodine receptor mutation in human malignant hyperthermia susceptible muscle. J. Biol. Chem. 272, 5256–5260.
28 Fardeau, M. (1969) Etude d’une nouvelle observation de ‘Nemaline Myopathy’ II—Données structurales. Acta Neuropathol., 13, 250–266.[Medline]
29 Brandt, A., Schleithoff, L., Jurkat-Rott, K., Klingler, W., Baur, C., and Lehmann-Horn, F. (1999) Screening of the ryanodine receptor gene in 105 malignant hyperthermia families: novel mutations and concordance with the in vitro contracture test. Hum. Mol. Genet., 8, 2055–2062.
30 Bhat, M.B., Zhao., J., Takeshima., H. and Ma., J. (1997) Functional calcium release channel formed by the carboxy-terminal portion of ryanodine receptor. Biophys. J., 73, 1329–1336.
31 Balshaw, D., Gao, L. and Meissner, G. (1999) Luminal loop of the ryanodine receptor: a pore-forming segment? Proc. Natl Acad. Sci. USA, 96, 3345–3347.
32 Chen, S.R., Leong, P., Imredy, J.P., Bartlett, C., Zhang, L. and MacLennan, D.H. (1997) Single-channel properties of the recombinant skeletal muscle Ca2+ release channel (ryanodine receptor). Biophys. J., 73, 1904–1912.
33 Chen, S.R., Ebisawa, K., Li, X. and Zhang, L. (1998) Molecular identification of the ryanodine receptor Ca2+ sensor. J. Biol. Chem., 273, 14675–14678.
34 Du, G. and MacLennan, D.H. (1999) Functional consequences of mutations of conserved, polar amino acids in transmembrane sequences of the Ca2+ release channel (ryanodine receptor) of rabbit skeletal muscle sarcoplasmic reticulum. J. Biol. Chem., 273, 31867–31872.
35 Takeshima, H., Nishimura, S., Matsumoto, T., Ishida, H., Kangawa, K., Minamino, N., Matsuo, H., Ueda, M., Hanaoka, M., Hirose, T. et al. (1989) Primary structure and expression from complementary DNA of skeletal muscle ryanodine receptor. Nature, 339, 439–445.[Medline]
36 Groh, S., Marty, I., Ottolia, M., Prestipino, G., Chapel, A., Villaz, M. and Ronjat, M. (1999) Functional interaction of the cytoplasmic domain of triadin with the skeletal ryanodine receptor. J. Biol. Chem., 274, 12278–12283.
37 Censier, K., Urwyler, A., Zorzato, F. and Treves S. (1998) Intracellular calcium homeostasis in human primary muscle cells from malignant hyperthermia-susceptible and normal individuals. Effect of overexpression of recombinant wild-type and Arg163Cys mutated ryanodine receptors. J. Clin. Invest., 101, 1233–1242.[ISI][Medline]
38 Fardeau, M. (1973) Caracteristiques cytochimiques et ultrasructurales des différents types de fibres musculaires squelettiques extra-fusales (chez l’homme et quelques mammiferes) Ann. d’Anat. Pathol., 18, 7–34.
39 Romero, N.B., Nivoche, Y., Lunardi, J., Bruneau, B., Cheval, M.A., Hillaire, D. and Fardeau, M. (1993) Malignant hyperthermia and central core disease: analysis of two families with heterogeneous clinical expression. Neuromusc. Disord., 3, 547–551.[Medline]
40 European Malignant Hyperpyrexia Group (1984) A protocol for the investigation of malignant hyperpyrexia (MH) susceptibility. Br. J. Anaesth., 56, 1267–1269.
41 Larach, M.G. (1989) Standardization of the caffeine halothane muscle contacture test. North American Hyperthermia Group. Anesthes. Analges., 69, 511–515.
42 Jeanpierre, M. (1987) A rapid method for the purification of DNA from blood. Nucleic Acids Res., 15, 9611.
43 Weissenbach, J., Gyapay, G., Dib, C., Vignal, A., Morissette, J., Millasseau, P., Vaysseix, G. and Lathrop M. (1992) A second-generation linkage map of the human genome. Nature, 359, 794–801.[Medline]
44 Chomczynski, P. and Sacchi, N. (1987) Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162, 156–159.[ISI][Medline]
45 Chen, S.R., Zhang, L. and MacLennan, D.H. (1993) Antibodies as probes for Ca2+ activation sites in the Ca2+ release channel (ryanodine receptor) of rabbit skeletal muscle sarcoplasmic reticulum. J. Biol. Chem., 268, 13414–13421.
46 Grynkiewicz, G., Poenie, M. and Tsien, R.Y. (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem., 260, 3440–3450.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  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]
|
|
|
|
 |
|
 D. Rossi, P. De Smet, A. Lyfenko, L. Galli, S. Lorenzini, D. Franci, F. Petrioli, A. Orrico, C. Angelini, V. Tegazzin, et al. A truncation in the RYR1 gene associated with central core lesions in skeletal muscle fibres J. Med. Genet., February 1, 2007; 44(2): e67 - e67. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. B. Shafi, J. C. Parker Jr., J. B. Atkinson, and J. R. Parker Novel Presentation of Central Core Disease with Nemaline Bodies (Rods) in the Setting of Diploid/Triploid Mosaicism Ann. Clin. Lab. Sci., January 1, 2007; 37(2): 177 - 181. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Fischer, M. Herasse, A. Ferreiro, H. M. Barragan-Campos, J. Chiras, L. Viollet, S. Maugenre, J. -P. Leroy, N. Monnier, J. Lunardi, et al. Muscle imaging in dominant core myopathies linked or unlinked to the ryanodine receptor 1 gene Neurology, December 26, 2006; 67(12): 2217 - 2220. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Zhou, N. Yamaguchi, L. Xu, Y. Wang, C. Sewry, H. Jungbluth, F. Zorzato, E. Bertini, F. Muntoni, G. Meissner, et al. Characterization of recessive RYR1 mutations in core myopathies Hum. Mol. Genet., September 15, 2006; 15(18): 2791 - 2803. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Wu, M C. A. Ibarra, M. C. V. Malicdan, K. Murayama, Y. Ichihara, H. Kikuchi, I. Nonaka, S. Noguchi, Y. K. Hayashi, and I. Nishino Central core disease is due to RYR1 mutations in more than 90% of patients Brain, June 1, 2006; 129(6): 1470 - 1480. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Polat, A. Tosun, Y. Ay, E. Ozer, G. Serdaroglu, S. Aydogdu, S. Gokben, and H. Tekgul Central Core Disease: Atypical Case With Respiratory Insufficiency in an Intensive Care Unit J Child Neurol, February 1, 2006; 21(2): 173 - 174. [Abstract] [PDF]
|
|
|
|
|
|
H. H. Goebel Congenital Myopathies in the New Millennium J Child Neurol, February 1, 2005; 20(2): 94 - 101. [Abstract] [PDF]
|
|
|
|
 |
|
 T D Graves and M G Hanna Neurological channelopathies Postgrad. Med. J., January 1, 2005; 81(951): 20 - 32. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A M Kaindl, F Ruschendorf, S Krause |