Human Molecular Genetics, 2001, Vol. 10, No. 22 2581-2592
© 2001 Oxford University Press
Familial and sporadic forms of central core disease are associated with mutations in the C-terminal domain of 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 de Pédiatrie, CHU Bicêtre, Le Kremlin-Bicêtre, France, 4Département d’Anesthésie, Hôpital Robert Debré, Paris, France and 5Laboratoire BECP/DBMS, EA 2943 UJF—CEA, Grenoble, France
Received July 16, 2001; Revised and Accepted August 29, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Central core disease (CCD) is an autosomal dominant congenital myopathy. Diagnosis is based on the presence of cores in skeletal muscles. CCD has been linked to the gene encoding the ryanodine receptor (RYR1) and is considered to be an allelic disease of malignant hyperthermia susceptibility. However, the report of a recessive form of transmission together with a variable clinical presentation has raised the question of the genetic heterogeneity of the disease. Analyzing a panel of 34 families exclusively recruited on the basis of both clinically and morphologically expressed CCD, 12 different mutations of the C-terminal domain of RYR1 have been identified in 16 unrelated families. Morphological analysis of the patients’ muscles showed different aspects of cores, all of them associated with mutations in the C-terminal region of RYR1. Furthermore, we characterized the presence of neomutations in the RyR1 gene in four families. This indicates that neomutations into the RyR1 gene are not a rare event and must be taken into account for genetic studies of families that present with congenital myopathies type ‘central core disease’. Three mutations led to the deletion in frame of amino acids. This is the first report of amino acid deletions in RYR1 associated with CCD. According to a four-transmembrane domain model, the mutations concentrated mostly in the myoplasmic and luminal loops linking, respectively, transmembrane domains T1 and T2 or T3 and T4 of RYR1.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Central core disease (CCD) was the first described hereditary congenital muscle disorder defined by structural changes of the muscle fibers, with a slow or non-progressive evolution (1). Transmission was autosomal dominant. Affected patients usually presented with diffuse muscle weakness and often hypotonia during infancy, delayed motor development and reduced muscle bulk. Histological examination of CCD skeletal muscles showed abnormal rounded areas (‘cores’) in type I muscles fibers, extending through the length of the fiber. The cores are clearly delineated from the normally structured zones of the fiber by a sharp border and are characterized by lack of oxidative activity, myofibrillar compaction with a variable degree of sarcomeric disorganization and Z line streaming; proliferation of sarcotubular profiles was often seen at the periphery of the core (reviewed in 2). However, CCD exhibits great variability both clinically and histologically. Severity of symptoms may vary from very mild to severe (3) even in the same family. Histologically, cases may be centrally located or eccentric, unique or multiple, eventually associated with rods (4,5). The distinction from other types of congenital myopathies, such as multicore disease, may be difficult (6). Importantly, the association of CCD with malignant hyperthermia susceptibility (MHS) was detected early (7) and repeatedly observed (8).
A gene associated to CCD was mapped to 19q12–q13.2 (9), a chromosomic region that contained the ryanodine receptor gene, RyR1 (10,11). Mutations of the RyR1 gene have since been associated with MHS, an autosomal dominant pharmacogenetic disorder of skeletal muscle Ca2+ regulation (12,13). The RyR1 gene encodes a calcium release channel located in the junctional terminal cisternae of the sarcoplasmic reticulum (SR) membrane of the skeletal muscle. 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 but one of the 23 MHS mutations that have been reported to date in RYR1 have been localized in the cytoplasmic domain (13–15).
Molecular studies have shown the presence of mutations in the first N-terminal half of the RYR1 in MHS/CCD families (16–20). This suggested that CCD and MHS were allelic disorders linked to the RyR1 gene. However, the situation was unclear since several MHS patients in the MHS/CCD families had a mutation in the RyR1 gene without clinical or histological signs of CCD. The involvement of RYR1 in congenital myopathies was also supported by the work of Takeshima et al. (21) who reported that mice homozygous for a targeted mutation in the skeletal muscle RyR1 gene died perinatally with gross abnormalities of skeletal muscle. More recently, mutations affecting the function of RYR1 were described in patients with a clear and documented clinical CCD phenotype (4,5,22). These data indicated that CCD was caused in most instances by dominant mutations in the RyR1 gene. However, Manzur et al. (23) described an autosomal recessive form of CCD, but did not determine the underlying cause of the disorder.
When expressed in a heterologous system the MHS and CCD mutants of RYR1 could not be distinguished on the basis of their sensitivity to caffeine and halothane (24). However, CCD RYR1 mutant channels appeared to be more permeable than MH mutant channels, raising resting Ca2+ concentrations and depleting Ca2+ stores (25). Expression studies conducted in HEK-293 cells for two mutations in the C-terminal end of the RYR1 protein that forms the transmembrane channel domain showed that the mutant channels were highly permeable to Ca2+ (4,22). Recently, expression studies performed in dyspedic myotubes from a RYR1-knockout mouse model indicated that the muscle weakness associated with the I4898T mutation involved a functional uncoupling of sarcolemmal excitation from SR Ca2+ release (26).
Because expression of the clinical phenotype is highly variable, the CCD diagnosis was based on histological signs as well as on clinical expression of the disease. To minimize the heterogeneity, we included in this study only patients who presented with both clinical symptoms of congenital myopathy and histological abnormalities in skeletal muscle. This paper shows that the disease condition is mostly associated with mutations of the C-terminal part of the RYR1 protein. Although different in their morphological aspects, all muscle specimens from affected patients harboring RYR1 mutations showed the presence of cores. Furthermore, the characterization of a significant number of neomutations may shed some light regarding recessive or sporadic forms of CCD.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Clinical and histological phenotypes
Most of the patients had a clinical onset ranging from birth to early childhood and the disease was characterized by a slow or non-progressive evolution (clinical data are summarized in Table 1). It should be noticed that a worsening of the clinical status from one generation to another was observed in a few families (CCD04, 06).
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The different morphological aspects of the observed cores are shown in Figure 1. Classical unique ‘central’ cores are shown in Figure 1A. Cores were present in a central position of almost every muscle type I fiber and the size of the fibers appeared to be quite homogeneous. Unique ‘eccentric’ cores corresponding to unique cores in the subsarcolemmal region are shown in Figure 1B. A ‘polymorph’ pattern with the presence of both single and multiple ‘peripheral’ cores in the same muscle biopsy is shown in Figure 1C. The particular pattern of unique cores in small fibers and multiple cores in large fibers observed in the muscle of patients is shown in Figure 1D.
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Haplotyping studies
When possible, families were haplotyped using flanking markers of the RyR1 gene on chromosome 19q13.1. Pedigrees presented in Figure 2 showed that the phenotype of ‘congenital myopathy with cores’ could be associated with a specific chromosome 19q13.1 haplotype or at least not excluded from the chromosome 19q13.1 locus in a significant number of families. This was taken as an indication for the involvement of the RyR1 gene regarding the CCD phenotype of the patients.
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Identification of mutations
Screening of our panel of CCD patients for the set of mutations previously described as responsible for the MHS/CCD phenotype, i.e. R163C, I403M, R2163H, G2434R, R2435H, proved to be unsuccessful. Nevertheless, a 6502 G
A mutation in exon 39, which led to a V2168M change previously identified in Swiss MHS families (19), was identified in a CCD patient (family CCD14) during this screening. This patient aged 52 years presented with mild proximal weakness and a myogenic EMG. She had a history of mild orthopedic problems during infancy. Her muscle biopsy showed typical pictures of single or multiple peripheral cores in type I fibers. Muscle symptoms observed in our panel of families suggested a possible abnormality in Ca2+ homeostasis. As the Ca2+ movements might depend upon the integrity of the C-terminal channel forming domain of RYR1, we focused our attention on this region. The 3' terminal part of the RyR1 gene was investigated through a combination of single-strand conformation analysis (SSCA) and sequencing analysis of exons 94–106 when using genomic DNA (see Materials and Methods). Alternatively, direct sequencing of cDNA spanning exons 87–106 was performed when a muscle biopsy was available. This led to the identification of nine new mutations with two of them present, respectively, in two and three unrelated families (Table 2). Furthermore, the I4898T mutation previously characterized by Lynch et al. (22) in a large Mexican family was also identified in two additional unrelated families.
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Among the novel mutations, three mutations led to in frame deletions: 12640–12648 del (12640 delCGCCAGTTC), 13938–13943 del (13938 delTCTGAC) and 14578–14580 del (14578 delTTC). These different mutations represent the first report of deletions in the RyR1 gene. The five other mutations were missense mutations in exon 100, L4793P, R4825C; in exon 101, R4861H; and in exon 102, R4893W, G4899E, R4914G.
The 12640–12648 del mutation identified in exon 91 in family CCD10 led to the loss of residues R4214, Q4215 and F4216. As shown in Figure 3, these amino acids and particularly the F4216 residue are well conserved among RYR isoforms. Furthermore, this mutation clearly segregated with the disease in the CCD10 pedigree (Fig. 4). Interestingly, patient III-2 of the CCD10 family who harbored the 12640–12648 del mutation was classified MHN when tested by in vitro contracture testing (IVCT). Although the 13938–13943 del deletion started at the third nucleic acid of the C4646 codon and spanned over three codons, it resulted only in the loss of amino acids L4647 and S4748 in exon 95. Because of the presence of a G at position 13944, the sense of the C4646 codon remained unchanged. This mutation segregated with the CCD phenotype in family CCD01 (Fig. 2). The 14578–14580 del mutation resulted in the loss of a phenylalanyl residue at position 4860 in family CCD02. This residue is well conserved among RYRs primary sequences. Mutation and haplotyping analysis of the CCD02 family showed that theF4860 del mutation occurred as a neomutation in patient CCD02 II-2 (Fig. 2).
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The 14378 T
C and the 14473 CT mutations identified in exon 100 corresponded, respectively, to L4793P and R4825C mutations in RYR1. Both L4793 and R4825 residues are well conserved among RYRs species. The R4825C mutation segregated with the disease in the CCD12 family. This mutation affected an arginyl residue located vicinal to the T4826I mutation described in a large MHS New Zealand Maori pedigree (15). This mutation was associated with the MHS phenotype in one patient of family CCD12 that underwent IVCT. A 14582 GA mutation leading to a R4861H change in the RYR1 protein was identified in three unrelated families: CCD07, CCD09 and CCD15. As shown in Figure 3, this mutation affected an amino acid located in a very well conserved region of RYR proteins. Genetic analysis of family CCD09 indicated that the R4861H mutation arose as a neomutation in patient CCD09 II-1. Interestingly the R4861 amino acid affected by this neomutation is the vicinal residue of the F4860 residue also affected by a neomutation in family CCD02. Three missense mutations were identified in close vicinity in exon 102. The 14677 CT, the 14696 GA and the 14740 AG transitions led, respectively, to the R4893W, G4899E and R4914G changes in RYR1. The R4893W mutation was identified in two unrelated families (CCD04 and CCD08) and it segregated with the pathological trait. The I4898T mutation, previously associated with CCD in a large Mexican kindred (22), was identified in two unrelated families. Family CCD05 originated from France and presented with a family history of CCD. The second situation (family CCD11) corresponded to a sporadic case that originated from North America. R4893, I4898T and G4899 residues belong to the GXRXGGGXGD pore-forming segment, which was shown to play a role in skeletal muscle Ca2+-release channel activity and conductance (27,28). The R4914G mutation affected a very well conserved arginyl residue located in very close vicinity to these mutations. None of the missense mutations described here was found in 100 independent alleles in the general population. All these mutations introduced significant changes in the structural or chemical characteristics of amino acids. A V4849I change was identified in one of our CCD patients. However, as this change was identified in one out of the 100 control chromosomes we screened, we did not include it in this study. It must be noted that the V4849I change has been previously described as associated with MH. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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MHS and CCD are usually considered as allelic diseases of the RyR1 gene. However, both diseases appeared to be heterogeneous from a genetic point of view. A mutation in the gene coding for the
1 subunit of the dihydropyridine receptor has been associated with MHS (29) whereas linkage studies indicated that the pathological trait was not linked to the RyR1 locus on chromosome 19q13.1 in a few CCD families (30) and in CCD associated to hypertrophic cardiomyopathy (31). MHS and CCD are also heterogeneous from a clinical point of view. MH crises range from fulminant to mild crisis and penetrance of the disease may vary from patient to patient (32). CCD presents with different levels of severity and cores that may vary in their extent or localization in the muscle fibers. As shown in Figure 1, the histological phenotype in our panel of CCD families varied significantly between central cores, eccentric cores, multiple cores, peripheral cores and a mixture of these. Yet each histological phenotype was associated with mutations in the C-terminal domain of RYR1. The presence of the same picture of cores either in young patients or in adults of the same family indicated that the age of the patients did not by itself introduce a critical bias in the morphological analysis. Noticeably, some unrelated patients bearing the same mutation, e.g. patients from families CCD07, CCD09 and CCD15, showed the same pictures of cores. MHS and CCD, and other related disorders, are likely to be associated with an alteration of the Ca2+ fluxes at the level of the skeletal muscle triadic junction. While alteration of the Ca2+ homeostasis might be caused by defects in the two channel proteins, i.e. RYR1 or DHPR, it is likely that defects in others proteins of the triade that interact with RYR1 or DHPR might also be involved. Therefore, we propose to refer to these diseases as ‘triadopathies’.
A few mutations of the RyR1 gene have been associated previously with both CCD and MHS phenotypes (13). However, the relationship between MHS and CCD is unclear. It is important to remember that in most published MHS/CCD cases the CCD phenotype was mostly based on the presence of cores in the skeletal muscle biopsy. Families were recruited on the basis of problems during anesthesia and not on the basis of clinical symptoms of congenital myopathy. This may lead to some confusion as cores have been described in the muscle of MHS patients who did not present with clinical symptoms of congenital myopathy. On the other hand, while the MHS status of patients is based on positive IVCT results, it is well recognized that several other muscle diseases, including congenital myopathies, may be responsible for positive IVCT (33). In order to minimize the heterogeneity of our patients’ population, all families and patients included in this study presented with clinical symptoms of a congenital myopathy. Furthermore, patients’ muscle showed the presence of cores in type I fibers.
Molecular analysis of the largest panel of CCD families investigated so far allowed the identification of nine new mutations and the characterization of two previously described recurrent mutations in the RyR1 gene in 16 families presenting with clinical and histological signs of CCD. This allowed the molecular characterization of >45% of the families and isolated patients included in our study which is a remarkable figure considering the complexity of the RyR1 gene and the possible genetic heterogeneity of the disease. Amazingly, none of the MHS/CCD mutations previously described in the literature (16–20) was identified in our families. This probably reflects the fact that our panel of families was recruited on the basis of a congenital myopathy. Interestingly, all but one of the mutations presented here clustered in the last 15 exons that code for the C-terminal transmembrane domains of the RYR1 protein. The V2168M mutation has been identified previously in MHS Swiss families who did not present with clinical or histological symptoms of CCD (19). The fact that the very same mutation is associated in the CCD14 family with a clinical CCD raised the question of a possible role of the individual genetic background in the expression of the RYR1 mutations.
Limitations in the yield of mutation screening had a different origin. First, the mutation screening was mostly performed using a SSCA approach. According to our experience, the efficiency of this screening method to detect mutations is in the 80–90% range depending on the nature of the nucleic acid sequence. Secondly, while the screening of the exons of the first half of RYR1 previously associated with MHS/CCD families proved to be quite unsuccessful, one cannot rule out the presence of mutations in one of the 80 exons that have not been investigated yet. Thirdly, one has to consider the possible genetic heterogeneity of the disease and mutations affecting other genes encoding proteins that form the triade cannot be ruled out. However, due to the size and structure of most families, informative linkage analysis aimed at the characterization of new genes associated with the disease could not be easily performed.
A major point raised by our results was the significant number of neomutations in the RyR1 gene that we have identified in our panel of 34 CCD families. Molecular proofs for the occurrence of a neomutation were documented in four families: F4860 del mutation in CCD02, R4861H in family CCD09, Y4796C in family CCD06 and R4914G in family CCD16. The presence of a fifth neomutation was also strongly suspected in patient II-1 of family CCD11. This patient originated from North America and presented as a sporadic clinical CCD case. The I4898T mutation was identified in RYR1 at a heterozygous level. Parents were not consanguineous and did not show clinical symptoms. This mutation has been described so far in three families (22; this study) and the pathogenic role of the mutation has been clearly documented (22,26). Besides the fact that all these mutations were not found in 100 unrelated chromosomes of the general population, the causative effect of the F4860 del and R4861H mutations is strongly supported by several arguments. One concerns the nature of the amino acid modification: deletion of a well conserved phenyl-alanyl residue and change of a conserved arginyl residue. A second point is the recurrence of the R4861H mutation that has been also identified in two other unrelated families (CCD07, CCD15). Third, deletion of a single amino acid in RYR1 has also been recently associated with a pathologic phenotype. However, in that case the deletion associated with MHS was localized in the first half of the protein at position 2347 (34).
Taking into account all the figures, we have found a neomutation in almost 10% of the CCD families investigated. This may have several implications for the genetics of CCD. First, when considering ‘recessive’ forms of CCD, one has to consider the presence of a neomutation in the RyR1 gene as a possible explanation. A second point that is raised by this significant figure of neomutations in the RyR1 gene is the incidence on linkage studies. There is growing evidence for the presence of two independent mutations in several MHS/CCD families (29; N.Monnier, R.Krivosic-Horber, J.F.Payer, G.Kozak-Ribbens, Y.Nivoche, P.Adnet, H.Reyford and J.Lunardi; manuscript in preparation). On some occasions and based on linkage data, this led to the exclusion of the RYR1 locus as being the disease locus. Therefore, it could be worth reconsidering some of the MHS/CCD pedigrees that have been excluded from an association with the chr19q13.1 locus using the hypothesis of two independent RYR1 mutations.
Two models for the membrane arrangement of the last fifth C-terminal domain of RYR1 suggested either 10, M1–M10 (35), or four, T1–T4 (36), transmembrane domains. However, based on sequence conservation, it was reported that segments M3 and M4 are not likely to represent transmembrane domains (37). Therefore, and as shown in Figure 5, the main differences between the two models concentrated in the last 473 amino acids. One model (Fig. 5A), based on the 10-transmembrane domain hypothesis, postulated six transmembrane segments in this region (M5–M10) while the second showed the presence of four transmembrane domains (Fig. 5B). As shown in Figure 5, the position of the mutations identified in this study may vary when considering their luminal, myoplasmic or membranous localization depending on the different models. Although no structural data are yet available, the four-transmembrane domain model is favored by data obtained through site-directed antibodies studies, single channel recordings with tryptic fragments and deletion mutants (38). Various data indicate that RYR1 interact with various proteins responsible for the Ca2+-release channel function at the level of the skeletal muscle triadic junction, i.e. dihydropyridine receptor, triadin, calmodulin. It must be noted that in the four-transmembrane domain model all but one of the mutations described so far are located in external domains while most of them would affect amino acids of membrane segments in the 10-transmembrane domain based model. Considering the clinical features associated with the reported mutations, these mutations are likely to affect the excitation–contraction (E–C) process. Therefore, the positioning of the mutations described in this paper in the external loops linking transmembrane domains may be of interest when considering the domains of RYR1 that play a critical role in the E–C coupling. Along this line, it has been recently shown that the I4898T mutation that is located in the luminal region linking transmembrane domains TM3 and TM4 uncouples the E–C process in dyspedic myotubes (26).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Patients and families
The study initially included 35 unrelated patients or families presenting with clinical symptoms of CCD. A mutation in the RyR1 gene was identified in 16 families originating from France (CCD01, 02, 03, 04, 05, 06, 07, 09, 10, 12, 13, 14, 15, 16) or North America (CCD08, 11). Pedigrees for documented families are shown in Figure 2 and clinical features are summarized in Table 1. Family CCD06 has been detailed previously (4).
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. Histo-enzymological protocols and ultrastructural studies were processed as described previously (39,40).
In vitro contracture testing
When indicated, IVCT was performed on patients using a quadriceps muscle biopsy. Muscle bundles were tested according to the caffeine–halothane contracture test of the European Malignant Hyperthermia Group (41). According to this protocol, a patient is diagnosed as susceptible to malignant hyperthermia if the 2 mN threshold value for muscle tension is attained at concentration of <2 mM caffeine and <2% halothane.
Haplotyping analysis
When possible, DNA from members of the different families was haplotyped as described previously using the following markers: D19S220, RYR1, D19S422 and D19S 417 (4). Pedigrees of families CCD01, 02, 05, 08, 09, 10 and 12 are presented in Figure 2.
DNA purification
Genomic DNAs were obtained either from the Genethon Bank (AFM, Evry, France) or extracted from whole blood using a rapid guanidine method (42). Total RNA was extracted from a frozen muscle specimen using an acid guanidinium thiocyanate–phenol–chloroform method (43). First-strand cDNA synthesis of the C-terminal domain was performed by using 1 µg of total RNA, 100 ng of oligo(dT), 40 pmol of a specific primer (nt 14443–14423) and 50 U Long Expand Reverse Transcriptase (Roche, Meylan, France) in a 20 µl reaction volume at 48°C for 1 h according to the manufacturer’s instructions. Bases were numbered from the first coding nucleotide according to Zorzato’s cDNA sequence (35) corrected by Philips et al. (44).
CCD mutation screening
R163C, I403M, R2163H, G2434R and R2435H mutations previously described in ‘MHS/CCD’ families were analyzed by restriction enzyme analysis (REA) for R163C and I403M or by direct sequencing of exons 39 and 45. As this screening proved to be fully inefficient in our patient’s population and considering the complexity of the RyR1 gene, we focused our attention toward the 3' end of the RyR1 gene. This region corresponds to the transmembrane channel-forming domains of RYR1, a region where mutations have been recently identified in three different symptomatic CCD families (4,5,22).
Exons 94–99 and 103–106 were analyzed by SSCA. Each exon was amplified using the specific set of primers described in Table 3 and in the presence of 1 µCi of 32P dCTP (10 mCi/mmol). PCR products were denatured in loading buffer (95% formamide, 0.01 M NaOH, 0.025% xylene cyanol, 0.025% bromophenol blue) at 95°C for 2 min, then cooled on ice and finally separated on a non-denaturing DNA-sequencing gel prior to autoradiography. Two separating conditions were used for each sample: a run on a 6% acrylamide gel at 4°C and 40 W for 1–4 h depending on the size of the amplified products and a run on a 6% acrylamide/10% glycerol gel at room temperature and 4 W overnight.
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Exons 100–101 and 102 were analyzed by direct sequencing. Alternatively, first-strand cDNAs obtained from frozen biopsies were amplified in eight 400–600 bp overlapping fragments spanning exons 87–106 (nt 11820–12364, 12331–12731, 12704–13121, 13021–13462, 13385–13936, 13864–14443, 14383–14941, 14772–15187). PCR-amplified fragments obtained from genomic DNA or cDNA were sequenced by use of an ABI 377 apparatus and the PCR primers as sequencing primers.
Extensive screening of the various novel mutations using REA or heteroduplex analyses was performed as described in Table 2. For restriction analysis, 5 µl of amplified products were digested by 5 U of the appropriate enzyme for 3 h under conditions recommended by the manufacturer and separated on an 8% acrylamide gel. For heteroduplex analysis, 5 µl of amplified products were incubated for 5 min at 95°C and 5 min at 65°C and were analyzed on an 8% acrylamide gel.
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ACKNOWLEDGEMENTS |
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We thank all family members for their participation in this study and Mrs De Carvahlo for critical reading of the manuscript. This work was supported in part by grants from AFM, INSERM, DRC CHU Grenoble and from Fondation Daniel Ducoin (to J.L.).
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FOOTNOTES |
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+ To whom correspondence should be addressed at: Laboratoire Biochimie de l’ADN, EA 2943 UJF, CHU Grenoble BP 217, 38043 Grenoble Cedex, France. Tel: +33 4 76 76 55 73; Fax: +33 4 76 76 58 37; Email: jlunardi@chu-grenoble.fr
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