Departments of 1Neuropathology and 2Anaesthesia, Royal Perth Hospital, Perth, Western Australia. 3Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand. 4Anaesthetic Department, Christchurch Hospital, Private Bag 4710, Christchurch, New Zealand. 5Centre for Neuromuscular and Neurological Disorders, University of Western Australia, Perth, Australia. 6Department of Anaesthesia and Intensive Care, Palmerston North Hospital, Palmerston North, New Zealand 7Present address: Department of Medical Genetics, Wellcome Trust/MRC Building, Addenbrookes Hospital, Cambridge CB2 2XY, UK 8Present address: Central Region Genetic Service, Wellington Hospital, Wellington South, New Zealand 9Present address: Department of Genetics, The University of Melbourne, Victoria, 3010, Australia*Corresponding author
Accepted for publication: November 16, 2001
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Abstract |
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Br J Anaesth 2002; 88: 50815
Keywords: genetic factors, hyperthermia; complications, malignant hyperthermia; complications, myopathy; complications, exercise-induced rhabdomyolysis; receptors, ryanodine
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Introduction |
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Methods |
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In vitro contracture testing
IVCT of muscle biopsies was carried out according to the European MH group protocol.28 IVCT is based on contracture tensions induced in muscle in vitro by caffeine and halothane. A diagnosis of MHS is made when contractures of 0.2 g or greater are induced individually by a halothane concentration of 2% or less and a caffeine concentration of 2 mM or less. A diagnosis of MH equivocal (MHE) is made when contractures of 0.2 g or greater are induced with either halothane or caffeine, but not both, at the above concentrations.
Muscle histology
Histological examination using both light microscopy and electron microscopy was performed in the Western Australian laboratory on samples from all patients tested for susceptibility to MH. Light microscopy only was carried out in the New Zealand laboratory. Ten-micron sections from frozen muscle specimens were stained or reacted for hematoxylin and eosin, modified Gomori trichrome, NADH-tetrazolium reductase, ATPase, acid phosphatase, periodic acidSchiff, and oil red O.29
Mutation screening by RT-PCR and DNA sequencing
Total RNA was extracted from 30100 mg frozen skeletal muscle tissue using TrizolTM RNA extraction reagent (Invitrogen, Groningen, The Netherlands). First strand synthesis was carried out using the Superscript reverse transcriptase (RT) pre-amplification system (Invitrogen) with 4 µg total RNA and either 50 ng random hexamers or 500 ng oligo(dT) in a 20 µl volume. Hot-start polymerase chain reaction (PCR) was performed using 1 µl of a 20-fold dilution of the first strand cDNA reaction in 50 µl reactions with 0.32 µM of each primer, 0.3 mM dNTPs, 1.5 mM MgCl2 and 1.5 U Taq polymerase (Invitrogen). MH/CCD region 1 was amplified in three overlapping 816 bp, 824 bp, and 578 bp fragments using the following primer pairs: CAGGAGGACGCAACAGGAGAG/GTTGTATAGGCC ATTGGTGCT; TCCAAGGAGAAGCTGGATGTGG/TG CTTGTCCAGGAGGGAGATG and CTCTCCATGGTC CTGAATTGCATAGAC/AGTCACCTCGTCCACCATC AC, respectively. MH/CCD region 2 was amplified in one 1101 bp fragment using the CAGTACGACGGGCTGGGTGAG/GGATGCTGACATCTTTGGCT primer pair. MH/CCD region 3 was amplified in one 630 bp region using the GAACCCGCCCTGCGCTGTCTG/GTAGACGACCACCGCCAGAAG primer pair. PCR products were purified using the ConcertTM rapid PCR product purification kit (Invitrogen) and then screened for the presence of mutations by automated DNA sequencing using an ABI 377-36 or 373 with Big-Dye terminator chemistry (Applied Biosystems, New Zealand). Exon 12 of RYR1 was amplified from genomic DNA using primers 1143F and 1213R,5 purified using the ConcertTM rapid PCR product purification kit (Invitrogen) and sequenced from the forward primer using an ABI 377-36 or 373 with Big-Dye terminator chemistry (Applied Biosystems).
SSCP analysis and allele-specific PCR
Genomic DNA was isolated using the Wizard DNA extraction kit (Promega Corporation, Madison, USA) according to the manufacturers instructions. Exon 12 of RYR1 was amplified as above and screened for the presence of the C1201T mutation by single-stranded conformation polymorphism (SSCP) analysis using 12% 29:1 polyacrylamide gel electrophoresis. Alternatively, an allele-specific probe (CAGGCCGCCCGCATGAT) labelled with fluorescein together with an anchor probe (CAGCACCAATGGCCTATACAACCAGTTCATCAAGTGA) labelled with LC-red 640 was used to detect the presence of the C1201T mutation by real-time amplification of genomic DNA using a Light Cycler (Roche Diagnostics, NZ Ltd, Auckland, New Zealand).30 31 The sequences of the flanking primers were as follows: CTCTGTCTCCCCACTCCTA (forward) and GGCAACAGAGGTAGAGATGAA (reverse). The oligonucleotide primers and probes were designed using the software MeltCalc (http://www. meltcalc.de). The following thermocycling protocol was used for amplification: denaturation at 95°C for 120 s, followed by 45 cycles of: 95°C for 0 s, 55°C for 10 s, 72°C for 11 s, with a temperature transition rate of 20°C s1 and data acquisition at the annealing step. The melting curve analysis was as follows: 95°C for 0 s, 45°C for 30 s, with a temperature transition rate of 20°C s1, followed by 75°C for 0 s at a temperature transition rate of 0.1°C s1 with continuous data acquisition. For each step in the protocol the fluorescence display was F2/1. The melting temperatures of the wild-type and mutant alleles were 64.5°C and 57.5°C, respectively.
Haplotype analysis
Haplotype analysis was carried out using the D19S22032 33 and D19S4734 chromosome 19q microsatellite repeat markers that flank the RYR1 locus. The forward primer of each pair was labelled with 6-FAM and the microsatellite regions were amplified independently using standard PCR reactions. The reactions were diluted 1:5 in distilled water, pooled, and analysed on an ABI 37736 using Genescan software (Applied Biosystems). Allele identities and frequencies were as published. Three intragenic restriction fragment length polymorphism (RFLP) markers,35 Ile1151 (Taq I), Asp2729 (Fok I) and Ser 2862 (Cfo I), were typed by restriction endonuclease digestion of the amplified polymorphic regions. By convention, alleles that lack the polymorphic restriction sites were denoted 1 and those with the polymorphic restriction endonuclease sites were denoted 2.
SNAPshotTM mutation detection
A SNAPshotTM (Applied Biosystems) dideoxy primer extension method was used as a diagnostic test for the presence of the C1201T mutation according to the manufacturers instructions. PCR products were amplified from genomic DNA using the exon 12 primers described above and purified using the ConcertTM rapid PCR product purification kit (Life Technologies, Auckland, New Zealand). Five nanograms of PCR product were used in the primer extension reaction with the primer TGGTGCTGTGGATCATGC and the reactions were analysed on an ABI 37764 using Genescan software (Applied Biosystems).
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Results |
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IVCT conducted 2 yr later recorded a contracture of 5.5 g with 2% halothane (normal value <0.2 g) and 2.7 g with 2 mM caffeine (normal value <0.2 g), indicative of a strong MHS response. Histology and histochemistry were reported as normal. IVCT carried out at a later date in both mother (II-1, Fig. 1A; 2% halothane 1.7 g, 2 mM caffeine 0.35 g) and one sibling (III-2, Fig. 1A; 2% halothane 2.8 g, 2 mM caffeine 1.9 g) were also indicative of MHS status.
The medical and anaesthetic histories of three siblings were also of interest. The older sibling (III-2, Fig. 1A) had undergone repair of harelip and cleft palate in infancy. However, anaesthetic records were unavailable. Two younger siblings (III-4 and III-5, Fig. 1A) had received multiple anaesthetics for clubfoot surgery. The history of MH was unknown for these anaesthetic procedures and both children had received trigger agents. Neither child has been biopsied because of age, but in one procedure the younger of the two (III-5, Fig. 1A) developed tachycardia, pyrexia towards the end of the operation and tachypnoea in recovery. The older of the two (III-4, Fig.1A) had similar findings, with increased E'CO2. In retrospect these may have been developing MH reactions.
Family 2
The proband (I-2, Fig. 1B) was referred for muscle biopsy because of a history of sporadically elevated CK levels (5212 and 203 i.u.) and intermittent muscle pain and muscle cramps, often precipitated by exercise. These episodes occurred several years apart but lasted for 2 weeks, with difficulty in walking because of muscle pain rather than weakness. She had no previous general anaesthetics and there were no anaesthetic records for other family members. IVCT was indicative of MHS status and demonstrated a static halothane threshold of 1% (maximum contraction of 1.8 g), a dynamic halothane threshold of 1% (maximum contraction of 1.3 g) and a caffeine threshold of 1.0 mM. Histology showed numerous basophilic fibres with prominent nuclei and non-specific necrosis, type 2 fibre predominance with type 1 grouping. Regeneration was in phase, suggesting recent specific muscle injury coinciding with the high CK (5212 i.u.) and her symptoms at referral. Electron microscopy showed a few atrophic fibres with a few areas of Z-band streaming. A neurological examination was normal. Her son (II-1, Fig. 1B), who routinely suffers from muscle cramps after playing sport, also tested MHS by IVCT, with a static halothane threshold of 1% (maximum contraction of 0.8 g), a dynamic halothane threshold of 0.5% (maximum contraction of 1.2 g) and a caffeine threshold of 1.5 mM. Histology and histochemistry were normal.
Family 3
The proband (II-1, Fig. 1C) was referred for muscle biopsy because of elevated serum enzymes after exercise in hot weather, and a history of muscle cramps. CK was recorded at 899 i.u.. He had previously undergone two general anaesthetics with no apparent problems. IVCT results were indicative of MHS status: static halothane threshold of 0.5% (maximum contraction of 1.4 g), a dynamic halothane threshold of 0.5% (maximum contraction of 2 g) and a caffeine threshold of 1.5 mM. Histology and histochemistry showed an increased variability in fibre size and an increased number of internal nuclei in 30% of 140 fibres. Two of his daughters (III-1 and III-2, Fig. 1C) tested MHE by IVCT with the following results. III-1: static halothane normal, dynamic halothane threshold at 0.5% (maximum contraction of 0.5 g) and caffeine normal; III-2: static halothane threshold of 2% (maximum contraction of 0.4 g), dynamic halothane threshold of 0.5% (maximum contraction of 0.5 g) and caffeine normal. A neurological examination showed no evidence of clinical signs that might indicate MH. One of the probands daughters (III-1, Fig. 1C) had had four uneventful general anaesthetics and the muscle was morphologically unremarkable, with no evidence of myositis or other recognizable pathology. The other daughter (III-2, Fig. 1C) had no anaesthetic history, and normal skeletal muscle by histology. Electron microscopy showed some atrophic fibres indicative of non-specific atrophy.
RYR1 mutation screening
The RYR1 gene contains 106 exons spanning 160 kb of DNA36 but most MH-associated mutations are located within three hot-spot regions, designated MH/CCD regions 1, 2 and 3.19 Each of these regions was amplified using RT-PCR from RNA prepared from muscle biopsy tissue from individual III-1 in family 1 (Fig. 1A) and sequenced in both directions. Two novel silent polymorphisms (cgc/t-R2403 and gcc/t-A2427) were detected and one missense C1201T mutation (in exon 12 of RYR1) that substitutes Arg401 for Cys. Exon 12 was amplified from genomic DNA from the probands mother (II-1, family 1) and each of her offspring (III-2, III-4 and III-5), and sequenced. The C1201T mutation was identified in each of these individuals.
The C1201T mutation was also identified in individuals I-2 (Fig. 1B) and II-1 (Fig. 1C) after sequencing the majority of the RYR1 cDNA as described previously.19 The mutation was also identified in individual II-1 (Fig. 1B) by sequencing exon 12 amplified by PCR using primers 1143F and 1213R.5 The C1201T mutation was not identified in the two MHE individuals (III-1 and III-2 from family 3; Fig. 1C).
As the C1201T transition does not alter any informative restriction sites, alternative methods were used to screen for the mutation in the normal population. A total of 200 unrelated DNA samples (400 chromosomes) were screened by SSCP analysis or allele-specific PCR using the Light Cycler by amplifying genomic DNA. The mutation was not detected in any normal chromosomes from unrelated control subjects of either Maori or Caucasian descent. The mutation was also absent from MHS individuals in 32 unrelated New Zealand MH pedigrees, including three of Maori origin, as well as 21 unrelated Western Australian pedigrees.
Haplotype analysis
Microsatellite markers flanking the RYR1 locus and three intragenic RFLP markers were used to analyse the chromosome 19q13.1 genotype in each of the three families.3234 This analysis revealed a common haplotype that segregated with MHS in family 1 (Fig. 1A) although the small size of the pedigree prohibited a statistical evaluation. The markers were less informative for the Australian families. However, the 7 allele of the D19S220 marker32 33 clearly segregated with MHS in the New Zealand kindred (Fig. 1A) but was not present in either of the Australian families (Fig. 1B and 1C). The D19S47 genotypes were identical (4,8) for the two Australian families and a common allele is shared at the D19S220 locus (6,6 and 3,6) for families 2 and 3, respectively. The RFLP markers were all homozygous for family 2 and all heterozygous for family 3. However, insufficient samples were available for families 2 and 3 to allow phasing of the alleles to be determined, so the genotyping was of limited value.
Diagnostic testing for the C1201T mutation
Dideoxy primer extension (SNAPshotTM) was used to distinguish between the normal and mutant allele for family 1. DNA from all individuals known to have the C1201T mutation, as well as from 20 normal samples, was analysed by this method. The heterozygote can be clearly distinguished from the normal homozygote (Fig. 2).
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Discussion |
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Sequencing of either the entire RYR1 cDNA or mutation hot-spot regions of the RYR1 cDNA in three individuals suspected of being MHS has identified a novel C1201T transition that results in an Arg401Cys substitution in the N-terminal region of the RYR. All individuals from the three families investigated who were classified as MHS by IVCT also had the C1201T mutation. This mutation was not found in any of 200 normal individuals or in 32 New Zealand or 21 Australian MH families. These observations strongly suggest that the C1201T mutation is causative of MH in these three families. The mutation is situated two residues upstream of an Ile403Met mutation that has been shown previously to be associated with both MH and CCD in an Italian family.5 There was no evidence of CCD in any of the pedigrees with the C1201T mutation. Residue Arg401 is strictly conserved across all known sequences from the RYR family (Fig. 3). This, together with the proximity of the mutation to the previously reported Ile403Met mutation5 implies that this residue is functionally significant and is an important region in the MH disorder. Indeed, studies of calcium release using normal and mutant RYR-transfected HEK-293 cells have shown that the Ile403Met mutation is significantly more sensitive to both caffeine and halothane38 and is associated with higher resting cytoplasmic calcium levels, implying that the mutant channel may be leaky.39
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In two recent reports EIR has been correlated with mutations in RYR1 that are also causative of MH.14 15 These are the first reports providing clear evidence of mutations that cause MH also being associated with EIR. Numerous earlier reports have linked EIR with MH susceptibility by demonstrating a positive IVCT.9 10 40 41 Here we report a novel RYR1 mutation that has been identified in three separate MHS families, including two where the mutation is associated with EIR. Three siblings of family 1 (Fig. 1A) also had congenital musculoskeletal or connective tissue abnormalities. It is interesting to ask why the C1201T RYR1 mutation appears to provide different phenotypes, that is MH alone, MH plus congenital abnormalities, or MH with EIR. It is possible that other genetic factors contribute to different MH phenotypes, as proposed by Robinson and colleagues.42 The RYR1 defect could account for the MHS phenotype, while a mutation within another gene could modify this phenotype to produce either EIR or other musculoskeletal abnormalities. Clear evidence of the same RYR1 defect producing different phenotypes has been provided in the case of CCD and MH.21 Considering the variable penetrance of clinical MH, it is highly likely that modifier genes have an important role to play in this complex genetic disorder.
Congenital musculoskeletal and connective tissue abnormalities have been linked previously to MH susceptibility.43 Britt and Kalow43 noted a high incidence of abnormalities, including cramps, strabismus, hernia, hare lip and cleft palate, clubfoot, kyphoscoliosis, recurrent dislocating patella and other orthopaedic abnormalities. More recently, however, a prospective Scandinavian study of 210 patients investigated for MH found no significant increase in the incidence of congenital musculoskeletal or connective tissue abnormalities.44 In addition, Larach compared 198 patients with positive MH biopsies with 849 normal individuals with no personal or family history of MH, and found a similar incidence of muscle cramps in the two groups.45 Larach and colleagues also analysed 178 patients experiencing very likely or almost certain MH events and found a 22% incidence of musculoskeletal abnormalities, which included problems with muscle tone.46 The findings from a study of 400 patients undergoing IVCT in New Zealand indicate a low incidence of abnormalities (Pollock N; unpublished data).
Many of the abnormalities reported by Britt and Kalow43 occur with some frequency in the general population, and results of the later studies question whether an increased incidence of abnormalities exists in MHS individuals. However, these were largely retrospective studies and a large prospective study comparing MHS and MH normal individuals is required. MH-type reactions have, however, been reported in rare skeletal and connective tissue syndromes with genetic abnormalities.4750 Reactions have also been reported in families with musculoskeletal and connective tissue problems that affect multiple individuals,5154 with possible associated genetic abnormalities, as in family 1. In addition, the non-specific myopathic changes observed in families 2 and 3, as well as the incidence of EIR where there was also no known family history of MH suggest that an awareness of the possibility of an MH reaction in each of these groups may need to be exercised.
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References |
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