Human Molecular Genetics, 2000, Vol. 9, No. 10 1515-1524
© 2000 Oxford University Press
A novel ryanodine receptor mutation and genotype–phenotype correlation in a large malignant hyperthermia New Zealand Maori pedigree
Institute of Molecular BioSciences, Massey University, Private Bag 11-222, Palmerston North, New Zealand, 1Department of Anaesthesia and Intensive Care, Palmerston North Hospital, Ruahine Street, Palmerston North, New Zealand, 2Department of Neurology, Auckland Hospital, Private Bag 92024, Auckland, New Zealand, 3Ngati Raukawa, Ki Te Tonga, Aoteoroa, New Zealand and 4Department of Biochemistry, University College Cork, Cork, Ireland
Received 14 February 2000; Revised and Accepted 14 April 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Malignant hyperthermia (MH) is a pharmacogenetic disorder that predisposes to a sometimes fatal hypermetabolic reaction to halogenated anaesthetics. MH is considered to originate from abnormal regulation of skeletal muscle Ca2+ release. Current diagnosis of MH susceptibility (MHS) relies on in vitro contracture testing (IVCT) of skeletal muscle. The ryanodine receptor (RYR1) encoding the major Ca2+ release channel in the skeletal muscle sarcoplasmic reticulum has been shown to be mutated in a number of MH pedigrees. The large Maori pedigree reported here is the largest MHS pedigree investigated to date and comprises five probands who experienced clinical episodes of MH and 130 members diagnosed by the IVCT. Sequencing of the 15 117 bp RYR1 cDNA in a MHS individual from this pedigree identified a novel C14477T transition that results in a Thr4826 to Ile substitution in the C-terminal region/transmembrane loop of the skeletal muscle ryanodine receptor. This is the first mutation in the RyR1 C-terminal region associated solely with MHS. Although linkage analysis showed strong linkage (max LOD, 11.103 at
= 0.133) between the mutation and MHS in the pedigree using the standardized European IVCT phenotyping protocol, 22 MHS recombinants were observed. The relationship between the IVCT response and genotype was explored and showed that as IVCT diagnostic cut-off points were made increasingly stringent, the number of MHS discordants decreased with complete concordance between the presence or absence of the C14477T mutation and MHS and MH normal phenotypes, respectively, using a cut-off of 1.2 g tension at 2.0 mM caffeine and 1.8 g tension at 2.0% halothane. Many MHS pedigrees investigated have been excluded from linkage to the RYR1 gene on the basis of a small number of recombinants; however, the linkage analysis reported here suggests that other recombinant families excluded from linkage to the RYR1 gene may actually demonstrate linkage as the number of members tested within the pedigrees increases. The high number of discordants observed using the standardized diagnostic cut-off points is likely to reflect the presence of a second MHS susceptibility locus in the pedigree. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Malignant hyperthermia (MH) is a dominantly inherited skeletal muscle disorder that predisposes susceptible individuals to a potentially fatal reaction during general anaesthesia (1). The reaction apparently results from a rapid, sustained rise in myoplasmic Ca2+ triggered by commonly used inhalational anaesthetics, and depolarizing muscle relaxants (2–4). A MH crisis constitutes a hypermetabolic state manifesting as metabolic and respiratory acidosis, hyperthermia, tachycardia, cardiac arrythmias, skeletal muscle rigidity and rhabdomyolysis (5). The formerly high morbidity rate (70%) has been ameliorated with the introduction of sodium dantrolene, a Ca2+ channel blocking agent that can usually halt the crisis if administered when early symptoms are recognized (6,7). ‘Awake’ reactions have been described in which MH-like events are triggered in MH-susceptible (MHS) subjects in the absence of anaesthesia (8). Although the only clinical myopathy consistently associated with MH is the inherited myopathy central core disease (CCD) (9,10), sudden infant death has been reported in connection with MH in Australian (11), Scandinavian (12) and American (13) studies.
Physiological and biochemical studies of skeletal muscle from MHS pigs and humans revealed abnormalities in the regulation of Ca2+ release from the sarcoplasmic reticulum (SR) Ca2+ channel (2,4). Muscle from MHS individuals is hypersensitive to the contracture-inducing effects of agents that stimulate Ca2+ release from the SR. Diagnosis of susceptibility relies on the measured in vitro contracture responses of skeletal muscle to caffeine (Caf) and halothane (Hal) (14). The standardized European in vitro contracture test (IVCT) allows the following diagnoses to be made: MH susceptible (MHS), MH equivocal (MHE) and MH normal (MHN). If a muscle biopsy strip produces a sustained increase in muscle tension of 0.2 g at 
2% Hal and independently at a Caf concentration of 2 mM, the patient is considered MHS. MHN is diagnosed if the 0.2 g threshold is not attained at these concentrations while MHE is diagnosed if the threshold tension is attained with Caf [MHE(c)] or with Hal [MHE(h)] but not with both.
Molecular genetic studies in humans and pigs have established the ryanodine receptor gene (RYR1) on chromosome 19 as the primary locus for MHS (15,16) (designated the MHS1 locus OMIM 145600). The RyR1 protein has a subunit size of 560 kDa and forms an elaborate tetrameric structure that acts as a Ca2+ release channel and forms a large myoplasmic ‘foot’ structure that bridges the gap between the SR and the t-tubule in skeletal muscle (reviewed in ref. 2). The C-terminal region of the receptor forms the transmembrane channel, which may contain between four (17) and 10 (18) transmembrane helices. The RYR1 gene encodes a 15.3 kb mRNA (17,18) and comprises 106 exons encompassing a total of 160 kb, and as such is one of the most complex genes characterized to date (19). In addition to the MHS1 locus, five other loci (MHS2–6, OMIM 154275, 154276, 600467, 601887 and 601888, respectively) have been tentatively identified by linkage analysis (20–23). However, outside of the MHS1 locus, only the MHS4 and MHS5 loci on chromosomes 3q13.1 and 1q, respectively, have generated LOD scores greater than 3.0 in favour of linkage to MHS. A mutation in the CACNL1A3 gene encoding the main subunit of the voltage-gated dihydropyridine receptor that interacts with the RyR1 channel has been confirmed for the 1q locus (24).
To date, 22 missense mutations in the 15 117 bp RYR1 cDNA have been found to segregate with the MHS trait while a much smaller number of these mutations is also associated with CCD. The majority of RYR1 mutations appear to be clustered between amino acid residues 35 and 614 (MH/CCD region 1) and amino acid residues 2163 and 2458 (MH/CCD region 2) and are predicted to reside in the myoplasmic foot region of the protein. Recent sequencing of the complete cDNA in a CCD/MHS family identified an Ile4898Thr mutation in the highly conserved C-terminus of the protein (25). This mutation, unlike other MHS/CCD mutations, is predicted to be located in the luminal/transmembrane region of the protein and represents a new region of the gene (MH/CCD region 3) where MHS/CCD mutations may cluster. All individuals bearing the mutation had clinical symptoms of CCD ranging from mild to severe and the two individuals tested within the family were MHS (25).
Reports of discordance between mutations and IVCT phenotype have questioned the causal role of some RYR1 mutations (26–28), though the lack of specificity of the IVCT (29–32) or the presence of additional MH mutations (26,32,33) may account for some discrepancies.
Here, we report a novel mutation in the C-terminal transmembrane channel domain of RyR1 that is associated with MHS but not with clinical CCD in a large Maori MH pedigree. We also show that the level of discordance between the MHS phenotype and the segregating mutation is high when the standardized MHS diagnostic criteria are applied but becomes successively smaller as the stringency of the diagnostic criteria is increased.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Linkage to chromosome 19q markers
MH in New Zealand has a national incidence of 1 in 80 000 anaesthetics. The frequency is somewhat elevated in the lower North Island due to the presence of a large MH Maori pedigree from which over 1500 members spanning seven generations have been traced. In the extended pedigree, 17 known MH reactions (including five fatalities) have been documented.
Members of one large branch of the Maori pedigree were sampled initially (Fig. 1A) and DNAs were genotyped for a microsatellite in the RYR1 gene (RYR1CA) (34), three RYR1 intragenic restriction fragment length polymorphisms (RFLPs) (35) (data from the three RFLP markers were combined and represented as one locus RFLP3) and RYR1-linked microsatellites D19S220 (36), D19S190 (37) and D19S47 (38). Pairwise LOD scores for linkage between MHS and RYR1-linked markers were calculated using the LINKAGE version 5.1 package (Table 1A). The maximum LOD score generated was Zmax = 4.6, = 0.031 for the marker D19S47 and this clearly shows that MHS is linked to the RYR1 region of chromosome 19. The inheritance of the ‘high-risk’ haplotype of alleles 6-1-4-1-3 for the markers D19S220, RYRCA, RFLP3, D19S190 and D19S47 respectively, coincides with the inheritance of the MHS phenotype in 12 of 14 MHS individuals in this branch of the MH1 pedigree and was not observed in any of the seven MHN subjects or seven MHE patients (Fig. 1A). However, two individuals (VII:41 and VII:47) diagnosed MHS according to European MH Group (EMHG) diagnostic criteria did not inherit the 6-1-4-1-3 haplotype. This result suggests that MHS in the MH1 pedigree arises from a mutation in a RYR1-linked gene distal to either D19S47 or D19S220 rather than within RYR1 itself. Since the specificity of the EMHG IVCT is <100% (39), an alternative explanation is that the individuals VII:41 and VII:47 may be false positives with respect to phenotyping for genetic analysis purposes. The latter possibility has been tested in other discordant families where alteration of the IVCT diagnostic cut-off points has permitted investigators to link MHS families tested using the European (31) or North American protocols (30,40) to the RYR1 gene, which were previously unlinked when conventional cut-off points were applied. In order to test this possibility, conventional cut-off points were raised to the highest level applied by Healy et al. (31), i.e. tensions 0.4 g at 2 mM Caf and 0.8 g at 2% Hal and pairwise LOD scores were recalculated. The maximum LOD score generated using the more stringent MHS phenotype was Zmax = 7.551, = 0.00 for the D19S47 marker (Table 1B) and the inheritance of the ‘high-risk’ haplotype 6-1-4-1-3 was concordant with the more stringent MHS phenotype in all individuals. Using the elevated cut-off points individuals VII:41 and VII:47 who were diagnosed as MHS according to the accepted standardized EMHG criteria were reclassified as a MHE(c) and MHE(h) phenotype, respectively, using the more stringent criteria (individuals VII:41 and VII:47: tension at 2 mM Caf/2% Hal: 0.6 g/0.4 g and 0.3 g/1.2 g respectively, Caf/Hal concentration where a threshold value of 
0.2 g tension was attained; 2 mM/2% and 2 mM/1% respectively).
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RYR1 mutation screening
Following the linkage analysis, the RYR1 gene from MHS individuals with the stringent phenotype was screened for the presence of 13 published RYR1 mutations (41). None of the published mutations were detected. The entire RYR1 coding region and 5' untranslated region (UTR) was amplified using RT–PCR on RNA isolated from skeletal muscle and sequenced in the search for a novel mutation. The cDNA sequence of MHS individual VII:44 bearing the high-risk 6-1-4-1-3 haplotype was compared with the cDNA sequence of his MHE(h) sibling (VII:42, Fig. 1A) who shared a non-disease-associated maternal haplotype but lacked the MH-associated paternal haplotype. This strategy aided the distinction of candidate mutations from non-disease-linked polymorphisms, since any sequence variants unique to the MHS individual could be directly attributed to the 6-1-4-1-3 high-risk haplotype. Fifteen sequence differences were detected that corresponded to previously published polymorphisms (35,42). None of the previously reported mutations or any candidate novel mutations were detected in the mutation-rich regions in the 5' or central region of the RYR1 gene from individual VII:44. One new sequence discrepancy was encountered (in both MHS and MHN patients) resulting from the resolution of a probable GCCG compression in the original sequence to GCGC, changing Val2550 to Leu. In addition, seven new silent polymorphisms (tgc/t-C2363, ccc/t-P2366, cgc/t-R2403, gcc/t-A2427, act/a-T3918, gcg/c-A4293 and aca/c-T4752) and two missense mutations (c/gag-Gln3756Glu and ac/tc-Thr4826Ile) were detected. The Gln3756Glu is a conservative substitution and was not considered to be a candidate MH mutation since the mutation was detected only in the MHE(h) sibling. Furthermore, the corresponding residue in the rabbit (17) and porcine (43) RYR1 sequences is also Glu. The second candidate mutation, a C14477 to T transition in exon 100 substitutes Ile for Thr at position 4826. This mutation was present only in the cDNA from the MHS sibling and is thus in phase with the MHS high-risk haplotype. The Thr4826Ile mutation was a strong candidate MH mutation, as T4826 was strictly conserved (Fig. 2) and was the only disease-linked missense mutation detected from the sequence analysis.
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Association of the Thr4826Ile mutation with CCD was investigated by a standard series of stains and reactions (44) in muscle biopsy specimens. None of the four specimens tested showed any evidence of central core disease.
Segregation of the Thr4826Ile mutation
As the C14477T transition does not alter any informative restriction sites, non-radioactive single-stranded conformational polymorphism analysis (SSCP) was employed to screen for the mutation in a 208 bp PCR fragment amplified from genomic DNA. The mutation was not detected in 220 normal chromosomes from unrelated control subjects of both Maori (n = 36) and Caucasian (n = 74) descent. The mutation was also absent from MHS individuals in 32 unrelated New Zealand MHS pedigrees, including three of Maori origin. A total of 212 members (including 130 members that were tested by IVCT) of the extended seven generation Maori pedigree (MH1) were sampled and typed for the C14477T mutation (Fig. 1B). Of the 130 members tested by IVCT in the extended pedigree, 94 are negative for the mutation and 36 are positive. All 36 mutation-positive subjects are MHS. Of the 94 mutation-negative individuals, 27 are MHN, 43 are MHE(h), two are MHE(c) and 22 are MHS. The mutation was detected in the DNA of five family members who survived clinical MH reactions, including one individual (VII:14, Fig. 1B) who suffered two MH crises and whose mother died of MH. Immediate relatives of the five probands also tested positive for the Thr4826Ile mutation, as did all obligate carriers of the disease in four major branches of the pedigree (Fig. 1B). Pairwise LOD scores were calculated (Table 2) and show that the maximum LOD score generated in favour of linkage between C14477T and MHS was Zmax = 11.103, = 0.133 and this clearly shows that the mutation is linked to MHS in the extended pedigree. The 22 MHS discordant individuals suggest that the mutation may not be causative of MHS; however, the conserved nature of the mutation and its absence from the normal population suggest that alteration in RyR1 function is causal of MHS in this pedigree.
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The IVCT phenotypes were reclassified as described by Healy et al. (31) to observe the impact of applying more stringent diagnostic criteria to the MHS phenotype on the linkage analysis. The maximum LOD score generated using the more stringent phenotype MHS(s) was Zmax = 23.725,
= 0.024 (Table 2). The number of discordant MHS individuals using the more stringent criteria decreased from 22 to nine.
Systematic elevation of the stringency of the criteria to assign a MHS phenotype with respect to tensions generated at 2.0 mM Caf and 2.0% Hal showed that as cut-off points were made increasingly more stringent with respect to tensions attained, the number of MHS discordants decreased (Table 3). Using a cut-off of 1.2 g tension at 2 mM Caf and 2% Hal, there was only one MHS discordant and no discordants for individuals who did not attain these elevated tensions. Using a cut-off of 1.2 g tension at 2.0 mM Caf and 1.8 g tension at 2.0% Hal, all individuals with the stringent MHS phenotype were positive for the C14477T mutation, whereas all individuals not attaining these tensions in the IVCT were negative for the mutation. According to the standardized criteria, MHS is diagnosed when a threshold tension of 0.2 g is recorded at or below 2 mM Caf and independently at or below 2% Hal. When the criteria for MHS diagnosis are made more stringent with respect to the concentration of Caf and Hal required to attain the threshold tension of 0.2 g, the number of MHS discordants using the more stringent phenotyping criteria also decreased systematically to the level where no discordants were observed using a concentration of 0.5 mM Caf and 0.5% Hal. However, at this concentration 21 individuals positive for the mutation did not have the stringent phenotype.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Linkage analysis in the large MH pedigree presented here showed strong linkage between the Thr4826Ile mutation identified and MHS despite the presence of 22 MHS recombinant individuals. This is the first pedigree described that has a significant number of recombinants that still display linkage to the RYR1 region of chromosome 19. The extensive size of the pedigree means that it can accommodate a sizeable number of recombinants without losing significant resolution in the linkage analysis and this is the most likely explanation as to why linkage between RYR1 and MHS is observed despite the large number of discordants. This suggests that other discordant families excluded from linkage to the RYR1 gene may actually demonstrate linkage as the number of members tested within the pedigrees grows. This would also explain why genome scans for new MH loci in several discordant families have only identified two single families definitely linked to loci other than RYR1 (22–24). Using the standard EMHG diagnostic threshold, the sensitivity and specificity was 100 and 76.6%. This is considerably different to the respective values of 99 and 93.6% reported by Ording et al. (39), and the respective values of 98.5 and 81.8% more recently reported (45). In all discordant cases, haplotype profiles are consistent with alleged paternity and key recombinant individuals tested negative for eight common RYR1 mutations and the reported CACNL1A3 mutation (24).
Exploration of the relationship between the IVCT response and genotype have been reported previously (30,40,46). In the pedigree presented here, increasing the stringency of the IVCT diagnostic cut-off points with respect to assignment of a MHS phenotype had the effect of decreasing discordance to the level where complete concordance was observed using a cut-off of 1.2 g tension at 2.0 mM Caf and 1.8 g tension at 2.0% Hal.
The contracture responses to 2% Hal are plotted against the 2 mM Caf-induced tensions (Fig. 3) for patients identified with and without the Thr4826Ile mutation. A linear correlation between the Caf and Hal data was found (r2 = 0.83) with mutation-positive individuals falling on the upper right quadrant of the graph. The average (±SEM) 2% Hal and 2 mM Caf induced IVCT contractures are 4.4 g (±0.24) and 2.88 g (±0.16), respectively, for the group heterozygous for the Thr4826Ile mutant allele and 0.45 g (±0.06) and 0.15 g (±0.03) for the mutation-negative group. Inspection of the Caf test responses indicate that a 2 mM Caf threshold of 1.2 g would distinguish the mutation-positive and mutation-negative genotype groups.
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There are several possible explanations for the high level of discordance observed in the Maori pedigree between the mutation genotype and the MHS phenotype assigned according to the standardized EMHG criteria. It is tempting to presume that the mutation-negative individuals diagnosed as MHS using the standardized EMHG IVCT criteria are false positives in this pedigree. However, this is not an acceptable conclusion because application of the stringent criteria necessary to achieve concordance in this pedigree would create a considerable number of false negative diagnoses in other pedigrees. In particular, the cut-off point of 1.2 g tension at 2.0 mM Caf and 1.8 g tension 2.0% Hal used for assignment of the MHS phenotype and where concordance was observed is higher than the average tensions generated in individuals with the known causal MHS mutations Cys35Arg, Arg614Cys, Val2168Met, Thr2206Met and Arg2458Cys (41). Thus, for clinical diagnosis of the MHS phenotype using the IVCT, it is critical that the standardized EMHG thresholds of 0.2 g tension at 2.0 mM Caf and 2.0% Hal be maintained.
The EMHG IVCT has proved itself well as a phenotyping method as judged by the success of genetic studies on the disorder, and the Caf portion of the test in particular is very robust with little indication of interlaboratory variation (41). Thus a likely explanation for the high level of discordance observed between MHS and the mutation genotype is that there are two MHS genetic susceptibility loci segregating with MHS in this family. The RYR1 defect could account for a strong IVCT response and susceptibility to clinical MH, a mutation/polymorphism at another locus could account for a weak MHS response and indeed, this kindred potentially constitutes a pedigree with the power to map a second locus. In such a model the penetrance of clinical MH might be highest if an individual inherited both loci. Such a model would explain several anomalies in MH literature such as: (i) the apparent high incidence (1.5%) of MHS in the low-risk population (39); (ii) the apparent low incidence of clinical MH in MHS individuals exposed to anaesthetic agents; and (iii) the apparent discordance between MHS and RYR1 mutations observed in several families.
The Thr4826Ile mutation and the associated chromosome 19q haplotype was detected in the DNA extracted from autopsy samples from two distantly related young children, aged 9 and 24 months (VII:59 and VIII:1, respectively) who have suffered sudden unexplained deaths in the absence of any known triggering agents. In both cases death was preceded by muscle rigidity and hyperthermia. In the case of the 24-month-old child, analysis of post-mortem eye fluid did not reveal raised creatine kinase (CK). The case of the 9-month-old infant whose closely related parents were both MHS and positive for the mutation was suggestive of the expression of a more severe phenotype, possibly resulting from the inheritance of two copies of the disease allele (25% likelihood). An analysis of the DNA extracted from paraffin-embedded autopsy spleen tissue stored for 30 years revealed that this was not the case as the infant was heterozygous for both the Thr4826Ile mutation and RYR1 RFLP markers, with maternal inheritance of the mutant allele.
The Thr4826 to Ile substitution identified in the large MH Maori pedigree presented here is in the C-terminal region/transmembrane loop of the RyR1 channel and satisfies the criteria expected of a causal mutation. Recently, the first C-terminal RyR1 (Ile4898Thr) mutation associated with CCD and MHS was reported (25). The Ile4898Thr mutation is considered to be located in the cytoplasmic loop between M2 and M3 transmembrane domains or the luminal loop between M7 and M8 transmembrane domains of the RyR1 channel according to the models by Takeshima et al. (17) and Zorzato et al. (18), respectively, and is tightly associated with symptomatic CCD in the Mexican pedigree where it was described (25). The mutation reported here in the Maori pedigree is relatively close to the Ile4898Thr mutation; however, clinical symptoms of CCD were not present in Thr4826Ile carriers in the pedigree. The mutation reported here is the second mutation in the C-terminal region of the RYR1 gene and confirms this as a third region for MH/CCD mutations.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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DNA and tissue samples
Blood and tissue samples were obtained with informed consent from 260 members of a large Maori MH family (MH1) participating in this study (Fig. 1). Procedures for the storage and analysis of samples were established in consultation with a Ngati Raukawa ki te Tonga tribal authority. Ethical approval was obtained from the Manawatu-Whanganui and Massey University ethics committees. Five members (individuals VI:27, VI:31, VI:41, VI:58 and VI:64) of the MH1 family suffered fatal MH reactions (Fig. 1). Siblings of individuals VI:31 and VI:58 and a son of individual VI:64 were subsequently diagnosed MHS by IVCT. Fulminant MH episodes have been documented in seven other family members (individuals VI:111, VII:14, VII:36, VII:63, VII:75 and VII:84). Susceptibility to MH was confirmed in the latter two individuals by positive IVCT. In retrospect, three individuals (VII:14, VII:36 and VII:84) had previously had at least one other abnormal reaction to anaesthesia. Members of the pedigrees presented to the New Zealand MH testing centre in Palmerston North and were tested for susceptibility to MH by the standardized European protocol (14), which has been adopted in New Zealand since it was established in 1984.
Two children from the MH1 pedigree (individuals VII:59 and VIII:1) aged 9 and 24 months died sudden unexplained deaths in the absence of known triggering agents. In both cases death was preceded by febrile episodes. Individual VIII:1 was suffering from influenza and leg pain. Medical attention was sought after the child became pyrexic and rigid. Resuscitation attempts were hindered by masseter muscle rigidity and excessive surface temperature was noted. Analysis of post-mortem eye fluid did not reveal elevated levels of CK. The mother of individual VIII:1 was diagnosed MHS. Individual VII:59 was born to a consanguineous partnership between two MHS individuals.
Isolation of genomic DNA
Genomic DNA was isolated using the Wizard DNA extraction kit (Promega Corp., Madison, WI) according to the manufacturer’s instructions. Paraffin-embedded autopsy spleen samples were obtained from infants belonging to the large Maori family (MH1) who suffered sudden unexplained deaths. DNA was prepared from autopsy tissue with prolonged treatment with proteinase K followed by phenol/chloroform extraction (47).
Screening for published mutations
DNA from MHS individuals from the large Maori family was screened for the presence of 13 published RYR1 mutations: Cys35Arg (48), Arg163Cys (49), Gly248Arg (35), Gly341Arg (50) Ile403Met (49), Tyr522Ser (51), Arg552Trp (52), Arg614Cys (53), Arg614Lys (54), Arg2163Cys, Arg2163His (41), Gly2435Arg (32,55), Arg2436His (42) and one CACNLA3 mutation (24) by PCR amplification of the appropriate regions from genomic DNA followed by RFLP analysis or direct sequencing.
Linkage analysis
Members of the Maori family were typed for the three RYR1 RFLP polymorphisms (53) Ile1151, Asp2729 and Ser2826 (analysed with TaqI, FokI and CfoI, respectively) and four chromosome 19 microsatellite repeat markers flanking the RYR1 locus [D19S220 (36,56), RYR1-CA (34), D19S190 (37) and D19S47 (38)]. Microsatellite markers were amplified by PCR on genomic DNA using 33P-labelled primers and resolved on denaturing 5–8% polyacrylamide gels, with sequenced M13 as a size marker. Two-point LOD scores for IVCT phenotype versus individual markers were calculated using Linkage version 5.1 applying parameters recommended by the EMHG genetics section (27). Allele frequencies for the chromosome 19 microsatellite markers were as published. Allele frequencies for the combined RYR1 RFLP locus were estimated from observed allele frequencies in 50 unrelated Caucasian members of the population.
Mutation screening by RT–PCR and sequencing
Total RNA was extracted from 30–100 mg of frozen skeletal muscle tissue using TrizolTM RNA extraction reagent (Life Technologies). Typical yields were 70–80 µg RNA per 100 mg of tissue. First strand synthesis was carried out using the Superscript reverse transcriptase pre-amplification system (Life Technologies) with 4 µg of total RNA and 50 ng of random hexamers or 500 ng oligo(dT) in a 20 µl volume. Hot-start PCR was performed using 1 µl of a 20-fold dilution of the cDNA template in 50 µl reactions with 0.32 µM of each primer, 0.3 mM dNTPs, 1.5 mM MgCl2 and 1.5 U of Taq polymerase (Life Technologies). The 630 bp region encompassing the C14477T mutation was amplified from cDNA using forward (5'-GAACCCGCCCTGCGCTGTCTG-3') and reverse (5'-GTAGACGACCACCGCCAGAAG-3') primers. PCR products amplified from cDNA and genomic DNA were screened for novel mutations in RYR1 by direct sequence analysis using an Applied Biosystems 377-18 automatic DNA sequencer with dye terminator chemistry (PE Corporation, Foster City, CA). The entire 15 117 bp coding region was amplified in overlapping fragments ranging from 300 to 1500 bp and sequenced to obtain 350–450 bp stretches of unambiguous sequence with at least a 50 bp overlap. RYR1 mutation-rich regions and areas of ambiguity were sequenced in both directions.
SSCP detection of the Thr4826Ile mutation
Non-radioactive SSCP was employed to detect the Thr4826Ile mutation. A 208 bp fragment within exon 100 of RYR1 was amplified by PCR using the forward (5'-ACCTGGGCTGGTATATGGTG-3') and reverse (5'-TTATCCCTTCACCACCCACT-3') primers in a 50 µl reaction volume with 200–500 ng genomic DNA, 0.32 µM of each primer, 0.3 mM dNTPs, 1.5 mM MgCl2 and 1.5 U of Taq polymerase (Life Technologies). The cycle consisted of an initial 3 min denaturation at 94°C followed by 33 cycles of 94°C for 20 s, 58°C for 30 s and a 72°C extension for 45 s. Ten microlitres of PCR product was diluted with 20 µl of 0.5 x TBE (44.5 mM Tris–HCl, pH 8.0, 44.5 mM boric acid and 0.1 mM EDTA) and 5 µl of 98% formamide dye. PCR products were denatured by heating at 95°C for 5 min and cooled rapidly by plunging into an ice–acetone bath. Twenty-five microlitres of the denatured DNA was resolved on a 16 x 16 cm polyacrylamide gel (monomer:bis, 19:1) at 280 V and 3 W constant power for 3.25 h at 4°C in 0.5 x TBE buffer. Gels were stained in 2 µg/ml ethidium bromide, and photographed under UV at 254 nm.
Linkage analysis was performed using two sets of criteria for establishing MHS phenotype. Standard EMHG IVCT thresholds (0.2 g) were applied in the initial analysis. Under the second diagnostic scheme, higher thresholds of 0.4 g Caf and 0.8 g Hal as described by Healy et al. (31) were applied to classify patients as MHS. Individuals with IVCT tensions greater than or equal to the assigned thresholds in response to only one of the test agents (MHE) were entered as ‘status unknown’ for linkage analysis under both sets of diagnostic criteria. Data from 26 individuals tested before the adoption of the standardized IVCT procedure in 1984 were excluded from the analysis.
Muscle histochemistry
Ten-micron sections from frozen muscle specimens were stained or reacted for haematoxylin and eosin; modified Gomori trichrome; NADH-tetrazolium reductase; ATPase (after preincubation at pH 4.3, 4.6 and 9.4); acid phosphatase; periodic acid-Schiff; and oil red O (44).
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ACKNOWLEDGEMENTS |
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We would like to express our gratitude to all the patients and relatives that took part in this study and thank Danielle James and Robyn Marston for technical assistance and Dr Richard Coutts for performing the muscle biospies. R.L.B was supported by a Massey University Doctoral Scholarship. This work was supported by grants from the Australian and New Zealand College of Anaesthetists, The New Zealand Lotteries Grants Board and the Palmerston North Medical Research Foundation.
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FOOTNOTES |
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+ Present address: Clinical Pharmacology Unit, Level 6, Centre for Clinical Investigation (ACCI), Addenbrooke’s Hospital, Box 110, Hill’s Road, Cambridge CB2 2QQ, UK
§ To whom correspondence should be addressed. Tel: +64 06 3505515; Fax: +64 06 350 5688; Email: k.m.stowell@massey.ac.nz
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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