Identification of genetic mutations in Australian malignant hyperthermia families using sequencing of RYR1 hotspots.
Advances in analysis of the RYR1 gene (which encodes the skeletal muscle ryanodine receptor) show that genetic Examination is a useful adjunct to the in vitro contracture test in the diagnosis of malignant hypothermia, as defects in RYR1 have been shown to be responsible for malignant hyperthermia susceptibility. DNA from 34 malignant hyperthermia susceptible individuals and four malignant hyperthermia equivocal subjects was examined using direct sequencing of 'hot-pots' in the RYR1 gene to identify mutations associated with malignant hyperthermia. Seven different causative mutations (as defined by the European Malignant Hyperthermia Group) in nine malignant hyperthermia susceptible individuals were identified. In another six malignant hyperthermia susceptible individuals, five different published but as yet functionally uncharacterised mutations were identified. A further three as yet unpublished and functionally uncharacterised (novel) mutations were identified in three malignant hyperthermia susceptible samples If the novel and previously published mutations prove to be functionally associated with calcium homeostasis, then this method of analysis achieved a mutation defection rate of 47910. Based on the number of relatives presenting to our unit in the study period, the muscle biopsy rate would have decreased by 25%. That we only identified a genetic defect in RYR1 in 47% of in vitro contracture test positive individuals suggests that there are other areas in RYR1 where pathogenic mutations may occur and that RYR1 may not be the sole gene associated with malignant hyperthermia. It may also reflect a less than 100 pecificity of the in vitro contracture test.
Key Words: malignant hypothermia, genetics, diagnosis, physiopathology, DNA sequencing, RYR1, ryanodine receptor, calcium release channel, in vitro contracture test, novel mutation, general anaesthesia, chromosome 19
Malignant hyperthermia (MH) is an acute pharmacogenetic disorder, which develops during or immediately after the application of general anaesthesia. The classic MH crisis consists of a hyper-metabolic state caused primarily by continued contraction of the skeletal muscles, which leads to massive C[O.sub.2] production, skeletal muscle rigidity, tachyarrhythmias, unstable haemodynamics, respiratory acidosis, cyanosis, hyperkalaemia, lactic acidosis, fever and eventually (if untreated) death'.
The disorder is essentially a result of a defect in calcium homeostasis(2), which can result from an alteration in DNA coding for the ryanodine receptor (RYR1) on chromosome 19q(3,4). Volatile anaesthetic agents and depolarising muscle relaxants interact with the calcium channel resulting in the clinical crisis.
The RYR1 gene is very large (contains a total of 106 exons and 15,000 bases of coding sequence). Mutations in three 'hot-spot' areas of the RYR1 gene have been shown to be functionally responsible for up to 50% of malignant hyperthermia susceptible (MHS) patients in European populations'. These areas include the N-terminal region between codons 34 and 614 (exons 2 to 19), the central region between codons 2163 and 2458 (exons 39 to 47) and the C-terminal region between codons 4136 and 4973 (exons 85 to 104). The European Malignant Hyperthermia Group (EMHG) Guidelines(6) state that patients displaying one of the causative mutations in the RYR1 gene can be diagnosed as MHS without the need for a muscle biopsy.
The aim of this study was to identify mutations associated with MH in Australian families by direct sequencing of these previously outlined 'hot-spots' in the RYR1 gene and to use this information to decrease the number of family members requiring muscle biopsy and in vitro contracture test (IVCT) for diagnosis of MH. Secondary aims were to add to the international pool of RYR1 defects that may be associated with MH and to identify those mutations occurring more commonly in the Australian population.
MATERIALS AND METHODS
Approval was granted for this project by our Institutional Human Ethics Committee. Participants were chosen from records of MH families at the Royal Melbourne Hospital between 1977 and 2004. The majority were referred after an episode of possible MH and as far as possible these episodes were given a clinical grading score 7. Clinical details are outlined in Tables 1 and 2.
The method of contracture testing changed in 2001 to the EMHG IVCT(8). Prior to this the criteria for defining MHS were >0.2 g response to 2 mmol caffeine and a > 1 g response to aeration with carbogen bubbled through a saturated solution of halothane in water (RMH protocol). Four patients were included who had positive caffeine halothane contracture tests(9) performed in another institution. While most subjects were MHS according to the above criteria, we also included four malignant hyperthermia equivocal (MHE) subjects. MHE was defined according to the EMHG criteria regardless of the test method used, so that a result was considered MHE if they had a threshold response to caffeine or halothane but not both.
After appropriate provision of information and written consent, blood was collected and DNA extracted.
Genomic DNA was extracted from unclotted blood using the Nucleon DNA extraction kit (Amersham Biosciences, U.K.). Amplification of genomic DNA was performed with a standard polymerase chain reaction (PCR) method using Hotstar TAQ (Qiagen, Valencia, CA), consisting of a denaturation at 95[degrees]C for 15 minutes, followed by 35 cycles of 95[degrees]C for 30 seconds, annealing at 55 or 59[degrees]C for 30 seconds (optimised for each primer pair), extension at 72[degrees]C for 90 seconds, with a final extension at 72[degrees]C for 10 minutes. The PCR mixture contained 50 ng DNA, 5 [micro]1 2 mM deoxyribonucleotide triphosphates (dNTPs) (lithium salts), 5 [micro]1 10x Hotstar Buffer, 3 or 5 [micro]1 additional 25 mM MgC[1.sub.2], 5 or 10 pmol of each primer (forward and reverse), 0.5 or 1 U Hotstar TAQ, in a final volume of 50 [micro]1. The exact composition of the PCR reaction mixture was optimised for each primer pair individually. Primer pairs were prepared for exons 1, 2, 3, 4-5, 6, 7, 8-9, 10-11, 12, 13, 14-16, 17, 18, 19, 20, 38, 39, 40-41, 42, 43, 44, 45, 46-47, 85-87, 98-99, 100-101, 102, 103-104 of the RYR1 gene which account for the 'hot-spots' of known MH causative mutations. Primer sequences are presented in Table 3.
The PCR products were sequenced in forward and reverse directions using the fluorescent DYEnamic ET dye terminator kit (Amersham Biosciences, U.K.). The sequencing PCR mixture contained 5 [micro]d of DNA product from the Hotstar amplification PCR, 4 [micro]1 ET Mix and 10 pmol of the same primer pairs used in the amplification PCR, in a final volume of 10 [micro]1. The reaction consisted of 35 cycles of 95[degrees]C for 20 seconds, 50[degrees]C for 15 seconds and 72[degrees]C for 120 seconds. The fluorescent products were purified using AutoSeg96 plates (Amersham Biosciences, U.K.) after the addition of 10 [micro]1 [H.sub.2]O to each well and sequenced on a MegaBACE 1000 (Molecular Dynamics, Sunnyvale, CA, U.S.A.). Electro-pherograms were analysed with Mutation Surveyor 3.01 software (SoftGenetics, State College, PA, U.S.A.).
Mutations were considered likely to be causative if the following criteria were met:
* An amino acid substitution was produced which substantially changed the polarity or the cross-linking ability of the amino acid residue in question.
* The amino acid residue changed by the mutation was preserved across vertebrate species down to at least mouse and rat.
* The mutation was not present in IVCT malignant hyperthermia negative (MHN) control samples from 100 unrelated individuals.
* The mutation segregated with MHS and MHN IVCT results, as known, within the appropriate family.
Despite meeting the above criteria, functional analysis of these novel mutations was beyond the scope of this project, so novel mutations cannot yet be proven as causative.
Haplotype analysis was carried out using the chromosome 19 microsatellite repeat sequence markers D19S228, D19S557, D19S897, D19S421, D19S422, D19S881(10), flanking the RYR1 locus. The forward primer of each pair was fluorescently labelled with either 6-carboxy Fluorescein (FAM) or hexachloroFluorescein (HEX) and amplified using the same Hotstar TAQ PCR method as for mutation detection. The primer sequences are presented in Table 4.
The reactions were analysed on the MegaBACE 1000 using 550 6-carboxy-X-rhodamine (ROX) as size marker, using Fragment Profiler 1.2 software (Amersham, U.K.).
Samples of genomic DNA from 38 individuals with known positive IVCTs (MHS or MHE) were sequenced over RYR1 exons 1 to 20, 38 to 47, 85 to 87, 98 to 104 covering the known hotspots. Seven different causative (as defined by EMHG) mutations were identified in nine individuals. In another six individuals, five different published but as yet functionally uncharacterised mutations were identified. Three further, as yet unpublished and functionally uncharacterised (novel), mutations were identified in four different samples. Two samples each contained two functionally uncharacterised mutations. The mutations are presented in Table 5. None of the mutations was observed in 100 unrelated MHN controls.
One hundred percent concordance was achieved in all families included in the study as shown in Table 6. Some families had more than one member with an IVCT result to use for concordance measurement but in half of the cases only one individual in the family had been tested. It is in no way implied that these numbers represent statistically significant concordance for the families with more than one member studied, but the lack of discordance is satisfying when all the mutations identified are taken in to account.
The numbers of IVCTs that could have been or were avoided were recorded for all referred family members in Table 7.
The six microsatellite markers flanking the RYR1 locus were used to determine the haplotypes of each of the families displaying identical mutations. The results are presented for families AH and M with the c.1615T>C mutation (Figure 1), families C and H with the c.7300G>A mutation (Figure 2) and families AD, I and F with the c.14210G>A mutation (Figure 3). Haplotypes were also determined for families AM and W with the c.487C>T mutation, although only a single individual from each family was available for testing.
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Families AH and M displayed a common haplotype which clearly segregated with the mutation in both families (212, 144, 89, 191, 185, 136 for markers D19S228, D19S557, D19S897, D19S421, D19S422, D19S881 respectively). However, this was not the case for the two families displaying the c.7300G>A mutation, the haplotypes being 210, 136, 89, 193, 195, 136 for family C and 206, 136, 90, 189, 197, 134 or 138 for family H, respectively. The data for the c.14210G>A mutation in families F and I (who would be considered the same family except that family F has a second causative mutation on a different allele) when compared with family AD is inconclusive, as a common haplotype is possible for D19S557-D19S421, with a crossover between markers D19S421 and D19S422 and the possibility that the mutation may lie between D19S421 and the crossover point. Finally, for the mutation c.487C>T, although the available information is limited to a single member for each of families W and AM, a common haplotype is probable, the allele sizes being 208/210, 132/144, 96/96, 189/197, 195/195, 134/140 for family W and 206/210, 124/144, 89/96, 191/197, 195/195, 136/140 for family AM, respectively.
Sequencing of 'hot-spot' areas proved to be an effective, if somewhat limited, way of examining for RYR1 mutations. Sequencing has revealed a number of previously identified mutations as well as some novel mutations.
Sequencing of the RYR1 gene in our patients has identified several families displaying known MH causative mutations which are common in the European population; a single family each with c.1021G>A, c.1840C>T, c.6617C>T and two families each with c.487C>T and c.7300G>A mutations plus a single family with the less common c.7373G>A mutation. Although the limited information available from haplotype analysis indicates that the two families with c.487C>T may not be distinct, the two families with c.7300G>A do have clearly distinct haplotypes. In addition, we have found a single family with a c.742G>C which produces the same amino acid substitution as the known MH causative c.742G>A mutation described in several North American and UX families.
Six of our families display mutations which have been published but not yet functionally characterised, two families with c.14210G>A having similar haplotypes and one each with the c.529C>T, c.1201C>T and c.14477C>T mutations each of which have been observed in several European families and one with the c.677T>A which has been described in one Swiss family to date.
Two families in this study displayed the c.1615T>C novel mutation which segregated with MHS and MHN in both families. Haplotyping in these families revealed a common allele, indicating that these families are highly likely to be related, although the common forebear is unknown at this stage. This mutation produces a phenylalanine to leucine substitution at amino acid residue 539, six amino acids along from the published mutations c.1597C>T(20) and c.1598G>A(21), also located in exon 15. This region is highly conserved, being consistent right down to at least fish(22) (Figure 4, middle panel).
The novel mutation c.11798A>G in exon 86 codes for a tyrosine to cysteine substitution at amino acid 3933. Although this is not a well-known region for MH causative mutations, there have been reports of mutations in adjoining exons(13) and this is also a highly conserved region of RYR1 (Figure 4, lower panel). The closest reported mutation is from the French population, where a c.11748T>G mutation has been described(23), which produces an amino acid substitution 17 residues earlier.
The final novel mutation found in this study is in exon 2, being c.152C>A, coding for a threonine to asparagine substitution at residue 51, six amino acids after the cysteine(20) and histidine(13) substitutions described for arginine at residue 44, and also in a highly conserved region of the gene (Figure 4, upper panel). Interestingly, this mutation was found in a family in combination with the previously described c.677T>A mutation. As these mutations were observed together in two generations of MHS individuals in the pedigree, and therefore reside on the same allele, it is difficult to determine what effect this mutation may have on MH sensitivity without independent functional analysis. While it appears that the c.677T>A mutation alone is sufficient to confer the MHS phenotype, it is possible that the c.152C>A may also confer MHS status and that the compound heterozygote state may behave differently to c.677T>A alone, perhaps increasing sensitivity to MH triggers.
Like the study by Monnier et al we have also found at least one family with more than one independent causative mutation, occurring on separate alleles, reinforcing the EMHG guidelines for the use of genetic testing to diagnose MHS but not MHN(24). Family F demonstrates two mutations (c.14210G>A and c.14477C>T), which can be tracked to both of the parents of the MHS patient studied. This was discovered by two distinct episodes of clinical MH occurring in relatives of both parents of the index case in this family.
While mutation identification and analysis has been significant for those families who have a mutation identified, it still leaves 53% of families with no mutation identified. There are several possible reasons for this:
* We know that mutations exist in regions other than the 'hot-spots' of the RYR1 gene(13, 25, 26).
* Linkage analysis indicates a high likelihood of the involvement of several other genes in the pathogenesis of MH. These may include SCN4A (chromosome 17g11.2-g24)(27), CACNA1S (alpha 1A subunit, dihydropyridine receptor) on Chromosome 1q32(28, 29), CACNA2 (chromosome 7g21-q22)(30) and other genes located on chromosome 3 and 5 of the human genome. These were not analysed in this study.
* The quoted specificity of the IVCT using the EMHG protocol is 94%(31). The specificity of the IVCT in our laboratory is difficult to determine as there were two different methods of IVCT testing, but the possibility exists that in families with only one positive IVCT and a mild or atypical clinical story that there could have been a false positive result. It is also possible that our MHE subjects fall in to this category.
We were able to recruit 47 relatives of mutation positive probands for analysis. Twenty-one of these (45%) would have been able to be diagnosed as MHS by mutation analysis alone if all mutations are proven functional, resulting in a total saving of A$76,000--equating to a 22% reduction in costs (total cost of all having IVCT with no genetic testing available =A$340,000, total cost with genetic testing available and IVCT testing for probands and negatives =A$264,000). These figures are based on an initial IVCT cost of A$4000.00 (combining day stay, surgical and anaesthetic costs) at our institution, initial proband DNA sequencing costs of A$500.00 and further screening for known mutations at A$100.00 per person.
The MH status of a child under the age of 10 years was able to be determined in four cases. As muscle biopsy is not performed under the age of 10 years, genetic testing facilitated early diagnosis. In addition, the MH status of two elderly patients who refused muscle biopsy on the grounds of ill health was able to be determined and five patients residing in states with no IVCT Unit were diagnosed as MHS avoiding travel related expenses and major inconvenience. The cost savings for future genetic testing would be amplified for larger families and for those remote from IVCT testing centres.
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A significant limitation of this study was the inclusion of only four MHE subjects (those with an IVCT responsive to caffeine or halothane but not both) as this is a problematic group of patients who need further definition in genetic analysis. Our follow up study sequencing the entire RYR1 using cDNA with the inclusion of more of our MHE subjects will aim to redress this limitation.
It may be significant that there is a higher mutation detection rate in those with more strongly positive contracture tests (Figures 5 and 6).
This may be a reflection of the highly sensitive but less highly specific dynamic test used for MHS diagnosis. Other potential explanations for this phenomenon are:
* Phenotypes with less strong contracture tests have mutations on other genes coding for different parts of the ryanodine receptor complex. Linkage studies in European and North American families have indicated that this may be one factor in the less than 100% mutation detection rate.
* There have been over 178 missense mutations(13) identified in all areas of RYR1. This project has been limited to the 'hot-spots'.
One of our secondary aims was to identify those mutations occurring more commonly in the Australian population. While further analysis of the RYR1 gene in patients not displaying a mutation in a 'hot-spot' region and RYR1 investigation of many more families will be needed to meet this aim, we have begun the process, which may help to decide on a limited screening test for RYR1 defects in MHS individuals in this country.
This project has achieved its aims of identifying mutations associated with MH and reducing the number of IVCTs required in those families for MHS diagnosis. We have also added to the pool of likely mutations in RYR1 related to MH.
The discovery of two mutations within one family has further emphasised the importance of only using genetic testing for diagnosis of MHS and not for MHN. Further analysis of the family with two possible causative mutations is required before any conclusions about compound heterozygosity can be made. This study has highlighted the need to functionally analyse new mutations so they can be added to the pool of mutations outlined by the EMHG and used for MH diagnosis. Future attempts at increasing the sensitivity of genetic testing should involve the study of other genes that code for proteins responsible for calcium channel activity as well as more detailed analysis of the RYR1 gene.
This project was funded in part by a grant-in-aid from the Australian and New Zealand College of Anaesthetists and supported by the Department of Anaesthesia and Pain Management at the Royal Melbourne Hospital. The authors would also like to thank the Murdoch Children's Research Institute and Victorian Genetic Health Services for the provision of facilities and clinical genetic advice essential for conducting this project.
Accepted for publication on January 10, 2008.
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R. L. GILLIES *, A. R. BJORKSTEN ([dagger]), M. DAVIS ([double dagger]), D. DU SART ([section])
Department of Anesthesia and Pain Management, Royal Melbourne Hospital, Parkville and Victorian Clinical Genetics Services Murdoch Children's Research Institute Melbourne Victoria, Australia
* M.B., B.S.(Hons.), F.A.N.Z.C.A., Head of Malignant Hyperthermia Diagnostic Unit, Royal Melbourne Hospital.
([dagger]) Ph.D., Medical Scientist, Department of Anaesthesia and Pain Management.
([double dagger]) Ph.D. (Distinction), F.H.G.S.A., Senior Medical Scientist, Department of Anatomical Pathology, Royal Perth Hospital, Perth, Western Australia.
([section]) Ph.D., F.H.G.S.A., Director, Molecular Genetics Laboratory, Victorian Clinical Genetics Services.
No reprints will be available from the authors. Electropherograms will be available from the authors upon request.
FIGURE 4: Conservation of the 20 amino acid residues on either side of the three novel mutations in RYR1 across species using the University of California, Santa Cruz (USCS) Multiz Alignment and PhastCons Conservation tools. The amino acid in bold is the novel mutation and other known MH causative mutations shown by residue number (15). Species included are human (Homo sapiens: hgl8), rhesus (Macaca mulatta: rheMac2), mouse (Mus musculus: mm8), dog (Canis familiaris: canFam2), horse (Equus caballus: equCabl), platypus (Ornithorhynchus anatinus: ornAnal), lizard (Anolis carolinensis: anoCarl), stickleback (Gasterosteus aculeatus: gasAcul). Exon 2 p.T51N p.C35R Human Q L K L C L A A Rhesus Q L K L C L A A Mouse Q L K L C L A A Do Q L K L C L A A Horse Q L K L C L A A Platypus Q Q K L C L A A Lizard Q L K L C L A T Stickleback I K L C L S C Exon 15 p.F539L p.Y522S, C Human N L L Y E L L A Rhesus N L L Y E L L A Mouse N L L Y E L L A Horse N L L Y E L L A Platypus N S L Y E L L A Lizard N L L Y E L L A Stickleback N L L Y E L L A Exon 86 p.Y3933C p.I3916M Human N I I I C T V D Rhesus N I I I C T V D Mouse N I I I C T V D Do N I I I C T V D Horse N I I I C T V D Platypus N I I I S T V D Lizard N I I I C T V D Stickleback N I I I C T V D Exon 2 p.T51N p.R44C Human E G F G N R L Rhesus E G F G N R L Mouse E G F G N R L Do E G F G N R L Horse E G F G N R L Platypus E G F G N R L Lizard E G F G N R L Stickleback E G F G N R L Exon 15 p.F539L p.R530H p.R533C Human S L I R G N R Rhesus S L I R G N R Mouse S L I R G N R Horse S L I R G N R Platypus A L I R G N R Lizard S L I R G N R Stickleback A L I R G N R Exon 86 p.Y3933C Human Y L L R L Q E Rhesus Y L L R L Q E Mouse Y L L R L Q E Do Y L L R L Q E Horse Y L L R L Q E Platypus Y L L R L Q E Lizard Y L L R L Q E Stickleback Y L L R L Q E Exon 2 p.T51N p.T51N Human C F L E P T S N A Rhesus C F L E P T S N A Mouse C F L E P T S N A Do C F L E P T S N A Horse C F L E P T S N A Platypus C F L E S T S N S Lizard C T L E P T S N A Stickleback C F L E T T S N A Exon 15 p.F539L p.F539L Human S N C A L F S T N Rhesus S N C A L F S T N Mouse T N C A L F S T N Horse T N C A L F S N N Platypus L N C A Q F S G S Lizard S N C A L F S N N Stickleback S N C A L F C D N Exon 86 p.Y3933C p.Y3933C Human S I S D F Y W Y Y Rhesus S I S D F Y W Y Y Mouse S I S D F Y W Y Y Do S I S D F Y W Y Y Horse S I S D F Y W Y Y Platypus S I S D F Y W Y Y Lizard S I S D F Y W Y Y Stickleback S I S D F Y W Y Y Exon 2 p.T51N p.D60N Human Q N V P P D L A I Rhesus Q N V P P D L A I Mouse Q N V P P D L A I Do Q N V P P D L A I Horse Q N V P P D L A I Platypus Q N V P P D L S I Lizard Q K V P P D L A I Stickleback L N V P P D L A I Exon 15 p.F539L Human L D W L V S K L D Rhesus L D W L V S K L D Mouse L D W L V S K L D Horse L D W L V S K L D Platypus L D W L I S R L E Lizard L D W L V S K L D Stickleback L D W L V S K L D Exon 86 p.Y3933C Human S G K D V I E E Q Rhesus S G K D V I E E Q Mouse S G K D V I E E Q Do S G K D V I E E Q Horse S G K D V I E E Q Platypus S G K D V I E E Q Lizard S G K D V I E E E Stickleback S G K D I I D G P Exon 2 p.T51N Human C C F V L E Q S Rhesus C C F V L E Q S Mouse C C F I L E Q S Do C C F V L E Q S Horse C C F V L E Q S Platypus C T F V L E Q S Lizard C C F S L E Q S Stickleback C S F V L V Q S Exon 15 p.F539L p.R552W Human R L E A S S G I Rhesus R L E A S S G I Mouse R L E A S S G I Horse R L E A S S G I Platypus R L E A S S G I Lizard R L E A S S G I Stickleback R L E A S S G I Exon 86 p.Y3933C Human G K R N F S K A Rhesus G K R N F S K A Mouse G K R N F S K A Do G K R N F S K A Horse G K R N F S K A Platypus G Q R N F S K A Lizard G K R N F S K A Stickleback G K R N F S K A TABLE 1 Clinical details of index cases of each of the families in whom a causative of putative causative mutation was found Proband Family Clinical Number of MH Other number code grading of positive status clinical notes IVCT MH episode in family 7321 A 50 1 MHS 7294 B 25 2 MHS 8370 C 1 MHS Death post GA in family 7380 D 43 4 MHS Death post GA in family 28603 E 32 1 MHS 32 8547 F 5 MHS Multiple MH episodes in family 7289 G 2 MHS 8318 H 35 1 MHS 35 7880 I 1 MHS Multiple MH episodes in family 28171 M 45 1 MHS 30274 0 38 2 MHS 30651 V 58 1 MHS 31303 W 6 MHS 1 MH episode and multiple MHS by IVCT in family 28234 AD 1 MHS Post op rhabdomyolysis 7370 AK 35 1 MHS 35 31304 AH 60 1 MHS 60 32810 AM 3 MHS 3 positive IVCT in family 8003 AC 50 1 MHS MH episode Proband Year IVCT (2% IVCT IVCT Test number of halothane) (2 mmol halothane protocol test caffeine) (bubbled or 3%) 7321 2001 1.2 g 1.3 g * EMHG 7294 1985 NA 2.7 g >1g ** RMH 8370 2003 1.85 g 2.5 g EMHG 7380 2001 0.45 g 2.15 g EMHG 28603 2000 NA 3 g >2g RMH 8547 1995 NA 1.2 g >2g *** CHCT 7289 2001 0.9 g 1.95 g EMHG 8318 2002 1.05 g 5.7 g EMHG 7880 2002 0.5 g 0.45 g EMHG 28171 1997 NA 1.3 g >2g RMH 30274 1996 NA 1.0 g >1.5 g RMH 30651 1997 NA 1.9 g >2g RMH 31303 2002 2.3 g 1.4 g EMHG 28234 2004 1.2 g 3.5 g EMHG 7370 2001 0.9 g 1.6 g EMHG 31304 2002 4 g 1.7 g EMHG 32810 1996 NA 2.5 g >5 g CHCT 8003 2002 1.1 g 0.6 g EMHG * EMHG=European Malignant Hyperthermia Group IVCT protocol, ** RMH=RMH protocol, ***CHCT=caffeine halothane contracture test. (See text for other abbreviations). TABLE 2 Clinical details of index cases of each of the families in whom no mutation has been found as yet Proband Family Number of MH Other clinical notes number code positive status IVCT in family 30524 AB 1 MHE Clinical grading=23 (4) 8469 AE 1 MHS Masseter spasm and 2 previous 'difficult anaesthetics', stiff baby 8546 AF 1 MHS Death in family ?MH 25077 AG 1 MHS EIR 28635 AI Isolated high temp PO 31052 AJ 1 MHS High CK and myonecrosis PO 31842 AL 3 MHS 9341 J 1 MHS Death in family ?MH 9020 K 1 MHS High temp, hypoxia, hyperpnoea PO 31301 L 1 MHS High temp, tachycardia and hypoxia PO 31344 N 1 MHE Rhabdomyolysis, high CK 8274 P 1 MHS Clinical grading=30 (4) 30593 Q 4 MHE Death in family and 3 other MHS 31305 R 1 MHS 1st degree relative MHS 9098 S 11 MHS 5 MH deaths in family 25088 T 2 MHE 1st degree relative MHS post viral myoglobinuria 7935 U 1 MHS Death in family ?MH 31302 X 3 MHS 2 other positive family members 7284 Y 1 MHS High CK and myonecrosis PO 31300 Z 3 MHS 2 other positive family members Proband IVCT (2% IVCT (2 mmol Year IVCT Test number halothane) caffeine) of test (bubbled protocol halothane) 30524 0.15 g 0.3 g 2005 EMHG 8469 0.45 g 0.5 g 2003 EMHG 8546 0.6 g 0.3 g 2002 EMHG 25077 0.2 g 0.3 g 2003 EMHG 28635 0.6 g 0.3 g 2004 EMHG 31052 1.5 g 0.6 g 2005 EMHG 31842 2.5 g 1993 >1g RMH 9341 1.0 g 0.5 g 2002 EMHG 9020 0.75 g 0.25 g 2003 EMHG 31301 0.65 g 2001 >1g 31344 0.1 g 0.2 g 2005 EMHG 8274 1.4 g 1.6 g 2003 EMHG 30593 NA 0.15 g 1982 0.4 g CHCT 31305 NA 0.3 g 2001 >1g RMH 9098 NA 1.0 g 1987 >1g 25088 NA 0.15 g 1991 0.55 g CHCT 7935 NA 1.6 g 1986 >1g CHCT 31302 NA 0.6 g 2002 1.0 g EMHG 7284 NA 0.5 g 1992 >1g RMH 31300 0.4 g 0.3 g 2001 EMHG ** European Malignant Hyperthermia Group IVCT protocol, ** RMH protocol, *** caffeine halothane contracture test, PO=post-operative. (See text for other abbreviations). EIR = excercise induced rhabdomyolysis. IVCT = in vitro contracture test TABLE 3 Primer sequences for ranodyne receptor (RYR1) from genomic DNA Exon Forward primer sequence 1 TGTAAAACGACGGCCAGTCGGGTGCCTCTGGTGTCT 2 GGCCTGTGGTCTGCAGTATT 3 TGTAAAACGACGGCCAGTGTGGCAGGGAATGTTGCT 4-5 TGTAAAACGACGGCCAGTACCTGTGACTAGGCCAGACC 6 GGAAATGGGAAATTGGGAGT 7 TGTAAAACGACGGCCAGTACCCTTGATTTCTGGCCTCT 8-9 CAGAGAGTTGGTGGCAGTGA 10-11 GACTGTAATGTCCATGGGAG 12 GGCAAGTGCAGAACTCAAGTC 13 TGTAAAACGACGGCCAGTGAGTGAGGTTGCGGCAGT 14-16 TTCGTGAATCCAAGAAAGACA 17 TCCACTTTCACCACCTCCTC 18 TGTAAAACGACGGCCAGTCTCTGGCTTCCCACCACTT 19 CCACAGGAGCCTCCAATATC 20 TGTAAAACGACGGCCAGTCATTGATCCCAGGACTGCTT 38 TGTAAAACGACGGCCAGTCCACTGCAACAGAACAAGACA 39 TTCACATTGTTCTGGTCCAA 40-41 TGTAAAACGACGGCCAGTTGTCACAGTGGTGGCTATGG 42 GTGGTCAGGGAGGGCTTC 43 TGTAAAACGACGGCCAGTCTCTGACAGGACCAGGGCTA 44 AGACCCCCTTTCCCCATGC 45 TGCCTCCCCAACCCACCCACCTTC 46-47 CCTGCTCTACCCTCCTGTGT 85-87 CCCACTGTTCATCTCCCCTA 98-99 GAGTTTGGAGTGTCCCCAGA 100- CTCCAGAGTGCTCCTCGTGT 101 102 CAGGAAACAGCTATGACCCCTGGGCCTGCATTCTTAG 103- AAGCCCTGGAGGTAGGTAGC 104 Exon Reverse primer sequence 1 CAGGAAACAGCTATGACCCCGGACAGGGACATTCAG 2 TCCTGTCAGCACCTCGTTC 3 CAGGAAACAGCTATGACCCACCGTGCCCTAAACGAC 4-5 CAGGAAACAGCTATGACCGGCGTTCTGCAGACCTTCTA 6 GGAAATGGGAAATTGGGAGT 7 CAGGAAACAGCTATGACCGTATGCCTGAAGGGCAACAT 8-9 GCCCCTGACTTCATCTTGG 10-11 ACTGCAGCCTTGACCTCCT 12 TGGGCAACAGAGGTAGAGATG 13 CAGGAAACAGCTATGACCCAGACCCTACTTCCCCAGGT 14-16 GGAAGTGAGGGAGTGAGCA 17 GGGGTGGGAGATTAAGGAAA 18 CAGGAAACAGCTATGACCAACCCCAAGGTGTATGGTCA 19 GGTGAGCTAGCCATCGAGAG 20 CAGGAAACAGCTATGACCATTACAGGCATGAACGATGG 38 CAGGAAACAGCTATGACCTCTGTCCTCATGCCCTCTTT 39 GGTGAGCTAGCCATCGAGAG 40-41 CAGGAAACAGCTATGACCAGTGAGCTGGTGGAAAGTGG 42 GAATCAGCAGGTGTCCCAAG 43 CAGGAAACAGCTATGACCGTCATCTCGAGGGAGGTGTG 44 GCGGGTTGCCCTATTGAG 45 TGCCTGCCCTCCCTTCCCATCCTG 46-47 GCTCTACCGTGACCCATCTC 85-87 GGCAAGACTTGGAAATGGAC 98-99 AGGGTCCCAGGAGTCATTCT 100- CGGCCTGGAGGTACCATTA 101 102 CAGGAAACAGCTATGACCCCTGGGCCTGCATTCTTAG 103- TGGACACTACACCCCCTTTC 104 TABLE 4 Primer sequences used for haplotyping of chromosome 19 Marker Forward primer sequence D19S228 AGCTAATATGGCAGAATCTTGAC D19S421 AGCCCTTGCTGGTTTATG D19S422 AGCTATGATCTCACCACTGTA D19S557 GAGATCAAGCCCAGGCAAC D19S881 TGAGATCACACGCCACTGC D19S897 AGGATTTCCCCAACAGC Marker Reverse primer sequence 5' label D19S228 GCAGGATCGCAGTGCTT HEX D19S421 GAGCCTGTGGTGGAGC FAM D19S422 CAGCATTTTGCCAGACTT FAM D19S557 AGCTGTGTTCATTCCCTTGG HEX D19S881 CTCTGCCCAGCCACAAAGGT HEX D19S897 TGCACATTACAGTGTGAGACAG FAM TABLE 5 EMHG causative and putative novel ranodyne receptor (RYR1) mutations DNA Family Mutation Exon Amino acid Ref EMHG sample code change causative 30651 V c.152C>A 2 p.T51N 32810 AM c.487C>T 6 p.R163C 11 Yes 31303 W c.487C>T 6 p.R163C 11 Yes 30274 0 c.529C>T 6 p.R177C 12 30651 V c.677T>A 8 p.M226K 13 7289 G c.742G>C 9 p.G248R 14 Yes 7380 D c.1021G>A 11 p.G341R 15 Yes 7294 B c.1201C>T 12 p.R401C 16 31304 AH c.1615T > C 15 p.F539L 28171 M c.1615T > C 15 p.F539L 8003 AC c.1840C>T 17 p.R614C 15 Yes 7321 A c.6617C>T 40 p.T2206M 17 Yes 8370 C c.7300G>A 45 p.G2434R 18 Yes 8318 H c.7300G>A 45 p.G2434R 18 Yes 28603 E c.7373G>A 46 p.R2458H 17 Yes 7370 AK c.11798A>G 86 p.Y3933C 28234 AD c.14210G>A 98 p.R4737Q 1,213 7880 1 c.14210G>A 98 p.R4737Q 1,213 8547 F c.14210G>A 98 p.R4737Q 1,213 8547 F c.14477C>T 100 p.T42861 19 TABLE 6 Concordance--RYR1 sequencing and in vitro contractivetest (IVCT) results Family Mutation Number Mutation Mutation Concordance code of IVCT (-) (+) V c.677T>A 100% 2 2 V c.152C>A AM c.487C>T 1 1 W c.487C>T 1 1 O c.529C>T 2 0 2 G c.742G>C 3 1 2 100% D c.1021G>A 7 3 4 100% B c.1201C>T 4 2 2 100% AH c.1615T>C 1 1 100% M c.1615T>C 1 1 AC c.1840C>T 1 1 A c.6617C>T 6 5 1 C c.7300G>A 1 1 100% H c.7300G>A 3 2 1 E c.7373G>A 1 1 AK c.11798A>G 1 1 AD c.14210G>A 1 1 I c.14210G>A 3 2 1 F c.14210G>A 3 5 F c.14477C>T 2 2 100% TABLE 7 Reduction of in vitro contracture tests (IVCTs) required for MH diagnosis Number of biopsies required Mutation testing No mutation testing available available Initial IVCT 38 38 IVCT required 26 * 47 Total 64 85 * Negative for familial mutation.
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|Author:||Gillies, R.L.; Bjorksten, A.R.; Davis, M.; Du Sart, D.|
|Publication:||Anaesthesia and Intensive Care|
|Date:||May 1, 2008|
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