Anesth Analg 2005;101:1401-1406
© 2005 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000180214.74580.39
TECHNOLOGY, COMPUTING, AND SIMULATION
Denaturing High Performance Liquid Chromatography Screening of Ryanodine Receptor Type 1 Gene in Patients with Malignant Hyperthermia in Taiwan and Identification of a Novel Mutation (Y522C)
Huei-Ming Yeh, MD*,
Mei-Chuan Tsai, BS
,
Yi-Ning Su, MD
,
Rong-Ching Shen,
Jeuy-Jen Hwang, MD
,
Wei-Zen Sun, MD*, and
Ling-Ping Lai, MD, PhD
Departments of *Anesthesiology, Medical Genetics, and Internal Medicine, National Taiwan University Hospital; and Institute of Pharmacology, National Taiwan University, Taipei, Taiwan
Address correspondence and reprint requests to Dr. Ling-Ping Lai, No. 1, Jen-Ai Rd., Section 1, Institute of Pharmacology, National Taiwan University, Taipei, Taiwan, 100. Address e-mail to lai{at}ha.mc.ntu.edu.tw.
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Abstract
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We performed the present study to identify the mutation in patients in Taiwan with malignant hyperthermia (MH). We also test the hypothesis that a denaturing high-performance liquid chromatography (DHPLC) protocol can be used for mutation detection in these patients. We identified five Taiwanese patients with typical clinical presentations of MH after general anesthesia. We also enrolled 50 healthy volunteers. Polymerase chain reaction was used to amplify the ryanodine receptor (RYR1) gene mutation hot spots and DHPLC techniques were used to screen for mutations. Upon detection of a heterozygous elution pattern in DHPLC analysis, DNA sequencing reaction was performed to identify the nucleotide variations. We identified a RYR1 mutation in all 5 patients with MH. There were 4 different mutations in the 5 patients: Tyr522Cys, Arg552Trp, Val2168Met, and Thr2206Arg. Among the 5 patients, 2 unrelated patients had the same Thr2206Arg mutation. Three of the mutations had been reported before, but the Tyr522Cys mutation was novel. None of the MH-related mutations were found in the control group. In conclusion, we identified RYR1 mutations in 5 Taiwanese patients with MH using a DHPLC-based approach. A DHPLC-based genetic test may be developed as a noninvasive and convenient test for MH.
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Introduction
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The present study was performed to identify the mutation in patients in Taiwan with malignant hyperthermia (MH). We also tested the hypothesis that a denaturing high-performance liquid chromatography (DHPLC) protocol can be used for mutation detection in these patients.
MH is a life-threatening skeletal muscle disorder that is triggered by depolarizing muscle relaxant and/or inhaled anesthetics in susceptible individuals (1,2). It is an autosomal-dominantly inherited, genetically heterogeneous myopathy (3). Its main symptoms include tachycardia, supraventricular or ventricular arrhythmia, tachypnea, muscle rigidity, hypercarbia, hyperthermia, rhabdomyolysis, and renal failure. The pathology of MH is an increase in the concentration of cytosolic calcium which is released from the sarcoplasmic reticulum via the ryanodine receptor type 1 (RYR1) (4). The increase of cytosolic calcium stimulates contracture and metabolic responses that finally create acid/base and electrolyte imbalances, causing cell damage and death.
In about 50% of the MH families, this disease is linked to the MHS-1 locus, which encodes the human skeletal muscle RYR1 (5,6). More than 40 RYR1 mutations have been described to associate with MH (7). Most RYR1 mutations appear to be clustered in 2–3 mutation hot regions, the N-terminal amino acid residues 35–614 region, the central amino acid residues 2162–2458 region, and the C-terminal amino acid residues 4000–4898 region (less frequent).
In vitro contracture test (IVCT) is a common way to detect MH-susceptible patients (8,9). However, IVCT is invasive, laborious and should be performed in specialized laboratories. With the advent of modern molecular biology techniques, screening for mutations in candidate genes is a possible alternative to identify MH-susceptible patients. Mutation detection in the RYR1 gene can be preformed using direct DNA sequencing techniques. However, the RYR1 gene is one of the largest genes in the human genome. Direct DNA sequencing is laborious. In the present study, we designed a DHPLC protocol to screen the DNA samples for sequence variations before sequencing. This method is rapid, inexpensive, and is high throughput. We also described the detailed protocol for DHPLC screening of the RYR1 mutation hot regions. We also reported the mutations, including a novel one, in five Taiwanese patients with MH.
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Methods
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We included 5 patients with MH and 50 volunteers. All the patients were identified after general anesthesia. All of them had typical clinical presentations of MH including high fever, marked increase of muscle enzymes, renal failure, and hyperkalemia. Dantrolene was used in all patients. One of the patients died despite dantrolene therapy and supportive treatment. None of the patients underwent IVCT because the diagnosis was well established. This study was approved by the IRB. Peripheral blood samples were collected after informed consent. Genomic DNA was extracted from leukocytes using a modified proteinase K method (Qiagen, Valencia, CA).
Primer pairs were designed to amplify the mutation hot regions of the RYR1 gene. Table 1 shows the exons selected, the primer sequences, and the PCR annealing temperature. PCR was performed in thin-walled PCR tubes in a total volume of 25 µL containing 100 ng of genomic DNA, 0.12 M of each primer, 100 M deoxynucleotide triphosphates, 0.5 U of AmpliTaq Gold enzyme (PE Applied Biosystems, Foster City, CA), and 2.5 µL of GeneAmp 10x buffer II (10 mM tris-HCl, pH = 8.3, 50 mM KCl), in 2 mM MgCl2 as provided by the manufacturer. Amplification was performed in a multiblock system thermocycler (ThermoHybaid, Ashford, UK). PCR amplifications were performed with an initial denaturation step at 95°C for 10 min, followed by 35 cycles consisting of denaturation at 94°C for 30 s, annealing for 30 s, and elongation at 72°C for 1 min. The annealing temperatures are shown in Table 1. A final extension step was applied at 72°C for 10 min after 35 cycles.
The DHPLC system used for detecting heteroduplexes was a Transgenomic Wave Nucleic Acid Fragment Analysis System (Transgenomic Inc., San Jose, CA). DHPLC was performed on automated HPLC instrumentation equipped with a DNASep column (Transgenomic Inc. San Jose, CA). DHPLC-grade acetonitrile (9017-03; JT Baker, Phillipsburg, NJ) and triethylammonium acetate (TEAA) (Transgenomic Inc., Crewe, UK) were used for the mobile phases, which comprised 0.05% acetonitrile in 0.1 M TEAA (eluent A) and 25% acetonitrile in 0.1 M TEAA (eluent B). For heteroduplex detection, crude PCR products, subjected to an additional 3-min, 95°C denaturing step, followed by gradual reannealing from 95° to 65°C over a period of 30 min before analysis, were eluted at a flow rate of 0.9 mL/min. The start- and end-points of the gradient by mixing eluents A and B and the temperature required for successful resolution of heteroduplex molecules were adjusted by using an algorithm provided by the WAVEmaker system control software, version 4.1.42 (Transgenomic Inc., San Jose, CA). Eight microliters of PCR product was injected for analysis in each running. Individual analytical gradient conditions for DHPLC running are described in Table 2 and are expressed as a percentage of eluent B. The flow rate was 0.9 mL/min, and the ultraviolet detector was set to 260 nm. Heterozygous profiles were identified by visual inspection of the chromatograms on the basis of the appearance of additional earlier eluting peaks. Corresponding homozygous profiles showed only one peak.
Amplicons were purified by solid-phase extraction and were bidirectionally sequenced with the PE Biosystems Taq DyeDeoxy terminator cycle sequencing kit (PE Biosystems) according to the manufacturer's instructions. Sequencing reactions were separated on a PE Biosystems 373A/3100 sequencer. The sequencing results were compared with RYR1 mRNA (GenBank accession number NM000540). The adenosine of the start codon (ATG) was numbered as the first nucleotide when expressing genetic variations.
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Results
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PCR amplification for all selected exons was successfully performed as revealed by agarose gel electrophoresis. In DHPLC analysis, 2 melting domains were predicted from the base sequence of exons 11, 12, 15, 17, 39, and 40. Two different conditions were applied to these exons to increase the sensitivity (Table 2). We identified 7 different heteroduplex patterns in amplicons for exons 6, 12, 14, 15, 17, 39, and 40, respectively. Figure 1 shows the DHPLC pictures for these heteroduplex patterns. Further DNA sequencing reaction identified the nucleotide variations in these heteroduplexes.
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Figure 1. Heteroduplex elution patterns identified by denaturing high-performance liquid chromatography (DHPLC) in the present study. Heteroduplex patterns are characterized by the appearance of early eluting peaks, whereas the homoduplex profile shows only one peak. DNA sequencing results are shown below the DHPLC heteroduplex patterns.
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The clinical information of the study patients is shown in Table 3. All patients received succinylcholine and volatile anesthetics for general anesthesia. All of them had fulminant clinical presentations of MH. We identified the mutation in all 5 patients with MH (Tables 3 and 4). There were 4 different mutations in 5 patients. All the mutations were mis-sense mutations and none were identified in the control group. These mutations were Tyr522Cys (A1695G in exon 14), Arg552Trp (C1784T in exon 15), Val2168Met (G6632A in exon 39), and Thr2206Arg (C6747T in exon 40). All the mutated amino acids were in sequences highly conserved among several species including nonmammal animals (Fig. 2). A comprehensive analysis of the evolutionary conserved sites in RYR1 is available at the following Web site: http://www-nbrf.georgetown.edu/cgi-bin/pirwww/nbrfget?uid=FA1808&db=A. Among the 4 mutations, Tyr552Cys was novel and has not yet been reported. Two unrelated patients had the same Thr2206Arg mutation.
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Figure 2. Comparison of the amino acids of ryanodine receptor type 1 among vertebrate species. All the mutations in the present study occur at amino acids, which are highly conserved among species.
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There were 3 heteroduplex patterns identified in the control group (Table 4). Further DNA sequencing reaction identified the nucleotide variations. These variations were an intronic single nucleotide polymorphism (SNP) in intron5, a synonymous SNP in exon 12 and a nonsynonymous SNP in exon 17. The nonsynonymous SNP has been reported and is not related to MH. None of the mutations identified in MH patients were identified in the normal controls. There was no heteroduplex pattern detected in exon 15, in which a common polymorphism (G1668A) had been reported by many other researchers. Further sequencing reaction showed that all the control patients were homozygous for 1668A.
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Discussion
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The RYR1 gene is one of the largest genes in human genome. It locates on chromosome 19q13.1, and comprises 106 exons encoding an mRNA longer than 15,000 base pairs (13–15). The RYR1 protein has a subunit size of 565 kDa and forms a homotetrameric structure that acts as a calcium release channel in the skeletal muscle. Its N-terminal region forms a large "foot" structure as a connector that bridges the gap between the sarcoplasmic reticulum and the T-tubule. Its C-terminal region forms the transmembrane channel. For mutation detection, a base-by-base screening method is laborious. There have been >40 mutations reported. Fortunately, these mutations clustered in several mutation hot regions. We therefore designed to screen the hot regions first. To further enhance the efficacy of screening, DHPLC technique was applied. We identified the mutations in all five patients with MH. The causal relationship between the base variations (mutations) and MH was supported by the following findings. First, the base variations all result in a change of amino acid, which is highly conserved among many species (Fig. 2). Second, the base variations were not identified in 50 control subjects (100 alleles). Therefore, the variations are unlikely simple SNPs. Third, the same mutation has been reported in the literature regarding MH. This is true for three of the mutations in the present study.
We identified a RYR1 mutation in all 5 patients. Therefore, the incidence of RYR1 mutation was 100% in the present study. In reports in Western countries, the incidence of RYR1 mutation in patients with MH was approximately 50%–70% (16). Tammaro et al. (7) also used DHPLC techniques for screening for RYR1 mutation in 52 Italian families. They reported that the incidence of detecting RYR1 mutation was 33% (11 of 33) in MH-susceptible families. In the present study, we reported a much more frequent incidence. There are several possible explanations. First, there might be ethnic differences, and Asian patients with MH have a more frequent incidence of RYR1 mutation. If the Asian patients had the same incidence, the chance of detecting RYR1 mutation in all 5 patients would be very small (1/25 = 1/32). Second, all the patients in the present study had fulminant clinical MH. The incidence of RYR1 mutation might be different between MH-susceptible subjects and patients with documented fulminant clinical disease. Third, different methods for mutation detection were used in different studies. We used DHPLC screening for mutation hot regions. In the DHPLC protocol, 2 conditions were used for 6 exons because 2 melting domains were predicted.
A novel mutation was identified in the present study. The index case was a 21-year-old woman who underwent a breast augmentation surgery. Succinylcholine was used for induction of general anesthesia with isoflurane. The Tyr522Cys mutation in the RYR1 gene was not found in the normal population. This amino acid is highly conserved across species including nonmammal animals. A different mutation at the same amino acid (Tyr522Ser) had been reported by Quane et al. (17). Therefore, we believe that Tyr522Cys is a disease-causing mutation. This mutation can be added to the list of mutation screening, especially for Asian patients.
There are screening methods other than DHPLC for detecting genetic variations. Single-strand conformation polymorphism analysis was the most frequently used method for mutation screening before DHPLC was available. However, it is time consuming, labor intensive, and has a sensitivity between 70%–90% (18–20). In the present study, we identified the mutation for all patients with MH. A sensitivity of 100% has been demonstrated in previous reports when DHPLC was used for mutation screening for many genes such as BRCA1, BRCA2, Notch3, MepC2, TSC1, and the entire mitochondria genome (21–25). Furthermore, the DHPLC elution patterns are specific for their underlying SNP or mutation. This further increases the power of this screening method in differentiating between common SNPs and mutations.
IVCT is often used for the diagnosis of MH susceptibility (15). However, it is invasive, costly, and should be performed in experienced laboratories. It has a sensitivity and specificity of 99% and 94%, respectively. Since the advent of modern molecular biology techniques and the discovery of responsible mutations for MH, noninvasive genetic tests are developing and guidelines for molecular genetic test have been published (26). However, the test is preliminary and has the following limitations. First, this test examines mutations within a list of known mutations. Mutations not on the list might have been missed by the test. Second, as suggested by the North America Working Group Meeting on Malignant Hyperthermia Genetic Testing in 2004, genetic testing is indicated for high-risk individuals such as probands with MH and their family members or subjects with positive IVCT (27). Genetic testing is not suitable for a general survey of patients before undergoing general anesthesia. Furthermore, if the result of a genetic test is negative, IVCT will still be performed. The DHPLC screening strategy has the potential of overcoming these problems. The DHPLC method can be used for the detection of novel mutations. It also has the potential for use in the general population because it is noninvasive, inexpensive, rapid, and is high throughput. With the discovery of more MH-responsible mutations and genes, these mutations and genes can be added to the list of DHPLC screening. If all or most of the responsible genes in a population are elucidated, a DHPLC-based genetic test may be developed as a noninvasive and convenient test for MH. Because the DHPLC test is a high-throughput and inexpensive procedure, it is possible to obtain peripheral blood samples and perform PCR and DHPLC screening before operation for all patients who need general anesthesia.
In conclusion, we applied the DHPLC mutation screening technique in patients with MH. We identified the responsible mutation in all five patients with MH and reported a novel disease-causing mutation. The DHPLC screening approach can be used for the development of a more powerful genetic test for MH.
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Footnotes
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This work was supported in part by Grant 93S74 from the National Taiwan University Hospital, Taipei, Taiwan.
Accepted for publication April 28, 2005.
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Abstract
Introduction
