North American Malignant Hyperthermia Population: Screening of the Ryanodine Receptor Gene and Identification of Novel Mutations

Skeletal muscle samples from 73 unrelated individuals were collected over a period of 3 yr and stored at −70°C. The samples were obtained from patients who had been diagnosed as MHS by the CHCT test at three North American MH diagnostic centers (Uniformed Services University of the Health Sciences, Bethesda, MD; Wake Forest University School of Medicine, Winston-Salem, NC; and Thomas Jefferson University–Jefferson Medical College, Philadelphia, PA). Patients had been referred for CHCT because of either a positive family history of MH or development of signs of MH during anesthesia. Diagnosis of MH was performed according to the North American CHCT 3protocol. Individuals were diagnosed as MHS if any one of three muscle strips exposed to halothane or caffeine exceeded diagnostic threshold. The threshold values for a positive test are greater than or equal to 0.7 g contracture response to 3% halothane or greater than or equal to 0.3 g contracture to 2 mm caffeine as recommended by the North American MH Group. 17Because this was a retrospective study on stored muscle samples, not all patients could be contacted to give consent for genetic analysis. Therefore, the institutional review boards at the three participating institutions (Uniformed Services University of the Health Sciences, Bethesda, MD; Wake Forest University School of Medicine, Winston-Salem, NC; and Thomas Jefferson University–Jefferson Medical College, Philadelphia, PA) determined that clinical and demographic data were permitted only in terms of reasons for referral, MH diagnostic status, and sample identification for patients who could not be contacted.

Genomic DNA was extracted from stored muscle tissue or peripheral blood taken from MHS patients using the QIAamp DNA mini kit (Qiagen, Valencia, CA) or the Wizard Genomic DNA Purification Kit (Promega, Madison, WI). Amplification was performed during standard polymerase chain reaction (PCR) conditions: denaturation at 95°C for 3 min followed by 28–35 cycles of 94°C for 30 s, annealing at primer specific temperatures for 30 s, extension at 72°C for 30 s, with a final 10-min extension at 72°C. The PCR mix contained 100 ng of genomic DNA, 1.5–3.0 mm MgCl², buffer (10 mm Tris-HCl [pH 8.3], 50 mm KCl; Perkin Elmer, Foster City, CA), 20 pmol of each primer, 250 mm of each dinucleotide triphosphate (dNTP), and 0.3 units of AmpliTaq (Perkin Elmer) in a final volume of 20–30 μl. For PCR we also used Master Amp PCR Pre-Mix D (Epicentre Technologies, Madison, WI) or Expand High Fidelity PCR System (Roche Diagnostics, Indianapolis, IN). The primer sets were either developed in the laboratory using Oligo (Molecular Biology Insights Inc., Plymouth, MN) software or chosen from the literature and covered the N-terminal and the central domains of the RYR1  gene. 18,19 

Screening for mutations was performed either by restriction fragment length polymorphism or single-strand conformation polymorphism analysis. PCR fragments were digested with 3–4 units of restriction enzyme (New England Biolabs, Beverly, MA) according to the manufacturer’s instructions. The digested products were resolved in a 3% NuSieve GTG^®low melting agarose gel (FMC BioProducts, Rockland, ME) or 20% polyacrylamide TBE gel (Novex, San Diego, CA) depending on their sizes. For single-strand conformation polymorphism analysis, PCR products were mixed with formamide in a ratio of 1:3 and, after denaturing for 5 min, were loaded onto 20% polyacrylamide gel. The samples were then run overnight at 45 V in a cold room, and the denatured single-stranded DNA bands were visualized by staining with 1 μg/ml ethidium bromide. Direct sequencing of PCR product was performed when changes in restriction site or single-strand DNA bands were detected. PCR products were purified using a QIAquick PCR Purification Kit (Qiagen, Valencia, CA) and directly sequenced using the Big DyeTerminator Cycle Sequencing kit on ABI 310 Genetic Analyzer (Perkin Elmer) as described by the manufacturer.

We initially screened 20 of 22 known RYR1  mutations using restriction fragment length polymorphism, single-strand conformation polymorphism, or both methods. 6,18,19The Tyr522Ser and Ile4898Thr mutations reported within the C-terminal region of the RYR1  gene were not analyzed in this study as they are associated with CCD and appear to be very rare. 19,20Of the 20 previously reported mutations in the RYR1  gene, seven were detected in North American MH population: Arg163Cys, Gly248Arg, Arg614Cys, Val2168Met, Thr2206Met, Gly2434Arg, and Arg2454His (table 1).

The central domain of the RYR1  gene, particularly exons 39, 40, and 44–46, are the most studied mutational sites within the RYR1  gene, and more than 50% of all known RYR1  mutations have been detected in these exons. 8,13We analyzed exons 12, 15, 39, 40, and 44–46 by single-strand conformation polymorphism and detected unique single-strand DNA patterns in three MH patients (table 1). Direct sequencing of the PCR products identified nucleotide substitutions at G6640A, G7099A, and G7291A, which results in amino acid changes Val2214Ile, Ala2367Thr, and Asp2431Asn. All of these alterations were sequenced in reverse directions, and nucleotide substitutions were confirmed. Segregation analyses of novel substitutions were not provided within the families as family members were not available for the study. All of the new substitutions could be detected using restriction enzymes Mae II for Val2214Ile, FauI for Ala2367Thr, and Taq I for Asp2431Asn. Analysis of these sites with restriction fragment length polymorphism was extended to 134–158 normal chromosomes, and none displayed the presence of novel substitutions.

Overall, 10 different RYR1  gene mutations were detected in 16 unrelated MHS individuals, which accounts for approximately 21.9% of the screened population (table 1). The most common mutation was Gly2434Arg, occurring in four patients and accounting for 5.5% of the population, followed by Arg2454His in three patients (4.1%) and Arg163Cys in two patients (2.7%). The other seven mutations were detected in single patients.

Caffeine–halothane contracture test data for MHS patients with RYR1  mutations are shown in table 2. Each subject bearing an RYR1  mutation showed strong positive CHCT results, particularly for 3% halothane. Four patients with RYR1  mutations were negative for the 2-mm caffeine test according to North American CHCT standards. 17Wide variations in contracture phenotype were observed within genotypes. For example, with the Gly2434Arg mutation, the halothane phenotype is similar among MHS subjects (3.4–7.4 g), whereas the caffeine sensitivity varies from 0.0 to 4.7 g (table 2).

Family members tested by CHCT were available for three MH patients with an RYR1  mutation, and complete phenotype and genotype correlation was seen in these families (table 2). In the family US40, Val2168Met (G6502A) mutation was segregated with CHCT results (fig. 1). The referred patient, US40 (III-1), a 30-yr-old man, underwent CHCT and was found to be positive for MHS. The mother (II-2) and brother (III-3) also had positive CHCT results in 1981 and 1988, respectively. There is a report of an intraoperative MH episode (death) in his maternal uncle (II-3). All three CHCT-positive individuals (II-2, III-1, and III-3) had Val2168Met (G6502A), whereas II-1 and III-2 (CHCT-negative) had no mutation (fig. 1). Gly2434Arg (G7303A) mutation was also cosegregated with MH phenotype in the family of BG21 (fig. 1). In the family BG21, both proband (III-1) and his mother (II-2) were CHCT-positive and showed the Gly2434Arg mutation. Proband’s father (II-1) and two brothers (III-2 and III-3) were negative for both CHCT and Gly2434Arg mutation. The same Gly2434Arg mutation also cosegregated with positive CHCT phenotype in two members of the family US47 (table 2, data not presented in fig. 1).

To assess the frequency and distribution of RYR1  gene mutations in a North American MHS population, we screened muscle samples from 73 unrelated individuals diagnosed as MHS according to the North American MH guidelines. We found 10 different RYR1  gene mutations in 16 unrelated MHS individuals; seven of the mutations had been previously described, but three are novel mutations identified for the first time in this study. Overall, these mutations account for 21.9% of the North American screened population, which is similar to results obtained in recent studies of 105 German and 57 Italian MHS individuals. 18,19 

All patients with an RYR1  mutation were highly positive for halothane in the CHCT, and most were positive to caffeine. However, variations in contracture phenotype, even within a genotype (e.g. , Gly2434Arg), were observed across the screened patients. Possible reasons for variability of CHCT data include diagnostic center-to-center test results, and other genetic or environmental factors. Statistical correlation between CHCT phenotypes and RyR1 genotype was not studied because of the small number of subjects carrying the same mutation or lack of family members for the study. Therefore, larger-scale studies are needed to assess genotype–phenotype relations for the North American MHS population.

We found three novel RYR1  missense mutations, G6640A (Val2214Ile), G7099A (Ala2367Thr), and G7291A (Asp2431Asn), in the RYR1 . Although linkage and gene expression studies are needed to define causality of mutations for hereditary diseases, we consider these novel amino acid substitutions as strong candidate mutations for MH susceptibility. The novel substitutions met two criteria that suggest they are not likely to be polymorphisms, namely, none were found among 134–158 normal control chromosomes, and each substitution occurred in conserved amino acid residues. 8 

Regional differences in frequencies for common MHS mutations are observed across Western Europe. 6,13Frequencies of RYR1  gene mutations detected in this North American MHS population were markedly different from those found in western Europe. Thus, the Arg614Cys and Val2168Met mutations, which are common in Germany (9%) and Switzerland (6%), respectively, were rare in our MHS population (only 1.4% each). Moreover, the Gly314Arg mutation, which is common in Ireland, England, and France (6%), was not detected in our population. In contrast, 4.1% of our MHS population had Arg2454His, which has been seen in only 1% of MHS families in Europe. The mutation common for both continents was Gly2434Arg, occurring in 4–7% of European and 5.5% of North American MHS families. It is likely that marked variability in frequency of RYR1  gene mutations between North American and European families is a result of regional and population differences. However, the North American MHS population was more heterogeneic in the types of observed RYR1  gene mutations (allelic heterogeneity), which may be explained by heterogeneity of North American population in general.

Although genetic linkage analysis shows that 50–80% of European MHS families are linked to the RYR1  gene, mutations are reported in only 25–40% of MHS families so far. 6,18,19The majority of RYR1  gene mutations are clustered in the N-terminal region with amino acid residues from 35 to 614 and in the central region from 2163 to 2458. Most genetic screening studies target these two regions, which account for only approximately one fourth of the entire coding region of the RYR1  gene. Thus, the absence of RYR1  mutations in the rest of the screened population might be explained either by a mutation located outside the two regions analyzed in the current study or by the involvement of another gene. Recent genetic studies reveal a new region of the RYR1  gene where MHS–CCD mutations may cluster. 20–23With regard to other MH candidate genes, only one other gene, that for the α¹-subunit of the dihydropyridine receptor, has mutations linked to MHS. 12Recent data obtained by Stewart et al.  24suggest that the dihydropyridine receptor gene Arg1086His mutation is present in only 1% of a large North American MHS population. The two mutations, Arg1086His and Arg1086Gly, reported in the dihydropyridine receptor gene 12,13were not detected in our 73 MHS individuals (data not shown). Thus, dihydropyridine receptor mutations associated with MH appear to be very rare.

Mutational screening analysis of MH candidate genes is an important step for the future development of simplified genetic testing and for understanding phenotype–genotype relation in MH. Continuous and collaborative efforts involving sharing of tissue samples, clinical data, and CHCT results are needed to achieve progress in MH genetics.

The authors thank Barbara W. Brandom, M.D. (Professor of Anesthesiology, Department of Anesthesiology, Children’s Hospital Pittsburgh, Pittsburgh, PA), for providing support for collecting DNA samples, and Michael N. Flora, B.S. (Technologist, Biomedical Instrumentation Center, Uniformed Services University of the Health Sciences, Bethesda, MD), for DNA sequencing.