Anesthesiology 2005; 102:515–21 © 2005 American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins, Inc. Screening of the Entire Ryanodine Receptor Type 1 Coding Region for Sequence Variants Associated with Malignant Hyperthermia Susceptibility in the North American Population Nyamkhishig Sambuughin, Ph.D.,* Heather Holley, B.S.,† Sheila Muldoon, M.D.,‡ Barbara W. Brandom, M.D.,§ Astrid M. de Bantel, B.S.,ʈ Joseph R. Tobin, M.D.,# Tom E. Nelson, Ph.D.,** Lev G. Goldfarb, M.D., Ph.D.†† Background: Malignant hyperthermia (MH) is a life-threaten- viewed as a genetic predisposition to MH and most ing and frequently fatal disorder triggered by commonly used commonly is inherited as an autosomal dominant trait. In anesthetics. MH susceptibility is a genetically determined pre- Downloaded from http://pubs.asahq.org/monitor/article-pdf/102/3/515/358264/0000542-200503000-00007.pdf by guest on 24 September 2021 disposition to the development of MH. Mutations in the ryano- the absence of the triggering agents, MH-susceptible dine receptor type 1 (RYR1) gene are the major cause of MH individuals are usually asymptomatic. The diagnosis of susceptibility. The authors sought to develop a reliable genetic MHS can be made by caffeine–halothane contracture screening strategy based on efficient and relatively inexpensive testing (CHCT),2 which is based on measurement of mutation-detection procedures. isometric tension changes of freshly biopsied skeletal of North American MH patients (30 ؍ Methods: A cohort (n and MH-susceptible individuals was studied. RNA and DNA ex- muscle in response to the ryanodine receptor agonists, tracted from muscle tissue or blood lymphocytes were used for caffeine and halothane. CHCT has been successfully ap- analysis. The entire RYR1 coding region was amplified in 57 plied for establishing the diagnosis of MHS, but it is not overlapping fragments and subjected to denaturing high-per- suitable for massive testing of the population at large or formance liquid chromatography analysis followed by direct nucleotide sequencing to characterize RYR1 alterations. members of the MH-affected families who may be under Results: Nine previously reported and nine unknown RYR1 increased risk of the development of MH if subjected to mutations were identified in 21 of 30 studied patients (70%). triggering anesthetics. Some of the new mutations were located outside of known Molecular genetic investigations have shown consider- mutational “hot spots,” suggesting that RYR1 contains previ- able locus and allelic heterogeneity with six genetic loci ously unknown mutation-prone areas requiring analysis. The 3,4 North American MH/MH-susceptible population is character- implicated in MH. However, accumulated data have ized by a high RYR1 allelic heterogeneity. clearly established that mutations in the RYR1 gene on Conclusions: Denaturing high-performance liquid chroma- chromosome 19 are the major cause of MHS.3,5 The tography analysis of RNA samples extracted from the biopsied RYR1 is one of the largest genes described in humans, skeletal muscle followed by DNA sequencing is a highly effi- cient methodology for RYR1 mutation detection. This approach spanning more than 159,000-nucleotides of genomic allows increasing the rate of mutation detection to 70% and DNA; the coding sequence contains 106 exons (of which identifying mutations in the entire RYR1 coding region. 2 are alternatively spliced) and transcribes a 15,117- nucleotide-long RNA molecule.6 The encoded 563-kD MALIGNANT hyperthermia (MH) manifests as a hyper- RYR1 protein forms a homotetrameric structure and ϩ metabolic crisis in susceptible individuals after exposure functions as a calcium-release channel regulating Ca2 to inhalational anesthetics or succinylcholine, a depolar- content in skeletal muscle during excitation–contraction izing muscle relaxant. This life-threatening reaction is coupling.7 Multiple RYR1 mutations known to cause MH caused by abnormally high release of myoplasmic Ca2ϩ are dispersed throughout the gene, and to complicate from the sarcoplasmic reticulum. The prevalence of MH the situation further, at least 16 silent DNA polymor- is estimated at 1 in 8,500.1 Susceptibility to MH (MHS) is phisms are present in the coding region.5,8 Because of the length and complexity of the RYR1 gene, efficient routine screening for mutations has been difficult. In * Biologist, ʈ M.D. Student Training Program, †† Medical Officer, National Institute of Neurological Disorders and Stroke, National Institutes of Health, addition, screening of the genetically heterogeneous Bethesda, Maryland. † Research Technician, Barrow Neurological Institute, North American population requires extensive knowl- Phoenix, Arizona. ‡ Professor, Uniformed Services University of the Health Sciences, Bethesda, Maryland. § Professor, Children’s Hospital and the Univer- edge of the expected spectrum of MHS-associated muta- sity of Pittsburgh, Pennsylvania. # Professor, ** Professor Emeritus, Wake Forest tions and data on allele frequency in the background University of Medicine, Winston Salem, North Carolina. population. Received from the National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland. Submitted for publication According to genetic linkage studies, MHS is linked to September 1, 2004. Accepted for publication October 22, 2004. Supported by the RYR1 locus in more than 50% of studied affected grants from the Anesthesia Patient Safety Foundation, Indianapolis, Indiana, and 9 the Malignant Hyperthermia Association of the United States, Sherburne, New families, whereas RYR1 mutations are found in only York. Presented at the Annual Meeting of the American Society of Anesthesiol- approximately 22–25% of individuals with positive ogists, Las Vegas, Nevada, October 26, 2004. CHCT results.10,11 Improvements of the screening tech- Address reprint requests to Dr. Sambuughin: Clinical Neurogenetics Unit, National 12 Institute of Neurological Disorders and Stroke, National Institutes of Health, 5625 niques increase the rate of mutation detection, sug- Fishers Lane, Rm. 4S14, Bethesda, Maryland 20892-9404. Address electronic mail to: gesting that the discrepancy reflects methodologic prob- [email protected]. Individual article reprints may be purchased through the Journal Web site, www.anesthesiology.org. lems of finding mutations in this large gene. Thus far, Anesthesiology, V 102, No 3, Mar 2005 515 516 SAMBUUGHIN ET AL. RYR1 screening studies targeted only three mutation- using the RNA-wiz reagent (Ambion, Austin, TX). Three clustered regions: the N-terminal region between codons micrograms of total RNA was used for reverse transcrip- 34 and 614, the central region between codons 2163 and tion reaction to synthesize complementary DNA. Avian 2458, and the C-terminal region between codons 4136 Myeloblastosis Virus Reverse Transcriptase (Promega, and 4973.3,5,13–17 The combined length of all these mu- Madison, WI) was used in the reaction as specified in the tational “hot spot” areas accounts for only approximately manufacturer’s protocol. Complementary DNA was sub- a third of the entire coding region of the RYR1 gene. The sequently used for amplification reactions. Genomic presence of MHS-causing mutations in other parts of the DNA was extracted from muscle tissue and anticoagu- gene has not been systematically investigated. lated blood using the Wizard Genomic DNA Purification The aim of this study was to develop an efficient Kit (Promega). methodology for identification of most, if not all, muta- tions in the RYR1 gene and to devise a strategy for Polymerase Chain Reaction Downloaded from http://pubs.asahq.org/monitor/article-pdf/102/3/515/358264/0000542-200503000-00007.pdf by guest on 24 September 2021 molecular diagnosis of MHS in the North American Complementary DNA (cDNA) and genomic DNA population. served as templates for PCR. The entire 15,117-nucleo- tide-long coding region of the RYR1 gene was amplified as 57 overlapping fragments, each approximately 350 Materials and Methods nucleotides long. Primers used for this purpose were designed using OLIGO software (Molecular Biology In- MH Patients, MH-susceptible Individuals, and sights, Cascade, CO). Intronic primers for amplification Population Controls from genomic DNA were designed for each exon within We performed genetic analysis of 30 MH/MH-suscep- the three mutational hot spots (exons 2–17, exons 39– tible individuals. The study was approved by the institu- 46, and exons 90–104), and subsequently for exons 34, tional review boards of all collaborating institutions. 51, and 67 in which novel mutations were detected Patients had been referred for CHCT because of devel- during screening. All primer sequences are available on opment of signs of MH during anesthesia or a positive request. Amplification from genomic DNA was per- family history of MH. In 28 at-risk family members, the formed using an optimal procedure designed for each diagnosis of MHS was established according to the North exon. Amplification was performed in a total volume of American CHCT protocol, which requires exposure of 20–25 ␮l with 1 ␮l single-stranded cDNA or 50–100 ng muscle strips to caffeine and halothane.2 Testing with genomic DNA, 0.5 ␮M of each primer, 125 ␮M of each each drug was performed on at least 3 muscle fascicles, dNTP, 1.5 mM of MgCl2,10mM Tris-HCl (pH 8.3), 50 mM and individuals were considered MH positive if any one KCl, and 0.6 U TaqDNA polymerase (Applied Biosys- of the three exposures exceeded the