1. Functional properties of RYR1 mutations identified in Swedish patients with malignant hyperthermia and central core disease.
- Author
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Vukcevic M, Broman M, Islander G, Bodelsson M, Ranklev-Twetman E, Müller CR, and Treves S
- Subjects
- Adult, Biopsy, Blood Specimen Collection, Calcium metabolism, Cell Line, Child, Preschool, Cresols pharmacology, DNA biosynthesis, DNA genetics, Dose-Response Relationship, Drug, Female, Genetic Testing methods, Heterozygote, Humans, Male, Malignant Hyperthermia blood, Malignant Hyperthermia diagnosis, Muscle Contraction drug effects, Muscle, Skeletal pathology, Myopathy, Central Core blood, Myopathy, Central Core diagnosis, RNA, Messenger biosynthesis, RNA, Messenger genetics, Sweden epidemiology, Malignant Hyperthermia genetics, Mutation physiology, Myopathy, Central Core genetics, Ryanodine Receptor Calcium Release Channel genetics
- Abstract
Background: A diagnosis of malignant hyperthermia susceptibility by in vitro contraction testing can often only be performed at specialized laboratories far away from where patients live. Therefore, we have designed a protocol for genetic screening of the RYR1-cDNA and for functional testing of newly identified ryanodine receptor 1 (RYR1) gene variants in B lymphocytes isolated from peripheral blood samples drawn at local primary care centers., Methods: B lymphocytes were isolated for the extraction of RYR1-mRNA and genomic DNA and for establishment of lymphoblastoid B cell lines in 5 patients carrying yet unclassified mutations in the RYR1. The B lymphoblastoid cell lines were used to study resting cytoplasmic calcium concentration, the peak calcium transient induced by the sarco(endo)plasmic reticulum Ca-ATPase inhibitor thapsigargin, and the dose-dependent calcium release induced by the ryanodine receptor agonist 4-chloro-m-cresol., Results: It was possible to extract mRNA for cDNA synthesis and to create B lymphocyte clones from all samples. All B lymphoblastoid cell lines carrying RYR1 candidate mutations showed significantly increased resting cytoplasmic calcium levels as well as a shift to lower concentrations of 4-chloro-m-cresol inducing calcium release compared with controls., Conclusions: Peripheral blood samples are stable regarding RNA and DNA extraction and establishment of lymphoblastoid B cell lines after transportation at ambient temperature over large distances by ordinary mail. Functional tests on B cells harboring the newly identified amino acid substitutions indicate that they alter intracellular Ca2+ homeostasis and are most likely causative of malignant hyperthermia.
- Published
- 2010
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