• Mirko Vukcevic, Marcus Broman, Gunilla Islander, Mikael Bodelsson, Eva Ranklev-Twetman, Clemens R Müller, and Susan Treves.
• Department of Anaesthesia and Biomedicine, Basel University Hospital, Basel, Switzerland.
• Anesth. Analg. 2010 Jul 1;111(1):185-90.

BackgroundA 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.MethodsB 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.ResultsIt 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.ConclusionsPeripheral 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.

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