Functional properties of RYR1 mutations identified in Swedish patients with malignant hyperthermia and central core disease.
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.
Vukcevic
Mirko
M
Department of Anaesthesia and Biomedicine, Basel University Hospital, Basel, Switzerland.
Broman
Marcus
M
Islander
Gunilla
G
Bodelsson
Mikael
M
Ranklev-Twetman
Eva
E
Müller
Clemens R
CR
Treves
Susan
S
D000328:Adult; D001706:Biopsy; D001800:Blood Specimen Collection; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002675:Child, Preschool; D003408:Cresols / Q000494:pharmacology; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D005260:Female; D005820:Genetic Testing / Q000379:methods; D006579:Heterozygote; D006801:Humans; D008297:Male; D008305:Malignant Hyperthermia / Q000097:blood / Q000175:diagnosis / Q000235:genetics*; D009119:Muscle Contraction / Q000187:drug effects; D018482:Muscle, Skeletal / Q000473:pathology; D009154:Mutation / Q000502:physiology*; D020512:Myopathy, Central Core / Q000097:blood / Q000175:diagnosis / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics*; D013548:Sweden / Q000453:epidemiology
D003408:Cresols; D012333:RNA, Messenger; D019837:Ryanodine Receptor Calcium Release Channel; C019292:4-chloro-2-cresol; D004247:DNA; D002118:Calcium