Scanning for mutations of the ryanodine receptor (RYR1) gene by denaturing HPLC: detection of three novel malignant hyperthermia alleles.
BACKGROUND
Malignant hyperthermia (MH) is a fatal autosomal dominant pharmacogenetic disorder characterized by skeletal muscle hypertonicity that causes a sudden increase in body temperature after exposure to common anesthetic agents. The disease is genetically heterogeneous, with mutations in the gene encoding the skeletal muscle ryanodine receptor (RYR1) at 19q13.1 accounting for up to 80% of the cases. To date, at least 42 RYR1 mutations have been described that cause MH and/or central core disease. Because the RYR1 gene is huge, containing 106 exons, molecular tests have focused on the regions that are more frequently mutated. Thus the causative defect has been identified in only a fraction of families as linked to chromosome 19q, whereas in others it remains undetected.


METHODS
We used denaturing HPLC (DHPLC) to analyze the RYR1 gene. We set up conditions to scan the 27 exons to identify both known and unknown mutations in critical regions of the protein. For each exon, we analyzed members from 52 families with positive in vitro contracture test results, but without preliminary selection by linkage analysis.


RESULTS
We identified seven different mutations in 11 MH families. Among them, three were novel MH alleles: Arg44Cys, Arg533Cys, and Val2117Leu.


CONCLUSIONS
Because of its sensitivity and speed, DHPLC could be the method of choice for the detection of unknown mutations in the RYR1 gene.
Tammaro
Angela
A
Malignant Hyperthermia Center, AO Cardarelli, via San Giacomo dei Capri 66, 80131 Napoli, Italy.
Bracco
Adele
A
Cozzolino
Santolo
S
Esposito
Maria
M
Di Martino
Antonietta
A
Savoia
Gennaro
G
Zeuli
Laura
L
Piluso
Giulio
G
Aurino
Stefania
S
Nigro
Vincenzo
V
D000483:Alleles; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004252:DNA Mutational Analysis / Q000379:methods; D006801:Humans; D008305:Malignant Hyperthermia / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D009691:Nucleic Acid Denaturation; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics*
D019837:Ryanodine Receptor Calcium Release Channel