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Abstract
Background:
Malignant hyperthermia (MH) 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. However, the search for known and novel mutations in the RYR1 gene is hampered by the fact that the gene contains 106 exons. We aimed to analyze mutations from the entire RYR1 coding region in Korean MH families.
Methods:
We investigated seven affected MH individuals and their family members. The entire RYR1 coding region from the genomic DNA was sequenced, and RYR1 haplotyping and mutational analysis were carried out.
Results:
We identified nine different RYR1 mutations or variations from seven Korean MH families. Among these, five previously reported mutations (p.Gly248Arg, p.Arg2435His, p.Arg2458His, p.Arg-2676Trp, and p.Leu4838Val) and four novel variations of unknown significance (p.Arg2508Cys, p.Met-4022Val, p.Glu2669Lys, and p.Ala4295Val) were identified. In two families, two variations (R2676W & M4022V, R2435H & A4295V, respectively) were identified simultaneously. Four of the observed nine mutations or variations were located outside the hotspot region of RYR1 mutations.
Conclusions:
These data indicate that RYR1 is a main candidate gene in Korean MH families, and that comprehensive screening of the entire coding sequence of the RYR1 gene is necessary for molecular genetic investigations in MH-susceptible individuals, owing to the presence of RYR1 mutations or variations outside of the hotspot region.
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Fig. 1.
Haplotype analysis and segregation of the RYR1 mutations and variations p.Arg2458His (in family 1), p.Arg2508Cys (in family 2), p.Met4022Val and p.Arg2676Trp (in family 3), p.Glu2669Lys (in family 4), and p.Arg2435His and p.Ala4295Val (in family 5). The arrow denotes the proband who experienced malignant hyperthermia crises. Black shapes indicate individuals with a past history of malignant hyperthermia attack, and white shapes with a question mark denote individuals for whom disease status is unknown. Segregation of the respective mutations and variations is also indicated: a plus sign (+) indicates that an individual is heterozygous for the mutant allele, and a minus sign (-) indicates that an individual is homozygous for the normal allele. The results of marker typing and mutation identification are shown in order: D19S191-D19S220-RYR1 mutations or variations-D19S422-D19S190-D19S223.
Fig. 2.
DNA sequencing of the entire coding region of the RYR1 gene, in which c.7373CGC>CAC∗ in exon 46 (from family 1); c.7522CGT> TGT in exon 47 (from family 2); c.12064ATG>GTG and c.8026CGG>TGG in exon 88 and 50, respectively (from family 3); c.8005GAG> AAG in exon 50 (from family 4); c.7304CGC>CAC and c.12884GCG>GTG in exon 45 and 91, respectively (from family 5); c.742GGG> AGG in exon 9 (from family 6); and c.14512CTG>GTG in exon 101 (from family 7) were detected. Individuals without a mutation or variation showed a single peak, whereas individuals with mutations or variations showed two superimposed peaks (arrows), indicating a heterozygous missense mutation or variation. ∗Residue numbering within the human RYR1 cDNA (accession number: NM_000540). Abbreviations: F, forward sequencing; R, reverse sequencing. DNA sequencing of the entire coding region of the RYR1 gene, in which c.7373CGC>CAC∗ in exon 46 (from family 1); c.7522CGT> TGT in exon 47 (from family 2); c.12064ATG>GTG and c.8026CGG>TGG in exon 88 and 50, respectively (from family 3); c.8005GAG> AAG in exon 50 (from family 4); c.7304CGC>CAC and c.12884GCG>GTG in exon 45 and 91, respectively (from family 5); c.742GGG> AGG in exon 9 (from family 6); and c.14512CTG>GTG in exon 101 (from family 7) were detected. Individuals without a mutation or variation showed a single peak, whereas individuals with mutations or variations showed two superimposed peaks (arrows), indicating a heterozygous missense mutation or variation. ∗Residue numbering within the human RYR1 cDNA (accession number: NM_000540).
Abbreviations: F, forward sequencing; R, reverse sequencing.
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