Sequence capture and massively parallel sequencing to detect mutations associated with malignant hyperthermia

Malignant hyperthermia (MH) is a potentially fatal pharmacogenetic disorder in which intracellular calcium homeostasis in the skeletal muscle of susceptible individuals is disrupted upon exposure to halogenated anaesthetics. While MH is linked to the ryanodine receptor (RYR1) on chromosome 19 and the α1S subunit of the voltage-dependent L-type calcium channel (CACNA1S) on chromosome 1, mutations have been found in only 50-70% of patients, and subsequently, there is a need for a more powerful screening tool.

Genomic DNA capture and next-generation sequencing was used to screen 32 genes involved in excitation-contraction coupling, skeletal muscle calcium homeostasis, or immune response in two MH patients. Lymphoblastoid cell lines were used to functionally characterize candidate RYR1 mutations in one family.

Sequence analysis revealed two putative causative mutations in RYR1 in one patient. Segregation analysis and functional analysis support a causative role of the detected variants. The amount of Ca(2+) released after stimulation with 4-chloro-m-cresol from B lymphocytes of the MH-susceptible patients in the family was significantly greater compared with that of Ca(2+) released from cells of an MH-negative family member. In the other patient, no causative mutations were identified in the 32 genes screened.

In this study, we successfully demonstrate the use of genomic DNA capture and next-generation sequencing for identification of putative mutations causing MH. We also suggest that whole exome sequencing may be necessary to identify MH causing mutations in patients where no mutations in RYR1 and CACNA1S have been identified thus far.