Functional characterization of the RYR1 mutation p.Arg4737Trp associated with susceptibility to malignant hyperthermia

Highlights

• Causative RYR1 mutations allow diagnosis of susceptibility to malignant hyperthermia.

• The RYR1 mutation p.Arg4737Trp has been recently detected in a German MH family.

• Functional properties of p.Arg4737Trp are consistent with malignant hyperthermia susceptibility.

Abstract

Aside from the in vitro contracture test, genetic screening for causative RYR1 mutations is the established procedure to diagnose susceptibility to malignant hyperthermia (MH). However, currently only 34 out of more than 300 known RYR1 mutations have been confirmed to be causative for MH by experimental studies addressing their functional impact on intracellular calcium homeostasis. The RYR1 mutation p.Arg4737Trp has been recently detected in a German MH family. To evaluate the effects of that mutation on intracellular calcium handling, the response after stimulation with the RYR1 agonist 4-chloro-m-cresol was investigated in immortalized B lymphocytes containing the p.Arg4737Trp mutation and compared to the response of wild type RYR1 from unaffected family members and unrelated controls. Intracellular resting calcium was slightly but significantly elevated in mutation positive cells. Calcium release following stimulation with 4-chloro-m-cresol was significantly increased in B lymphocytes carrying the p.Arg4737Trp mutation compared to mutation negative controls. Hence, the functional properties of the RYR1 mutation p.Arg4737Trp are consistent with susceptibility to MH. Together with previously published data, the mutation has now been reported in three independent MH positive families.

Introduction

Malignant hyperthermia (MH) is a rare but life-threatening pharmacogenetic disorder of excitation contraction coupling and calcium homeostasis in skeletal muscle [1], [2]. Exposure to halogenated volatile anesthetics as well as to depolarizing muscle relaxants may induce a hypermetabolic syndrome characterized by uncontrolled calcium release from the sarcoplasmic reticulum via functionally altered ryanodine receptors 1 (RYR1) or dihydropyridine receptors. Cardiac arrhythmia, hypercapnia, muscle rigidity, hyperkalemia and hyperthermia may result and are some of the typical symptoms of an acute MH reaction. Presymptomatic identification of individuals with susceptibility to MH is an important task in order to prevent this dangerous anesthetic complication [3].

According to the guidelines issued by the European and the North American MH Groups, the in vitro contracture test (IVCT) and the caffeine halothane contracture test (CHCT), respectively, are gold standard for identification of malignant hyperthermia susceptible (MHS) individuals. This invasive technique requires a surgical muscle biopsy usually taken from the quadriceps femoris muscle [4], [5], [6]. DNA analysis and searching for mutations is less invasive and has led to the identification of more than 300 different mutations in RYR1 so far [7], [8]. However, the pathogenic impact of most of these mutations is yet to be determined and so far only 34 mutations have been acknowledged to be causative for MH after functional evaluation [9]. Further expansion of this list by evaluation of every mutation is important in order to increase the number of patients and families who could be offered genetic rather than surgical testing [10].

In the present study we characterize a novel RYR1 mutation that was identified in a German family. The index patient survived an acute MH reaction in the 1960s at the age of 10 years during general anesthesia for eye surgery. After inhalational induction with halothane he received 1 mg/kg succinylcholine for intubation. Immediately afterward masseter spasm as well as generalized muscular rigidity was observed. Ten minutes later he developed tachycardia (170 bpm), rapid temperature increase to 39.1 °C and hypercarbia which required repeated exchange of the CO₂ absorber in short time intervals. Although not all necessary items have been recorded, the Larach score for this episode reached at least 48 points, indicating an MH event to be “very likely” [11]. The incident was published as a case report in a regional medical journal at that time, when knowledge about the underlying pathomechanism of the so-called anesthesia associated hyperpyrexia was still very limited [12]. Many years later, the predisposition of the index patient to MH was confirmed by the IVCT, which showed a clearly positive result. Screening of the patient's RYR1 for the most common causative mutations was negative but sequencing of the patient's entire RYR1 gene identified the heterozygous variant c.14209C>T (p.Arg4737Trp) in exon 98.

Epstein–Barr virus immortalized human B lymphocytes have been shown to express the skeletal muscle isoform of RYR1 and have been successfully used to examine the functional effect of several RYR1 mutations [13], [14]. Here, we present the results of IVCT, the genetic segregation of the p.Arg4737Trp mutation in the pedigree and measurements of intracellular calcium levels in human B lymphocytes derived from family members and controls with and without the mutation in order to characterize the functional effect of the mutation.