MH is an autosomal-dominant genetic disease that affects calcium release from the sarcoplasmic reticulum (SR) by skeletal muscle. It is characterized by a cause of hypermetabolism when exposed to irritating agents, such as anesthesia. European Malignant Hyperthermia Group (EMHG) guidelines indicate that to classify a pathogenic variant, variants need to be genetically and functionally characterized(3, 14, 15). Recent evidence has also confirmed the foresight of requiring functional analysis of missense variants before their adoption for diagnostic use(14). Currently, the golden standard for diagnosing MH is an in IVCT that is based on the contraction of muscle fibers in the presence of halothane or caffeine (16, 17). According to the EMHG guidelines, an individual is under suspicion to MH when both caffeine and halothane test results are positive. However, IVCT is expensive, confined to specialized testing centers, and requires a surgical procedure. Some research has supported DNA screening as a viable alternative primary diagnostic approach to IVCT(6). The potential expense in these additional variants has been more than compensated by the advances in genetic technology.
As the cost of targeted DNA sequencing decreases, next-generation sequencing (NGS) has become the method of choice for variant detection and has been used for the diagnosis of many disease causing variants(3). However, one of the bottlenecks in data analysis is the large number of variants identified in each individual. Common variants can be filtered based on their minor frequencies, but all of the other rare variants need to be investigated.
Genetic analysis of healthy volunteers in this study found that among variants in the RYR1, CACNA1S, and JSRP1 genes, variants in RYR1 had the largest number, with three identified variants causing amino acid sequence changes. Four JSRP1 gene variants caused amino acid sequence changes, and three CANA1S gene variants caused amino acid sequence changes. There were only 2 site changes in STAC3, and none of them caused amino acid sequence changes.
Combined with variants in the literature, there are more than 400 variants of unknown significance in RYR1, and 48 have been shown to be associated with MH(18–20). Changes in the amino acid sequences in proteins may disrupt the binding sites of other proteins, catalyzing residues and protein folding (structure), but not all of the amino acid sequence changes result in changes in protein function. Experimental evidence clearly indicates that the signs and symptoms of MH are associated with the uncontrolled release of intracellular Ca2+ from the skeletal muscle SR(21). Enhanced intracellular Ca2+ leads to abnormal metabolism of skeletal muscle, manifested by activation of muscle contraction, oxygen consumption and CO2 production, ATP hydrolysis and heat generation. Other proteins that have a potential or known role in the function of RyR1 include intact SR membrane proteins and proteins that link between the stable plasma membrane and the sarcoplasmic reticulum(22). Because RyR1 plays a vital role in maintaining Ca2+ homeostasis and excitation−contraction coupling in skeletal muscle cells, MH susceptible individuals carrying RYR1 variants may have skeletal muscle metabolism abnormalities even without triggering by anesthesia(23, 24).
The second gene related to MH sensitivity is CACNA1S, which encodes one of the dihydropyridine receptor (DHPR) subunits of skeletal muscle, and is found at locus 1q32(25). Currently, there were a number of variants (T1354S, A1086H, A1086S, R174W, and R1086H) that have been found in the CACNA1S gene in MH patients(26–30), but only two CACNA1S variants have been sufficiently characterized to be regarded as pathogenic for MH(20). These variants have not been functionally characterized as MH pathogenic factor by EMHG criteria(25).
JP-45 encoded by JSPR1 is another intact SR protein that has been shown to co-localize with RyR1. Some studies suggest that individuals with JSPR1 variants and pathogenic RYR1 variants will have a lower overall phenotype than those expressing RYR1 variants alone(4, 31). These observations highlight the possibility that polymorphic variants regulate RYR1 function and may help explain observed MH-sensitive variable phenotypes(32).
There are many free prediction tools that can be used to predict the pathogenicity of variants. Although pathogenic and common sequence variants in certain genes have been used to test the accuracy of computer-based prediction methods, variantal genes for this disease has not been performed yet. There have been reports in the literature that the prediction tools of the website used here have a total sensitivity of 84%−100% and a specificity of 25%−83%(3). Prediction is very useful for selecting variants for further functional analysis. There is always a certain degree of computer analysis in variant identification, but it is too early to begin using it for clinical diagnosis of MH sensitivity. No program yet can correctly predict all MH pathogenic variations. According to the literature, MutPred, SNPs & GO, PhD-SNP and CADD have the highest sensitivities (true positives), and SIFT and MutPred have the highest specificities (true negatives)(3). In this study, G1530T in CACNA1S was suggested to be pathogenic by 6 prediction tools, and C4615T was suggested to be pathogenic using 5 prediction tools. The results of the different prediction software were different, and how to increase the sensitivity and specificity of each prediction software by expanding the database and improving the algorithm is an issue that also needs attention.
Numerous RRY1 or CACNA1S variants have been identified by next-generation sequencing (33–35). However, none of the currently available platforms for sequencing, chemistry for sample preparation, or analysis software, are able to yield 100% coverage of all exons in the human genome(36). Individuals carrying one of the MH pathogenic variants are considered to be susceptible to MH, meaning that they have an increased risk of developing MH. When a familial pathogenic variant is identified, genetic testing can be used on extended family members, and all of the members of a family carrying the variant should be considered MH susceptible(30). However, the susceptibility to MH in individuals who do not carry familial variations cannot be ruled out because multiple pathogenic variations may exist in the same family(37, 38). There is evidence that there are multiple genetic factors that affect the MH-sensitive phenotype, because defects in more than one gene work together to produce a phenotype, or actually work opposite each other to modify or mask the MH phenotype(38).
It is worth noting that the variant of RYR1 also causes a variety of congenital myopathies with different clinical manifestations, but it is characterized by histological changes in muscle morphology(39). For clinical safety, these patients should be considered as having MH. Although variant screening of MH genes using the latest techniques has become feasible and cost-effective, the genetic heterogeneity of MH still prevents a‘quick and cheap’ MH test that can be done the night before anesthesia to prevent manifestation of MH(40).