Application of Mutation Sites in the Diagnosis of Malignant Hyperthermia

Abstract

The present invention discloses the application of mutation sites in the diagnosis of malignant hyperthermia, belonging to the field of biotechnology. The present invention provides the application of a reagent for detecting mutation sites in a subject sample in the preparation of a product for diagnosing malignant hyperthermia, specifically involving a mutation site including the change of the 2730th amino acid of the RYR1 protein from Asp to Tyr. The present invention also provides the application of the mutation site in constructing a scoring device for the risk of a subject suffering from malignant hyperthermia and a product under calcium homeostasis conditions. The present invention also provides corresponding products and devices. The present invention provides a method for regulating calcium homeostasis in vitro cells.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to the application of variant sites in the diagnosis of malignant hyperthermia. Background Art

[0002] Malignant hyperthermia was previously considered a serious complication of general anesthesia and has now been confirmed as a rare autosomal dominant genetic disease. In 2023, malignant hyperthermia was included in the second batch of rare disease catalogs in China. The main pathogenic genes of malignant hyperthermia are the RYR1 and CANCA1S genes. The full length of the coding region of the RYR1 gene exceeds 15 kb, which is one of the largest calcium channel proteins in mammalian bodies. The pathogenesis of malignant hyperthermia mainly involves the conformational changes of calcium channel proteins in skeletal muscle cells caused by mutations in the RYR1 and CANCA1S genes. When induced by certain anesthetics, stress, strenuous exercise, high temperature and other inducing factors, it shows generalized skeletal muscle rigidity and a systemic hypermetabolic response, with a very high mortality rate. Currently, more than 400 RYR1 gene variant sites have been reported, but the pathogenicity of most variants is not clear. 66 RYR1 gene mutation sites and 2 CANCA1S gene mutation sites with definite pathogenicity have been published in European Malignant Hyperthermia. Summary of the Invention

[0003] In order to solve the technical problems existing in the prior art, the present invention provides the following technical solutions.

[0004] The first aspect of the present invention provides the application of a reagent for detecting variant sites in a subject sample in the preparation of a product for diagnosing malignant hyperthermia, wherein the variant sites include the change of the amino acid at position 2730 of the RYR1 protein from Asp to Tyr.

[0005] Furthermore, the variant sites include the change of the base at position 8188 of the RYR1 gene from G to T.

[0006] Furthermore, the mutation sites further include the following one, more than one or all of the alterations in the RYR1 protein: p.Leu13Arg, p.Cys35Arg, p.Arg44Cys, p.Arg163Cys, p.Arg163Leu, p.Arg177Cys, p.Gly248Arg, p.Gly248Arg, p.Gly341Arg, p.Gly341Arg, p.Arg401Cys, p.Arg401His, p.Tyr522Ser, p.Tyr522Cys, p.Arg533Cys, p.Phe539Leu, p.Arg552Trp, p.Arg614Cys, p.Arg614Leu, p.Ser1728Phe, p.Arg2163Cys, p.Arg2163His, p.Arg2163Pro, p.Val2168Met, p.His2204Gln, p.Thr2206Arg, p.Thr2206Met, p.Val2210Phe, p.Arg2336His, p.Glu2348del, p.Ala2350Thr, p.Arg2355Trp, p.Phe2364Val, p.Gly2375Ala, p.Ala2428Thr, p.Asp2431Tyr, p.Gly2434Arg, p.Arg2435His, p.Arg2435Leu, p.Ala2437Val, p.Arg2452Trp, p.Arg2454Cys, p.Arg2454His, p.Arg2458Cys, p.Arg2458His, p.Arg2508Cys, p.Arg2508His, p.Arg2676Trp, p.Glu3104Lys, p.Arg3772Gln, p.Arg3903Gln, p.Gly3990Val, p.Gly4178Val, p.Val4234Leu, p.Val4234Leu, p.Arg4737Trp, p.Arg4737Gln, p.Leu4824Pro, p.Thr4826Ile, p.His4833Tyr, p.Leu4838Val, p.Val4849Ile, p.Arg4861His, p.Lys4876Arg, p.Pro4973Leu, p.Arg174Trp, p.Arg1086His.

[0007] Furthermore, the mutation sites also include one, more than one, or all of the following alterations in the RYR1 gene: c.38T>G, c.103T>C, c.130C>T, c.487C>T, c.488G>T, c.529C>T, c.742G>A, c.742G>C, c.1021G>A, c.1021G>C, c.1201C>T, c.1202G>A, c.1565A>C, c.1565A>G, c.1597C>T, c.1615T>C, c.1654C>T, c.1840C>T, c.1841G>T, c.5183C>T, c.6487C>T, c.6488G>A, c.6488G>C, c.6502G>A, c.6612C>G, c.6617C>G, c.6617C>T, c.6628G>T, c.7007G>A, c.7042_7044del, c.7048G>A, c.7063C>T, c.7090T>G, c.7124G>C, c.7282G>A, c.7291G>T, c.7300G>A, c.7304G>A, c.7304G>T, c.7310C>T, c.7354C>T, c.7360C>T, c.7361G>A, c.7372C>T, c.7373G>A, c.7522C>T, c.7523G>A, c.8026C>T, c.9310G>A, c.11315G>A, c.11708G>A, c.11969G>T, c.12533G>T, c.12700G>C, c.12700G>T, c.14209C>T, c.14210G>A, c.14471T>C, c.14477C>T, c.14497C>T, c.14512C>G, c.14545G>A, c.14582G>A, c.14627A>G, c.14918C>T, c.520C>T, c.3257G>A.

[0008] The term "diagnosis" as used in the present invention refers to determining the health status of a subject, covering aspects such as detecting the presence or absence of a disease, response to treatment, assessment of recurrence risk, risk and degree of malignant hyperthermia, and prognosis judgment. In some cases, the term "diagnosis" refers to being used as a single factor to determine, verify, or confirm the clinical status of a patient.

[0009] The 20 conventional (e.g., naturally occurring) amino acids and their abbreviations used herein follow conventional usage. See Immunology--A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, eds., Sinauer Associates, Sunderland, Mass. (1991)), which is hereby incorporated by reference for any purpose. Stereoisomers of the 20 conventional amino acids (e.g., D-amino acids), unnatural amino acids such as α,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other non-conventional amino acids may also be suitable components of the genes or proteins of the present invention. Examples of non-conventional amino acids include: 4-hydroxyproline, γ-carboxyglutamic acid, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline).

[0010] The second aspect of the present invention provides a product for diagnosing malignant hyperthermia, the product comprising a reagent for detecting a variant site in a subject sample in the application described in the first aspect of the present invention.

[0011] Furthermore, the product includes a kit, a chip, and a test strip.

[0012] Furthermore, the kit may further include one or more substances selected from the group consisting of: a container, an instruction manual, a positive control, a negative control, a buffer, an adjuvant, or a solvent.

[0013] Furthermore, the kit includes a suitable container, which generally includes at least one vial, test tube, flask, bottle, syringe, or other container in which a component can be placed, and preferably, can be appropriately aliquoted. When there is more than one component in the kit, the kit will generally also include a second, third, or other additional container in which the additional components are placed separately. However, different combinations of components can be included in one vial. The kit of the present invention will generally also include a container for containing the reactants, sealed for commercial sale. Such a container may include a molded or blow-molded plastic container in which the required vials can be retained.

[0014] Further, as an alternative embodiment, the chip includes: a solid-phase carrier, and oligonucleotide probes orderly immobilized on the solid-phase carrier. The solid-phase carrier can adopt various common materials in the field of gene chips, such as, including but not limited to, plastic products, microparticles, membrane carriers, etc. The plastic products can be combined with antibodies or protein antigens through non-covalent or physical adsorption mechanisms. The most commonly used plastic products are small test tubes, beads and microtiter plates made of polystyrene; the microparticles are microspheres or particles polymerized from high-molecular monomers, with a diameter mostly in micrometers. Due to having functional groups capable of binding to proteins, they are easy to form chemical conjugates with antibodies (antigens) and have a large binding capacity; the membrane carriers include microporous filter membranes such as nitrocellulose membranes, glass fiber membranes and nylon membranes.

[0015] Further, the reagents include reagents commonly used in PCR reactions, RT-PCR derived reactions, 3SR amplifications, LCR, SDA, NASBA, TMA, SYBR Green, TaqMan probes, molecular beacons, dual hybridization probes, composite probes, ISH, microarrays, Southern blots, Northern blots, multi-analyte profiling tests, enzyme-linked immunosorbent assay, radioimmunoassay, immunofluorescence assay, enzyme immunoassay, immunoprecipitation assay, chemiluminescence assay, immunohistochemical assay, dot blot assay or slot blot assay; the reagents are used to detect the protein type or genotype of the variant site marker.

[0016] Further, the reagents further include visualization reaction reagents, RNA extraction reagents, reverse transcription reagents, cDNA amplification reagents, and / or standards used for preparing standard curves.

[0017] The term "reagent" as used in the present invention includes compounds that induce desired pharmacological and / or physiological effects. This term also encompasses pharmaceutically acceptable and pharmacologically active components of those compounds specifically mentioned herein, including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs, etc. When using the above term, it should be understood that this includes the active agent itself as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs, etc. The term "reagent" should not be interpreted narrowly, but should be extended to small molecules, protein molecules such as peptides, polypeptides and proteins, and compositions containing them, as well as genetic molecules such as RNA, DNA and their mimetics and chemical analogs and cell agents.

[0018] Further, the samples include blood, serum, plasma, tissue, blood cells, bone marrow, ascites, fine needle biopsy samples, body fluids containing cells, cell-free floating nucleic acids, sputum, saliva, urine, cerebrospinal fluid peritoneal fluid, pleural fluid, feces, lymph, skin swabs, oral swabs, nasal swabs or lavage fluids.

[0019] In some embodiments, the sample taken directly from the subject is not further processed. For example, blood can be obtained from the subject's peripheral circulatory system (e.g., by a blood collection needle). The sample can include, for example, blood, urine, feces, saliva, cerebrospinal fluid, and sweat. Non-limiting examples of the sample include blood (or components of blood - e.g., white blood cells, red blood cells, platelets) obtained from any anatomical location of the subject (e.g., tissue, circulatory system, bone marrow), cells obtained from any anatomical location of the subject, skin, heart, lung, kidney, exhaled breath, bone marrow, feces, semen, vaginal fluid, tissue fluid derived from tumor tissue, breast, pancreas, cerebrospinal fluid, tissue, throat swab, biopsy, placental fluid, amniotic fluid, liver, muscle, smooth muscle, bladder, gallbladder, colon, intestine, brain, cavity fluid, sputum, pus, microbiota, meconium, milk, prostate, esophagus, thyroid, serum, saliva, urine, gastric juice and digestive fluid, tears, eye fluid, sweat, mucus, earwax, oil, glandular secretions, spinal fluid, hair, nails, skin cells, plasma, nasal swab or nasopharyngeal wash, spinal fluid, cord blood, lymph fluid, and / or other excretions or body tissues.

[0020] Furthermore, the types of the sample include tissue, blood, plasma, and blood cells.

[0021] Furthermore, the types of the sample include tissue, blood, and plasma.

[0022] Furthermore, the types of the sample are tissue and blood.

[0023] Furthermore, the type of the sample is tissue.

[0024] Furthermore, the subject includes a human or a non-human mammal.

[0025] Furthermore, the subject is a human.

[0026] The third aspect of the present invention provides the use of the variant site in constructing a scoring device for the risk of malignant hyperthermia in a subject, and the variant site is the variant site in the use described in the first aspect of the present invention.

[0027] Furthermore, the device contains a memory and a processor, and the memory includes a computer-readable storage medium.

[0028] The fourth aspect of the present invention provides a method for regulating calcium homeostasis in vitro cells, and the method includes the step of mutating the amino acid at position 2730 of the RYR1 protein in the cell from Asp to Tyr.

[0029] Furthermore, the method includes the step of mutating the base at position 8188 of the RYR1 gene in the cell from G to T.

[0030] Furthermore, the method is for non-therapeutic purposes.

[0031] Furthermore, the cell is a human or non-human mammalian cell.

[0032] Furthermore, the cell is a human or mouse cell.

[0033] Furthermore, the cell is a human cell.

[0034] The application of the reagent for forming a mutation site in the fifth aspect of the present invention in the preparation of a product for regulating intracellular calcium homeostasis, wherein the mutation site is the change of the 2730th amino acid of the RYR1 protein from Asp to Tyr.

[0035] Furthermore, the mutation site is the change of the 8188th base of the RYR1 gene from G to T.

[0036] The sixth aspect of the present invention provides a scoring device for evaluating the risk of malignant hyperthermia in a subject, and the scoring device includes the following units:

[0037] An acquisition unit: to acquire the RYR1 protein or gene sequence data of the subject;

[0038] A processing unit: the device retrieves the information of the mutation site described in the first aspect of the present invention from the acquired RYR1 protein or gene data, and matches the retrieved information with an existing database;

[0039] An output unit: when the information of the mutation site matches successfully, the device outputs an evaluation result.

[0040] The device or unit described above can be specifically implemented by a computer chip or an entity, or by a product with a certain function. A typical implementation device is a computer. Specifically, the computer can be, for example, a personal computer, a laptop computer, a cellular phone, a camera phone, a smart phone, a personal digital assistant, a media player, a navigation device, an email device, a game console, a tablet computer, a wearable device, or a combination of any of these devices.

[0041] In the present invention, the RYR1 gene refers to the gene with Gene ID: 6261 in NCBI, and the specific sequence code is NM_000540.3; in the present invention, the RYR1 protein refers to the specific sequence number NP_000531.2.

[0042] In some embodiments, the c.8188G>T, p.Asp2730Tyr mutation in the human RYR1 gene corresponds to the c.8191G>T, p.Asp2731Tyr mutation in the mouse RYR1 gene, where the Gene ID of the mouse RYR1 gene in NCBI is 20190, the specific gene sequence code is NM_009109.2, and the specific RYR1 amino acid sequence number is NP_033135.2.

[0043] In some embodiments, the above units or steps can be implemented by a computing device. In some embodiments, the units or steps can be concentrated on a single computing device. In some embodiments, the units or steps can be distributed among multiple computing devices, and the multiple computing devices are linked by a network or other connection means. In some embodiments, the computing device stores program codes for executing the above units or steps, and the program codes are stored in a storage device. In some embodiments, the units or steps can be separately fabricated into integrated circuits or combined into a single integrated circuit. In the specific embodiments of the present invention, no specific implementation manner is limited, whether it is hardware or software.

[0044] The term "unit" used in the present invention refers to an application specific integrated circuit (ASIC), an electronic circuit, a processor (such as a shared processor, a dedicated processor or a group of processors, etc.) for executing one or more software or firmware programs, a memory, a combined logic circuit and / or other suitable components that support the described functions.

[0045] The term "processor" used in the present invention refers to any type of processor, including more than one processor, such as a multi-core design or multiple processors each having a multi-core design. The processor can be configured to execute a sequence of computer program instructions, such as those instructions stored in the memory, to perform various operations, processes and methods according to the exemplary embodiments of the content of the present invention.

[0046] The term "memory" used in the present invention refers to any type of long-term, short-term, volatile, non-volatile or other memory, and should not be limited to any specific type of memory or any specific number of memories or the type of media for storing the memories.

[0047] In the context of the present invention, the term "subject" as used herein refers to any animal, including both humans and non - human animals. Non - human animals include all vertebrates, such as mammals, e.g., non - human primates (especially higher primates), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cows, and any domestic or pet animals; and non - mammals, such as amphibians, reptiles, etc. In a preferred embodiment, the subject is a human. Suitable vertebrates falling within the scope of the present invention include, but are not limited to, any member of the subphylum Chordata, including primates (e.g., humans, monkeys, and apes, and including monkey species (e.g., genus Macaca (e.g., Macaca mulatta such as Macaca fascicularis, and / or Macaca mulatta), and baboons (Papio ursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri), and tamarins (species from the genus Saguinus), and species of orangutans, such as chimpanzees (Pan troglodytes)), rodents (e.g., mice, rats, guinea pigs), Leporidae (e.g., rabbits, hares), Bovidae (e.g., cows), Ovis (e.g., sheep), Capra (e.g., goats), Sus (e.g., pigs), Equus (e.g., horses), Canis (e.g., dogs), Felidae (e.g., cats), Aves (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars, etc.), marine mammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizards, etc.), and fish.

[0048] The term "comprising / including" as used in the present invention means that a composition or method of one or more named elements or steps is open - ended, meaning that the named elements or steps are required, but other elements or steps can be added within the scope of the composition and method. To avoid prolixity, it should also be understood that any composition or method described as "comprising / including" one or more named elements or steps also describes the corresponding more limited composition or method "consisting essentially of the same named elements or steps", meaning that the composition or method includes the named required elements or steps and may also include other elements or steps that do not substantially affect the basic and novel features of the composition or method. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 It is a diagram of a patient's gene detection report.

[0050] Figure 2 It is a diagram of the gene detection reports of the patient's parents.

[0051] Figure 3It is a genetic result diagram of the patient's family.

[0052] Figure 4 It is a curve diagram of the effect of intracellular calcium ion concentration. Detailed implementation manners

[0053] The present invention will be further described below in conjunction with specific embodiments, which are only used to explain the present invention and should not be construed as limiting the present invention. Those of ordinary skill in the art can understand that: without departing from the principles and purposes of the present invention, various changes, modifications, substitutions, and variations can be made to these embodiments, and the scope of the present invention is defined by the claims and their equivalents. The experimental methods used in the following embodiments are all conventional methods unless otherwise specified; the reagents, biological materials, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0054] Embodiment

[0055] 1. Patient information and diagnosis of malignant hyperthermia

[0056] Basic patient information: male, 23 years old, with manifestations of malignant hyperthermia found during surgical treatment.

[0057] The specific manifestations of malignant hyperthermia are as follows: The patient was under general anesthesia during the operation, and the following symptoms and signs appeared 3 hours after anesthesia:

[0058] (1) Ventricular tachycardia, with a maximum heart rate of 168 beats per minute;

[0059] (2) General muscle stiffness and masseter spasm;

[0060] (3) Elevated end-tidal CO2 partial pressure, with a maximum value of 99 mmHg;

[0061] (4) Body temperature increase, with a maximum of 44.2 °C;

[0062] (5) Serum K+ 6.5 mmol / L;

[0063] (6) Arterial blood pH 6.7.

[0064] The clinical score of malignant hyperthermia was 53 points, evaluated as grade 6, and clinically diagnosed as malignant hyperthermia.

[0065] 2. Gene detection of the patient himself and his parents

[0066] Gene detection after the patient's operation found a mutation in the RYR1 gene c.8188G>T (p.Asp2730Tyr), and the specific results are as Figure 1 shown.

[0067] Subsequently, the gene conditions of the patient's parents were detected, and the same mutation site was found in the gene detection of the patient's mother. The specific results are asFigure 2 as shown

[0068] The pedigree of the patient and his / her parents was drawn, and the results are as Figure 3 shown

[0069] 3. Mouse experiments

[0070] The mouse skeletal muscle RYR1 gene was cloned into the pcDNA3.1-3Myc-His plasmid to construct the pcDNA3.1-3Myc-His-RYR1-WT plasmid, which was verified by Sanger sequencing. A plasmid carrying the RYR1-p.Asp2730Tyr mutation was constructed by site-directed mutagenesis (construct pcDNA3.1-3Myc-His-RYR1-Asp2730Tyr), which was verified by Sanger sequencing. Among them, the c.8188G>T, p.Asp2730Tyr mutation in the human RYR1 gene corresponds to the c.8191G>T, p.Asp2731Tyr mutation in the mouse RYR1 gene.

[0071] pcDNA3.1-3Myc-His-RYR1-WT and

[0072] pcDNA3.1-3Myc-His-RYR1-Asp2730Tyr were respectively transfected into 293T cells. After 48 hours, the changes in intracellular calcium ion concentration were measured by Fluo-4 calcium imaging experiment when stimulated with caffeine solutions at different concentrations. The results were respectively fitted to dose-effect curves, and the EC50 values of the two groups were calculated and the differences were compared. The EC50 value of the RYR1-WT group was 2.56±0.04 mM, and the EC50 value of the RYR1-Asp2730Tyr group was 1.12±0.09 mM, P<0.001. The results are as Figure 4 shown. Mouse experiments have proved that Asp2730Tyr can have a significant impact on intracellular calcium homeostasis, leading to malignant hyperthermia.

[0073] 4. Malignant hyperthermia pathogenicity detection chip

[0074] The newly discovered and verified mutation site (RYR1-p.Asp2730Tyr) and the known mutation sites with clear malignant hyperthermia pathogenicity published on the European Malignant Hyperthermia website (https: / / www.emhg.org / diagnostic-mutations). A gene detection chip was designed to quickly detect high-risk patients or patients with a positive family history before surgery to clarify whether there are malignant hyperthermia-related gene mutations and the risk of onset. When one or more mutation sites appear, the risk of malignant hyperthermia attack in the subject is high, and the more mutation sites, the higher the risk. The specific mutation sites are shown in Table 1.

[0075] Table 1

[0076]

[0077]

[0078]

Claims

1. A RYR1 protein related to malignant hyperthermia, the specific sequence number of the RYR1 protein is NP_000531.2, the RYR1 protein includes mutation sites, and the mutation sites include the change of the 2730th amino acid of the RYR1 protein from Asp to Tyr.

2. The RYR1 protein according to claim 1, wherein the mutation sites further include one, more or all of the following changes in the RYR1 protein: p.Leu13Arg, p.Cys35Arg, p.Arg44Cys, p.Arg163Cys, p.Arg163Leu, p.Arg177Cys, p.Gly248Arg, p.Gly248Arg, p.Gly341Arg, p.Gly341Arg, p.Arg401Cys, p.Arg401His, p.Tyr522Ser, p.Tyr522Cys, p.Arg533Cys, p.Phe539Leu, p.Arg552Trp, p.Arg614Cys, p.Arg614Leu, p.Ser1728Phe, p.Arg2163Cys, p.Arg2163His, p.Arg2163Pro, p.Val2168Met, p.His2204Gln, p.Thr2206Arg, p.Thr2206Met, p.Val2210Phe, p.Arg2336His, p.Glu2348del, p.Ala2350Thr, p.Arg2355Trp, p.Phe2364Val, p.Gly2375Ala, p.Ala2428Thr, p.Asp2431Tyr, p.Gly2434Arg, p.Arg2435His, p.Arg2435Leu, p.Ala2437Val, p.Arg2452Trp, p.Arg2454Cys, p.Arg2454His, p.Arg2458Cys, p.Arg2458His, p.Arg2508Cys, p.Arg2508His, p.Arg2676Trp, p.Glu3104Lys, p.Arg3772Gln, p.Arg3903Gln, p.Gly3990Val, p.Gly4178Val, p.Val4234Leu, p.Val4234Leu, p.Arg4737Trp, p.Arg4737Gln, p.Leu4824Pro, p.Thr4826Ile, p.His4833Tyr, p.Leu4838Val, p.Val4849Ile, p.Arg4861His, p.Lys4876Arg, p.Pro4973Leu, p.Arg174Trp, p.Arg1086His.

3. A RYR1 gene related to malignant hyperthermia, wherein the RYR1 gene refers to the gene with Gene ID: 6261 in NCBI, the RYR1 gene includes mutation sites, and the mutation sites include the change of the 8188th base of the RYR1 gene from G to T.

4. The RYR1 gene according to claim 3, wherein the mutation sites further include one, more or all of the following changes in the RYR1 gene: c.38T>G, c.103T>C, c.130C>T, c.487C>T, c.488G>T, c.529C>T, c.742G>A, c.742G>C, c.1021G>A, c.1021G>C, c.1201C>T, c.1202G>A, c.1565A>C, c.1565A>G, c.1597C>T, c.1615T>C, c.1654C>T, c.1840C>T, c.1841G>T, c.5183C>T, c.6487C>T, c.6488G>A, c.6488G>C, c.6502G>A, c.6612C>G, c.6617C>G, c.6617C>T, c.6628G>T, c.7007G>A, c.7042_7044del, c.7048G>A, c.7063C>T, c.7090T>G, c.7124G>C, c.7282G>A, c.7291G>T, c.7300G>A, c.7304G>A, c.7304G>T, c.7310C>T, c.7354C>T, c.7360C>T, c.7361G>A, c.7372C>T, c.7373G>A, c.7522C>T, c.7523G>A, c.8026C>T, c.9310G>A, c.11315G>A, c.11708G>A, c.11969G>T, c.12533G>T, c.12700G>C, c.12700G>T, c.14209C>T, c.14210G>A, c.14471T>C, c.14477C>T, c.14497C>T, c.14512C>G, c.14545G>A, c.14582G>A, c.14627A>G, c.14918C>T, c.520C>T, c.3257G>A.

5. A scoring device for evaluating the risk of malignant hyperthermia in a subject, the scoring device comprising the following units: An acquisition unit: acquiring RYR1 protein or gene sequence data of the subject, and the specific sequence number of the RYR1 protein is NP_000531.2; A processing unit: the device retrieves the information of the mutation sites described in any one of claims 1-4 from the acquired RYR1 protein or gene data, and matches the retrieved information with an existing database; Output unit: When the information of the mutation site matches successfully, the output device evaluates the result.

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