Application of variation site in diagnosis of malignant hyperpyrexia

Abstract

The invention discloses application of a variation site in diagnosis of malignant hyperpyrexia, belongs to the technical field of biology, provides application of a reagent for detecting the variation site in a subject sample in preparation of a product for diagnosing malignant hyperpyrexia, and particularly relates to application of the variation site including variation of the 2730th amino acid of RYR1 protein from Asp to Tyr. The invention also provides an application of the variation site in constructing a scoring device for the risk of the subject suffering from malignant hyperpyrexia and a product for calcium homeostasis conditions, also provides a corresponding product and device, and provides a method for regulating calcium homeostasis in in-vitro cells.

Description

Technical Field

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

[0002] Malignant hyperthermia was previously considered a serious complication of general anesthesia. It has now been confirmed to be a rare disease with autosomal dominant inheritance. my country will include malignant hyperthermia in the second batch of rare disease catalogs in 2023. The main pathogenic genes of malignant hyperthermia are RYR1 and CANCA1S genes. The RYR1 gene coding region is more than 15kb in length and is one of the largest calcium channel proteins in mammals. The pathogenesis of malignant hyperthermia mainly involves mutations in the RYR1 and CANCA1S genes, which lead to changes in the conformation of calcium channel proteins in skeletal muscle cells. When induced by certain anesthetics, stress, strenuous exercise and high temperature, the whole body skeletal muscle tonic contraction and whole body hypermetabolic response are manifested, and the mortality rate is extremely high. More than 400 RYR1 gene mutation sites have been reported, but the pathogenicity of most mutations and malignant hyperthermia is still unclear. European malignant hyperthermia has now announced that 66 RYR1 gene mutation sites and 2 CANCA1S gene mutation sites have clear pathogenicity. 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 use of a reagent for detecting a variation site in a subject sample in the preparation of a product for diagnosing malignant hyperthermia, wherein the variation site includes a change in which the amino acid at position 2730 of the RYR1 protein changes from Asp to Tyr.

[0005] Furthermore, the mutation site includes a change in the 8188th base of the RYR1 gene from G to T.

[0006] Furthermore, the variant sites also include one, more or all of the following changes in 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.Phe539Le u, p.Arg552Trp, p.Arg614Cys, p.Arg614Leu, p.Ser1728Phe, p.Arg2163Cys, p.Arg2163His, p.Arg2163Pro, p.Val2168Met, p.His22 04Gln, 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.Arg2 454Cys, 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.Thr 4826Ile, p.His4833Tyr, p.Leu4838Val, p.Val4849Ile, p.Arg4861His, p.Lys4876Arg, p.Pro4973Leu, p.Arg174Trp, p.Arg1086His.

[0007] Furthermore, the variant sites also include one, multiple 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.16 15T>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.6 612C>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.117 08G>A, c.11969G>T, c.12533G>T, c.12700G>C, c.12700G>T, c.14209C>T, c.14210G>A, c.14471T>C, c.1447 7C>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" used in the present invention refers to determining the health status of a subject, including detecting the presence or absence of a disease, response to treatment, recurrence risk assessment, assessment of the risk and degree of malignant hyperthermia, prognosis, etc. In some cases, the term "diagnosis" refers to a single factor used 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, ES Golub and DR Gren, eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference for any purpose. Stereoisomers (e.g., D-amino acids) of the 20 conventional amino acids, unnatural amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components of the genes or proteins of the present invention. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, 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, which comprises a reagent for detecting a variant site in a sample of a subject in the application described in the first aspect of the present invention.

[0011] Furthermore, the products include test kits, chips, and test strips.

[0012] Furthermore, the kit may also include one or more substances selected from the following group: a container, instructions for use, a positive control substance, a negative control substance, a buffer, an adjuvant or a solvent.

[0013] Further, the test kit includes a suitable container, which usually includes at least one vial, test tube, flask, PET bottle, syringe or other container, in which a component can be placed, and preferably, can be appropriately divided. When there is more than one component in the test kit, the test kit will usually also include a second, third or other additional container, in which additional components are placed separately. However, the components of different combinations can be contained in a vial. The test kit of the present invention will also usually include a container for accommodating reactants, sealed for commercial sale. This container can include a plastic container of injection molding or blow molding, in which the required vial can be retained.

[0014] Further, as an optional embodiment, the chip includes: a solid phase carrier, and oligonucleotide probes fixed in order on the solid phase carrier. The solid phase carrier can be made of various commonly used materials in the field of gene chips, such as 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 micro-reaction plates made of polystyrene; the microparticles are microspheres or particles polymerized from high molecular monomers, and their diameters are mostly micrometers. Because they carry functional groups that can bind to proteins, they are easy to form chemical couplings with antibodies (antigens) and have large binding capacity; the membrane carriers include microporous filter membranes such as nitrocellulose membranes, glass cellulose membranes and nylon membranes.

[0015] Furthermore, the reagents include reagents commonly used in PCR reaction, RT-PCR derivatization reaction, 3SR amplification, LCR, SDA, NASBA, TMA, SYBR Green, TaqMan probe, molecular beacon, double hybridization probe, composite probe, ISH, microarray, Southern blot, Northern blot, multi-analyte spectrum test, enzyme-linked immunosorbent assay, radioimmunoassay, immunofluorescence assay, enzyme immunoassay, immunoprecipitation assay, chemiluminescence assay, immunohistochemistry 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] Furthermore, the reagents also include visualization reaction reagents, RNA extraction reagents, reverse transcription reagents, cDNA amplification reagents, and / or standards for preparing standard curves.

[0017] The term "agent" used in the present invention includes compounds that induce the desired pharmacological and / or physiological effects. The term also covers 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 the above terms are used, 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 "agent" 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 cellular agents.

[0018] Furthermore, the sample includes blood, serum, plasma, tissue, blood cells, bone marrow, ascites, fine needle biopsy samples, body fluids containing cells, free floating nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, stool, lymph, skin swab, oral swab, nasal swab or lavage.

[0019] In some embodiments, the sample is taken directly from the subject without further processing, for example, blood can be obtained from the subject's peripheral circulatory system (e.g., via a blood collection needle). The sample may include, for example, blood, urine, feces, saliva, cerebrospinal fluid, and sweat. Non-limiting examples of such samples include blood (or components of blood - e.g., white blood cells, red blood cells, platelets) obtained from any anatomical location of a subject (e.g., tissue, circulatory system, bone marrow), cells obtained from any anatomical location of a subject, skin, heart, lung, kidney, exhaled breath, bone marrow, stool, semen, vaginal fluid, tissue fluid 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, breast milk, prostate, esophagus, thyroid, serum, saliva, urine, gastric and digestive fluids, tears, ocular fluids, sweat, mucus, earwax, oils, glandular secretions, cerebrospinal fluid, hair, nails, skin cells, plasma, nasal swab or nasopharyngeal wash, cerebrospinal fluid, umbilical cord blood, lymphatic fluid, and / or other excreta or body tissues.

[0020] Furthermore, the sample types include tissues, blood, plasma, serum, and blood cells.

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

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

[0023] Furthermore, the sample type 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 an application of a variant site in constructing a scoring device for a subject's risk of developing malignant hyperthermia, wherein the variant site is the variant site in the application described in the first aspect of the present invention.

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

[0028] A fourth aspect of the present invention provides a method for regulating calcium homeostasis in cells in vitro, the method comprising the step of causing the 2730th amino acid of the intracellular RYR1 protein to mutate from Asp to Tyr.

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

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

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

[0032] Furthermore, the cells are human or mouse cells.

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

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

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

[0036] A sixth aspect of the present invention provides a scoring device for assessing the risk of a subject suffering from malignant hyperthermia, the scoring device comprising the following units:

[0037] Acquisition unit: acquiring RYR1 protein or gene sequence data of the subject;

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

[0039] Output unit: When the information of the variant site is matched successfully, the output device evaluates the result.

[0040] The above-mentioned devices or units can be implemented by computer chips or entities, or by products with certain functions. 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, with a specific sequence code of NM_000540.3; in the present invention, the RYR1 protein refers to the specific sequence number of 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, wherein 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-mentioned units or steps can achieve the effect through 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 on multiple computing devices, and multiple computing devices are linked by a network or other linking methods. In some embodiments, the computing device stores a program code for executing the above-mentioned units or steps, and the program code is stored in a storage device. In some embodiments, the units or steps can be prepared into integrated circuits respectively, or they can be combined into a single integrated circuit. In the specific embodiments of the present invention, no implementation method 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 processor, etc.) and memory for executing one or more software or firmware programs, a combined logic circuit and / or other suitable components supporting 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 with a multi-core design for each processor. The processor can be configured to execute a sequence of computer program instructions, such as a sequence of those instructions stored in a memory, to perform various operations, processes and methods according to the exemplary embodiments of the content of the present invention.

[0046] The term "memory" as used in this disclosure 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 types of media storing memory.

[0047] In the context of the present invention, the term "subject" used refers to any animal, including humans and non-human animals. Non-human animals include all vertebrates, for example, mammals, such as non-human primates (particularly higher primates), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and any livestock or pets; 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., macaques, such as cynomolgus monkeys (Macaca fascicμLaris), and / or rhesus monkeys (Macaca mμLatta) and baboon (Papioursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri) and tamarins (species from the genus Saguinus), as well as species of apes, such as chimpanzees (Pantroglodytes), rodents (e.g., mice, rats, guinea pigs), Leporidae (e.g., rabbits, hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), avians (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgies, 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 the 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 may be added within the scope of the composition and method. To avoid redundancy, it should also be understood that any composition or method described as "comprising / including" one or more named elements or steps also describes a 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 characteristics of the composition or method. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is the patient's genetic testing report.

[0050] Figure 2 This is the genetic test report of the patient's parents.

[0051] Figure 3This is the genetic results chart of the patient's family.

[0052] Figure 4 It is the intracellular calcium ion concentration effect curve. DETAILED DESCRIPTION

[0053] The present invention is further described below in conjunction with specific embodiments, which are only used to explain the present invention and cannot be understood as limiting the present invention. It can be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purpose of the present invention, and the scope of the present invention is defined by the claims and their equivalents. The experimental methods used in the following embodiments are conventional methods unless otherwise specified; the reagents, biological materials, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial sources.

[0054] Example

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

[0056] Patient's basic information: Male, 23 years old, symptoms of malignant hyperthermia were found during surgical treatment.

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

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

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

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

[0061] (4) fever, up to 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, the assessment was level 6, and the clinical diagnosis was malignant hyperthermia.

[0065] 2. Genetic testing of the patient and his parents

[0066] The patient's postoperative genetic testing revealed a RYR1 gene c.8188G>T (p.Asp2730Tyr) mutation. The specific results are as follows Figure 1 shown.

[0067] The genetic status of the patient's parents was then tested, and the same mutation site was found in the patient's mother's genetic test. The specific results are as follows Figure 2 shown.

[0068] Draw a pedigree chart of the patient and his parents. Figure 3 shown.

[0069] 3. Mouse Experiment

[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 (construction of pcDNA3.1-3Myc-His-RYR1-Asp2730Tyr), which was verified by Sanger sequencing, where 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 was transfected into 293T cells, and the changes in intracellular calcium ion concentration were measured by Fluo-4 calcium imaging experiment 48 hours later when stimulated by different concentrations of caffeine solution. The results were fitted with dose-effect curves, and the EC50 of the two groups was calculated to compare the differences. The EC50 value of the RYR1-WT group was 2.56±0.04mM, and the EC50 value of the RYR1-Asp2730Tyr group was 1.12±0.09mM, P<0.001. The results are shown in Figure 4 Mouse experiments have shown that Asp2730Tyr can significantly affect intracellular calcium homeostasis, leading to malignant hyperthermia.

[0073] 4. Malignant hyperthermia disease detection chip

[0074] Newly discovered and verified mutation sites (RYR1-p.Asp2730Tyr) and known mutation sites with clear pathogenicity for malignant hyperthermia published on the European Malignant Hyperthermia website (https: / / www.emhg.org / diagnostic-mutations). Design a gene detection chip that can quickly detect high-risk patients or patients with a positive family history before surgery to determine whether there are malignant hyperthermia-related gene mutations and the risk of disease. When one or more mutation sites appear, the subject has a high risk of malignant hyperthermia. 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. Use of a reagent for detecting a mutation site in a subject's sample in the preparation of a product for diagnosing malignant hyperthermia, wherein the mutation site includes a change in which the 2730th amino acid of the RYR1 protein changes from Asp to Tyr.

2. The use according to claim 1, wherein the mutation site includes a change in the 8188th base of the RYR1 gene from G to T.

3. The use according to claim 1, wherein the mutation site also includes one, more or all of the following changes in 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.Phe236 4Val, 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.Arg3772G ln, p.Arg3903Gln, p.Gly3990Val, p.Gly4178Val, p.Val4234Leu, p.Val4234Leu, p.Arg4737Trp, p.Arg4737Gln, p.Leu4824Pro, p.Th r4826Ile, p.His4833Tyr, p.Leu4838Val, p.Val4849Ile, p.Arg4861His, p.Lys4876Arg, p.Pro4973Leu, p.Arg174Trp, p.Arg1086His.

4. The use according to claim 1, wherein the variant 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.1597 C>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.6502 G>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.144 77C>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 product for diagnosing malignant hyperthermia, comprising a reagent for detecting a variant site in a subject's sample in the application of any one of claims 1 to 4.

6. The product according to claim 5, comprising a test kit, a chip, and a test paper; Preferably, the sample comprises blood, serum, plasma, tissue, blood cells, bone marrow, ascites, fine needle biopsy sample, body fluid containing cells, free floating nucleic acid, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, stool, lymph, skin swab, oral swab, nasal swab or lavage; Preferably, the subject comprises a human or non-human mammal; Preferably, the subject is a human.

7. Application of a variant site in constructing a scoring device for a subject's risk of malignant hyperthermia, wherein the variant site is a variant site in the application according to any one of claims 1 to 4; Preferably, the device comprises a memory and a processor, and the memory comprises a computer-readable storage medium.

8. A method for regulating calcium homeostasis in cells in vitro, the method comprising the step of causing the amino acid 2730 of the RYR1 protein in the cell to mutate from Asp to Tyr; Preferably, the method comprises the step of causing a mutation from G to T at base 8188 of the RYR1 gene in the cell.

9. Use of a reagent that forms a mutation site in the preparation of a product for regulating intracellular calcium homeostasis, wherein the mutation site is a change in which the amino acid at position 2730 of the RYR1 protein changes from Asp to Tyr; Preferably, the mutation site is a change in which the base at position 8188 of the RYR1 gene changes from G to T.

10. A scoring device for assessing a subject's risk of malignant hyperthermia, the scoring device comprising the following units: Acquisition unit: acquiring RYR1 protein or gene sequence data of the subject; Processing unit: the device retrieves the information of the variant site described in any one of claims 1 to 4 from the acquired RYR1 protein or gene data, and matches the retrieved information with the existing database; Output unit: When the information of the variant site is matched successfully, the output device evaluates the result.

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