Gene mutations used to diagnose multiple epiphyseal dysplasia

Abstract

This invention discloses gene mutations for diagnosing multiple epiphyseal dysplasia. Through Sanger sequencing, this invention identifies mutation sites in the SLC26A2 gene: c.1262T>C: p.Ile421Thr and / or c.1020_1022delTGT: p.Val341del. The mutants provided by this invention can be used to diagnose multiple epiphyseal dysplasia, enabling early diagnosis and providing a new direction for its diagnosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to gene mutations used for diagnosing multiple epiphyseal dysplasia. Background Technology

[0002] Multiple epiphyseal dysplasia is a rare congenital bone dysplasia, also known as hereditary endochondral skeletal dysplasia. Its clinical manifestations and inheritance patterns are heterogeneous. Its main clinical features include: normal at birth, slow growth typically appearing after age 2; pain and stiffness in large joints such as the knees, hips, hands, and shoulders; unsteady gait; and gradually developing deformities such as short, thick fingers (toes), genu varum / valgum, etc. The spine may be mildly affected or normal. The head and face are normal, and intelligence is unaffected. The final height is mild to moderate short stature or normal, generally 145cm-170cm. Clinical diagnosis relies mainly on medical history, clinical manifestations, and X-ray characteristics; there is currently no effective treatment.

[0003] With the rapid development of molecular biology techniques and the widespread application of methods such as DNA polymorphism linkage analysis and direct sequencing, significant progress has been made in the genetic research of this disease. Polymorphic markers and gene loci closely linked to MED have been continuously discovered, providing new directions for the diagnosis of multiple epiphyseal dysplasia. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, this invention provides gene mutations for diagnosing multiple epiphyseal dysplasia.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] The first aspect of the present invention provides the use of a reagent for detecting SLC26A2 gene mutations in a sample in the preparation of a product for diagnosing multiple epiphyseal dysplasia, wherein the SLC26A2 gene mutation sites include c.1262T>C: p.Ile421Thr and / or c.1020_1022delTGT: p.Val341del.

[0007] Furthermore, the reagents include primers and probes for detecting the gene mutation sites c.1262T>C and / or c.1020_1022delTGT of SLC26A2, and / or specific antibodies for detecting the protein mutation sites p.Ile421Thr and / or p.Val341del of SLC26A2.

[0008] Furthermore, the primers or probes are labeled with one or more of a radioactive isotope, a fluorescent substance, or an enzyme.

[0009] Furthermore, the reagents also include reagents used to detect the SLC26A2 gene mutation site using PCR-SSCP, heteroduplex analysis, denaturing gradient gel electrophoresis, chemical cleavage mismatch analysis, allele-specific oligonucleotide analysis, DNA microarray technology, ligase chain reaction, allele-specific amplification, and sequencing.

[0010] Furthermore, the sequencing methods include Sanger sequencing, next-generation sequencing, and amino acid sequencing.

[0011] Furthermore, the sequencing method is the Sanger sequencing method.

[0012] Furthermore, the products include reagent kits, test strips, and chips.

[0013] Furthermore, the kit also includes genomic DNA extraction reagents, PCR reaction system reagents, DHPLC-related reagents, or protein extraction reagents.

[0014] Furthermore, the genomic DNA extraction reagent includes phenol, chloroform, isoamyl alcohol, or ethanol.

[0015] Furthermore, the PCR reaction system reagents include fluorescent dyes, Taq DNA polymerase, and dNTPs.

[0016] Furthermore, the sample was selected from peripheral blood.

[0017] Furthermore, the multiple epiphyseal dysplasia includes overt multiple epiphyseal dysplasia and occult multiple epiphyseal dysplasia.

[0018] Furthermore, the multiple epiphyseal dysplasia mentioned is selected from occult multiple epiphyseal dysplasia.

[0019] A second aspect of the present invention provides a diagnostic reagent for multiple epiphyseal dysplasia, the reagent comprising a reagent for detecting SLC26A2 mutation sites c.1262T>C: p.Ile421Thr and / or c.1020_1022delTGT: p.Val341del.

[0020] Furthermore, the reagents include primers and probes for detecting the gene mutation sites c.1262T>C and / or c.1020_1022delTGT of SLC26A2, and / or specific antibodies for detecting the protein mutation sites p.Ile421Thr and / or p.Val341del of SLC26A2.

[0021] Furthermore, the reagents also include dNTPs, Mg2+, and PCR reaction buffer.

[0022] A third aspect of the present invention provides a diagnostic kit for multiple epiphyseal dysplasia, the kit comprising the reagents described in the second aspect of the present invention.

[0023] The fourth aspect of the present invention provides the application of the mutant SLC26A2 gene in constructing a system for diagnosing multiple epiphyseal dysplasia, wherein the mutation sites of the mutant SLC26A2 gene are c.1262T>C: p.Ile421Thr and / or c.1020_1022delTGT: p.Val341del.

[0024] Furthermore, the multiple epiphyseal dysplasia includes overt multiple epiphyseal dysplasia and occult multiple epiphyseal dysplasia.

[0025] Furthermore, the multiple epiphyseal dysplasia mentioned is selected from occult multiple epiphyseal dysplasia.

[0026] A fifth aspect of the present invention provides a system for diagnosing multiple epiphyseal dysplasia, the system comprising:

[0027] 1) Sample extraction device, used to extract samples from organisms;

[0028] 2) A sequence determination device, connected to the sample extraction device, for analyzing the sample and determining the sequence of the sample;

[0029] 3) Result determination device, connected to the sequence determination device, compares the sequence of the sample with the wild-type SLC26A2 gene sequence to determine whether there are mutation sites c.1262T>C: p.Ile421Thr and / or c.1020_1022delTGT: p.Val341del in the biological sample, and then determines whether the biological sample is a biological sample with multiple epiphyseal dysplasia.

[0030] The sixth aspect of the present invention provides the application of the mutant SLC26A2 gene in constructing a computational model for diagnosing multiple epiphyseal dysplasia, wherein the mutation sites of the mutant SLC26A2 gene include c.1262T>C: p.Ile421Thr and / or c.1020_1022delTGT: p.Val341del.

[0031] Furthermore, the multiple epiphyseal dysplasia includes overt multiple epiphyseal dysplasia and occult multiple epiphyseal dysplasia.

[0032] Furthermore, the multiple epiphyseal dysplasia mentioned is selected from occult multiple epiphyseal dysplasia.

[0033] The seventh aspect of the present invention provides the use of an inhibitor of SLC26A2 gene mutation in the preparation of a medicament for treating multiple epiphyseal dysplasia, wherein the SLC26A2 gene mutation sites include c.1262T>C: p.Ile421Thr and / or c.1020_1022delTGT: p.Val341del.

[0034] Furthermore, the drug also includes a pharmaceutically acceptable carrier.

[0035] Furthermore, the multiple epiphyseal dysplasia includes overt multiple epiphyseal dysplasia and occult multiple epiphyseal dysplasia.

[0036] Furthermore, the multiple epiphyseal dysplasia mentioned is selected from occult multiple epiphyseal dysplasia.

[0037] Advantages and beneficial effects of the present invention:

[0038] This invention identified the mutation sites c.1262T>C: p.Ile421Thr and / or c.1020_1022delTGT: p.Val341del in the SLC26A2 gene using Sanger sequencing. The mutants provided by this invention can be used to diagnose multiple epiphyseal dysplasia, enabling early diagnosis of the disease and providing a new direction for the diagnosis of multiple epiphyseal dysplasia. Attached Figure Description

[0039] Figure 1 These are family pedigrees and clinical phenotypes of the proband. Among them, 1A is a pedigree chart, 1B is a diagram of widened distal femoral epiphysis in patient II1, 1C is a diagram of dysplasia of the proximal left fibular and tibial epiphysis in patient II1, and 1D is a diagram of dysplasia of the right femoral and tibial and fibular epiphysis in patient II1.

[0040] Figure 2 This is a Sanger sequencing result image of the proband's family;

[0041] Figure 3 It is an immunofluorescence image;

[0042] Figure 4 These are mRNA expression levels related to cartilage tissue proliferation and differentiation. Among them, 4A is the ACAN mRNA expression level, 4B is the MMP13 mRNA expression level, 4C is the COL10A1 mRNA expression level, and 4D is the RUNX2 mRNA expression level. Detailed Implementation

[0043] The following provides definitions for some of the terms used in this specification. Unless otherwise stated, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0044] This invention provides the application of reagents for detecting SLC26A2 gene mutations in samples in the preparation of products for diagnosing multiple epiphyseal dysplasia. The SLC26A2 gene mutation sites include c.1262T>C: p.Ile421Thr and / or c.1020_1022delTGT: p.Val341del.

[0045] SLC26A2 includes wild-type, mutant, or fragments thereof. The term encompasses full-length, unprocessed SLC26A2, any form of SLC26A2 derived from cell-processed SLC26A2, and naturally occurring variants of SLC26A2 (e.g., splice variants or allelic variants). The term covers, for example, human SLC26A2 and SLC26A2 from any other vertebrate source, including mammals such as primates and rodents (e.g., mice and rats), gene ID: 1836.

[0046] In this invention, diagnosis includes the prediction of disease risk, the diagnosis of whether a disease will occur, and the assessment of disease prognosis.

[0047] In this invention, mutation refers to an alteration in the wild-type polynucleotide sequence, resulting in a variant, which can be naturally occurring or non-naturally occurring. The mutation in this invention refers to the change of nucleotide 1262 of the SLC26A2 gene from T (thymine) to C (cytosine), leading to a mutation of amino acid 421 of the encoded SLC26A2 protein from Ile (isoleucine) to Thr (threonine) and / or the deletion of nucleotides 1020-1022 of the SLC26A2 gene from T (thymine)G (guanine)T (thymine), leading to the deletion of amino acid 341 of the encoded SLC26A2 protein from Val (valine).

[0048] In this invention, a sample can refer to a biological sample, including but not limited to peripheral blood, urine, and saliva; as well as a solid tissue sample, such as a biopsy specimen.

[0049] Samples also include samples that have been manipulated or processed in any appropriate manner after purchase, including but not limited to centrifugation, filtration, precipitation, dialysis, chromatography, treatment with reagents, washing, or enrichment of a component of the sample, such as a cell population.

[0050] In a specific embodiment of the present invention, the sample is selected from peripheral blood.

[0051] The reagents include primers, probes for detecting the gene mutation sites c.1262T>C and / or c.1020_1022delTGT of SLC26A2, and / or specific antibodies for detecting the protein mutation sites p.Ile421Thr and / or p.Val341del of SLC26A2.

[0052] In this invention, primers represent oligonucleotides, which can be natural RNA, DNA, or any form of natural nucleotide. Primers can even be non-natural nucleotides such as LNA or ZNA. Primers are substantially or essentially complementary to a specific sequence on one strand of the template. Primers must be fully complementary to one strand of the template to initiate extension, but the primer sequence does not need to be perfectly complementary to the template sequence. For example, adding a non-complementary sequence to the 5' end of a primer complementary to the template at its 3' end will still result in a primer that is substantially complementary to the template. Even imperfectly complementary primers can form primer-template complexes with the template, thereby enabling amplification, provided the primer is long enough to bind sufficiently to the template. Primers can be single-stranded or double-stranded and must be long enough to initiate the synthesis of the desired extension product in the presence of an inducer. The exact length of the primer depends on many factors, including temperature, primer source, and method of use. For example, for diagnostic applications, depending on the complexity of the target sequence, oligonucleotide primers typically contain 15-25 or more nucleotides, although they can contain fewer. The factors involved in determining the appropriate primer length are readily known to those skilled in the art.

[0053] In this invention, a probe refers to a molecular probe capable of binding to a specific sequence, subsequence, or other portion of another molecule, unless otherwise specified. A probe typically refers to a polynucleotide probe capable of binding to another polynucleotide (often called a target polynucleotide) through complementary base pairing. Depending on the stringency of the hybridization conditions, the probe can bind to a target polynucleotide that lacks complete sequence complementarity with the probe. Probes can be labeled directly or indirectly, including primers. Hybridization methods include, but are not limited to, solution phase, solid phase, mixed phase, or in situ hybridization assays. Exemplary probes in this invention include PCR primers and gene-specific DNA oligonucleotide probes, such as microarray probes immobilized on a microarray substrate, quantitative nuclease protection assay probes, probes linked to molecular barcodes, and probes immobilized on beads. These probes have a base sequence complementary to a specific base sequence of the target gene. Here, complementarity is used in the context of hybridization and does not necessarily mean complete complementarity. These polynucleotides typically have at least 80%, preferably at least 90%, more preferably at least 95%, and particularly preferably 100% homology with respect to the specific base sequence. These probes can be DNA or RNA, or they can be polynucleotides obtained by artificial nucleic acid substitution with PNA, LNA, ENA, GNA, TNA, etc., in part or all of their nucleotides.

[0054] The primers or probes of this invention can be chemically synthesized using phosphorimide solid-phase support or other well-known methods. The nucleic acid sequences can also be modified using many techniques known in the art. Non-limiting examples of such modifications include methylation, capping, or labeling.

[0055] The primers or probes are labeled with one or more of a radioactive isotope, a fluorescent substance, or an enzyme.

[0056] In this invention, the fluorescent material includes, but is not limited to, TAMRAT. TM Alexa555, Alexa647, Cy3 and Cy5 of the cyanine dye series, and fluorescein.

[0057] Radioactive isotopes including but not limited to 32 P, 33 P, 35 S.

[0058] Enzymes include, but are not limited to, alkaline phosphatase and horseradish peroxidase.

[0059] As for the method of labeling the primers or probes involved in this invention with radioactive isotopes, any method known in itself and commonly practiced in the field can be used. Specifically, for example, methods of labeling by incorporating nucleotides labeled with radioactive isotopes can be cited. Specifically, random primer methods, nick translation methods, 5' end labeling methods based on T4 polynucleotide kinase, and 3' end labeling methods based on terminal deoxynucleotidyl transferase can be cited.

[0060] As a method for labeling the primers or probes involved in this invention with fluorescent substances, it is sufficient to follow the methods known in the art, specifically, for example, a method of incorporating nucleotides labeled with fluorescent dyes into primers or probes according to methods known in the art.

[0061] As for the method of labeling the primers or probes involved in this invention with enzymes, it is sufficient to follow the methods known in themselves that are usually carried out in the field. Specifically, for example, a direct labeling method in which enzyme molecules such as alkaline phosphatase and horseradish peroxidase are directly covalently bound to the primers or probes to be labeled can be cited.

[0062] The reagents also include those used to detect the SLC26A2 gene mutation site using PCR-SSCP, heteroduplex analysis, denaturing gradient gel electrophoresis, chemical cleavage mismatch analysis, allele-specific oligonucleotide analysis, DNA microarray technology, ligase chain reaction, allele-specific amplification, and sequencing.

[0063] The CR-SSCP method involves short single-stranded DNA and RNA molecules forming different conformations on a non-denaturing polyacrylamide gel. A change in even a single base will affect this conformation, thus altering their migration speed on the gel. The basic principle is that single-stranded DNA forms secondary structures under neutral conditions. These secondary structures depend on its base composition; even a single base difference will result in different secondary structures and thus different migration rates.

[0064] Heterologous double-strand analysis (HA) is a method for directly separating hybridized mutant-wild-type DNA double strands on a denaturing gel. The basic principle is that heterologous hybrid double-stranded DNA formed from mutant and wild-type DNA will form protrusions at their mismatch sites, resulting in a different migration rate compared to the corresponding homologous double DNA during electrophoresis on a non-denaturing gel.

[0065] Denaturing gradient gel electrophoresis (DGGE) is a gel system that separates DNA fragments based on their melting properties. The double helix structure of nucleic acids can unwind under certain conditions, a process known as denaturation. The temperature at which 50% of nucleic acids are denatured is called the melting temperature (Tm). The Tm value primarily depends on the GC content in the DNA molecule. DGGE sets the gel under dual denaturation conditions: temperature 50-60°C, denaturant concentration 0-100%. When a double-stranded DNA fragment passes through a gel with a gradient of increasing denaturant concentration, the fragment migrates to a point where the denaturant concentration corresponds to the Tm value of the low-melting-point region of the DNA fragment, causing this region to begin melting, while the high-melting-point region remains double-stranded. This localized unwinding alters the migration rate of the DNA molecule, achieving separation. Changes in Tm are sequence-dependent; even a single base substitution can cause an increase or decrease in the Tm value. Therefore, DGGE can detect any single-base substitution, frameshift mutation, or deletion mutation of fewer than 10 bases in a DNA molecule.

[0066] Chemical cleavage mismatch assay (CCM) is a mutation detection technique developed based on Maxam-Gilbert sequencing. Its accuracy in detecting mutations is comparable to that of DNA sequencing. The basic principle involves mixing the DNA fragment to be tested with a corresponding wild-type DNA fragment or a DNA and RNA fragment for denaturation hybridization. In the heterozygous double-stranded nucleic acid molecule, mismatched C and T molecules can be cleaved. The presence of a mutation can be determined by denaturing gel electrophoresis. This method has a very high detection rate.

[0067] Allele-specific oligonucleotide (ASO) assay is a hybridization-based technique for detecting known mutations. It combines PCR and ASO, designing an approximately 20 bp oligonucleotide fragment containing the mutation site. This fragment serves as a probe, hybridizing with PCR-amplified sample DNA immobilized on a membrane. Various mutation types of oligonucleotide probes can be used, with a wild-type probe as a control. A positive hybridization band indicates the presence of a point mutation corresponding to the ASO probe in the sample.

[0068] DNA chip technology works by arranging many oligonucleotide DNA sequences with known sequences on an integrated circuit board, overlapping each other by one base and covering all the genes to be detected. Fluorescently labeled normal DNA and mutant DNA are hybridized with two DNA chips respectively. Since there is at least one base difference, the normal and mutant DNA will produce different hybridization patterns. By detecting the fluorescence signals produced by the two DNA molecules using a co-concentration microscope, it can be determined whether a mutation exists.

[0069] Allele-specific amplification (ASA) involves designing two 5' primers, one complementary to normal DNA and the other to the mutant DNA. For homozygous mutations, two parallel PCRs are performed using both primers and a 3' primer, respectively. Only the primer that is completely complementary to the mutant DNA will extend and produce the PCR amplification product. If the mismatch is located at the 3' end of the primer, the PCR will not extend.

[0070] The sequencing methods include pyrosequencing, Taqman technology, microsequencing, Sanger sequencing, second-generation sequencing, Taqman sequencing, cyclic sequencing, semiconductor sequencing, and amino acid sequencing.

[0071] The amino acid sequencing methods include N-terminal sequencing and C-terminal sequencing.

[0072] Among them, N-terminal sequencing is based on the Edman chemical degradation method, and the main methods include the dinitrofluorobenzene method (FDNB, DNFB), the cyanate method, and the dimethylaminonaphthalenesulfonyl chloride method.

[0073] C-terminal sequencing methods include hydrazine hydrolysis, the most commonly used method for determining the C-terminus. The peptide is dissolved in anhydrous hydrazine and reacted at 100°C. The C-terminal amino acid is released as a free amino acid, while the remaining peptide chain reacts with the hydrazine to form amino acid hydrazine. In addition, there is carboxypeptidase hydrolysis, where carboxypeptidases specifically hydrolyze C-terminal amino acids. Based on their specificity, carboxypeptidases can be classified as A, B, and C. When using carboxypeptidases to determine the C-terminus, enzyme kinetic experiments must be performed beforehand to select appropriate enzyme concentrations and reaction times, ensuring that the released amino acids are primarily C-terminal amino acids.

[0074] In an embodiment of the present invention, the reagents include those used to detect the SLC26A2 gene mutation sites c.1262T>C: p.Ile421Thr and / or c.1020_1022delTGT: p.Val341del using sequencing methods.

[0075] In a specific embodiment of the present invention, the reagents include those used for detecting the SLC26A2 gene mutation sites c.1262T>C: p.Ile421Thr and / or c.1020_1022delTGT: p.Val341del using Sanger sequencing.

[0076] The products include reagent kits, test strips, and chips.

[0077] The kit also includes genomic DNA extraction reagents, PCR reaction system reagents, DHPLC related reagents, or protein extraction reagents.

[0078] The genomic DNA extraction reagents include, but are not limited to, phenol, chloroform, isoamyl alcohol, or ethanol.

[0079] The PCR reaction system reagents include dNTPs, MgCl2, Taq DNA polymerase, and fluorescent dyes.

[0080] The kit also includes routine enzyme digestion components, including reaction buffer and deionized water.

[0081] The buffer solutions include, but are not limited to, polymerase buffer and Tris hydrochloric acid buffer.

[0082] The kit also includes suitable containers, typically including at least one vial, test tube, long-necked flask, PET bottle, syringe, or other container, for holding one component, and preferably, for appropriate aliquoting. When more than one component is present in the kit, the kit will also typically include second, third, or other additional containers for separately holding the additional components. However, different combinations of components may be contained in a single vial. The kit of the present invention will also typically include a container for containing the reactants, sealed for commercial sale. Such a container may include injection-molded or blow-molded plastic containers, in which the desired vials can be retained. In some embodiments, the kit also includes instructions for using the kit. The kit can be customized for home use, clinical use, or scientific research use.

[0083] In this invention, a chip, also referred to as an array, refers to a solid support containing linked nucleic acid or peptide probes. Arrays typically contain a variety of different nucleic acid or peptide probes attached to a substrate surface at different known locations. These arrays, also called "microarrays," can typically be produced using mechanosynthesis or photoguided synthesis methods, which combine photolithography and solid-phase synthesis. Arrays can comprise flat surfaces or can be nucleic acids or peptides on beads, gels, polymer surfaces, fibers such as optical fibers, glass, or any other suitable substrate. Arrays can be packaged in a manner that allows for diagnostic or other manipulation of a fully functional device.

[0084] In some embodiments of the invention, the microarray is a hybridization array of elements arranged in an ordered manner on a matrix, such as polynucleotide probes (e.g., oligonucleotides) or binders (e.g., antibodies). The matrix can be a solid matrix, such as a glass or silica slide, beads, fiber optic adhesive, or a semi-solid matrix, such as a nitrocellulose membrane. The nucleotide sequence can be DNA, RNA, or any arrangement thereof.

[0085] This invention provides a system for diagnosing multiple epiphyseal dysplasia, the system comprising:

[0086] 1) Sample extraction device, used to extract samples from organisms;

[0087] 2) A sequence determination device, connected to the sample extraction device, for analyzing the sample and determining the sequence of the sample;

[0088] 3) Result determination device, connected to the sequence determination device, compares the sequence of the sample with the wild-type SLC26A2 gene sequence to determine whether there are mutation sites c.1262T>C: p.Ile421Thr and / or c.1020_1022delTGT: p.Val341del in the biological sample, and then determines whether the biological sample is a biological sample with multiple epiphyseal dysplasia.

[0089] When the sample contains c.1262T>C: p.Ile421Thr and / or c.1020_1022delTGT: p.Val341del mutations, it is diagnosed as multiple epiphyseal dysplasia.

[0090] The present invention also provides the use of an inhibitor of SLC26A2 gene mutation in the preparation of a drug for treating multiple epiphyseal dysplasia, wherein the SLC26A2 gene mutation sites include c.1262T>C: p.Ile421Thr and / or c.1020_1022delTGT: p.Val341del.

[0091] In this invention, treatment refers to a clinical intervention that attempts to alter the natural course of the disease in the treated individual, which may be for prevention or in the course of clinicopathology. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and eliminating or improving prognosis.

[0092] In this invention, the inhibitor is an inhibitor that prevents the occurrence of multiple epiphyseal dysplasia. Specifically, the inhibitor can induce mutations in the SLC26A2 gene, preferably inhibiting the c.1262T>C: p.Ile421Thr and / or c.1020_1022delTGT: p.Val341del mutations.

[0093] Inhibitors are agents that alleviate the symptoms of multiple epiphyseal dysplasia, including those used to induce further mutations in the SLC26A2 gene following mutations in c.1262T>C: p.Ile421Thr and / or c.1020_1022delTGT: p.Val341del.

[0094] Specifically, the further mutation of the SLC26A2 gene after the c.1262T>C:p.Ile421Thr mutation refers to a C (cytosine) mutation at nucleotide position 1262 of the mutated SLC26A2 gene, which can be mutated to any nucleotide, preferably T (thymine); resulting in a mutation at amino acid position 421 (threonine) of the encoded SLC26A2 protein, which can be mutated to any amino acid, preferably Ile (isoleucine); and / or an insertion of nucleotides at positions 1020-1022 of the SLC26A2 gene, which can be any combination of nucleotides, preferably T (thymine)G (guanine)T (thymine), resulting in an insertion of amino acid position 341 of the encoded SLC26A2 protein, which can be any amino acid, preferably Val (valine).

[0095] The drug also includes a pharmaceutically acceptable carrier.

[0096] In this invention, pharmaceutically acceptable means, to the extent of reasonable medical judgment, those compounds, materials, compositions, and / or dosage forms suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers include any solvent, diluent or other liquid solvent, dispersant or suspending agent, surfactant, isotonic agent, thickener or emulsifier, preservative, solid binder, lubricant, provided it is suitable for the desired specific dosage form. The use of any conventional carrier medium is also considered within the scope of this invention unless it is incompatible with the medicament of this invention, for example, due to any undesirable biological effects or otherwise incompatible with one or more other components of the pharmaceutically acceptable composition in a harmful manner.

[0097] The invention is further illustrated below with reference to specific embodiments. It should be understood that the specific embodiments described herein are by way of example and are not intended to limit the invention. The main features of the invention can be used in various embodiments without departing from the scope of the invention.

[0098] Example

[0099] 1.1 Experimental Methods

[0100] First, clinical data (5 individuals across 2 generations, including 2 patients) and peripheral blood samples from the MED family were collected. Whole-exome sequencing, bioinformatics analysis, and variant database filtering were performed on the proband to identify candidate pathogenic variants. Second, PCR-Sanger assay was used to verify genotype-phenotype co-segregation among family members. Next, wild-type and mutant pcDNA3.1 eukaryotic expression plasmids for the SLC26A2 gene were constructed and transfected with commercially available human primary chondrocytes using lipo3000. After 48 hours, cellular RNA was extracted, and qPCR was used to detect the effect of the mutant expression vector on the expression of human chondrocyte differentiation-related genes ACAN, MMP13, COL10A1, and RUNX2.

[0101] 1.2 Experimental Results

[0102] X-ray of the proband showed distal epiphyseal dysplasia of the tibia and fibula. Figure 1 ).

[0103] The proband carries a complex heterozygous mutation consisting of a deletion mutation c.1020_1022delTGT (p.Val341del) in exon 2 of the SLC26A2 gene and a missense mutation c.1262T>C (p.Ile421Thr). His father is a carrier. Figure 2 ).

[0104] Immunofluorescence revealed that, compared to the wild-type plasmid, the mutant plasmid showed reduced localization and expression levels in the cytoplasm. Figure 3 ).

[0105] qPCR analysis showed that, compared with wild-type, the expression of ACAN, a chondrogenesis marker, was significantly increased in human chondrocytes transfected with the SLC26A2 mutant group. Figure 4 A) The expression levels of chondrocyte differentiation markers MMP13, COL10A1, and RUNX2 were significantly reduced. Figure 4 B-4D).

[0106] In summary, this study shows that SLC26A2 gene mutations alter the intracellular expression and localization of SLC26A2 in in vitro cell experiments. Furthermore, the mutations promote chondrocyte proliferation and inhibit chondrocyte differentiation.

[0107] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. Application of reagents for detecting SLC26A2 gene mutations in samples in the preparation of products for diagnosing multiple epiphyseal dysplasia, where SLC26A2 gene mutation sites include c.1262T>C: p.Ile421Thr and / or c.1020_1022delTGT: p.Val341del.

2. The application according to claim 1, characterized in that, The reagents include primers and probes for detecting the gene mutation sites c.1262T>C and / or c.1020_1022delTGT in SLC26A2.

3. The application according to claim 2, characterized in that, The primers or probes are labeled with one or more of a radioactive isotope, a fluorescent substance, or an enzyme.

4. The application according to claim 2, characterized in that, The reagents also include those using PCR. The reagents used to detect the SLC26A2 gene mutation site include SSCP, heteroduplex analysis, denaturing gradient gel electrophoresis, chemical mismatch cleavage, allele-specific oligonucleotide analysis, DNA microarray technology, ligase chain reaction, allele-specific amplification, and sequencing.

5. The application according to claim 4, characterized in that, The sequencing methods include Sanger sequencing, next-generation sequencing, and amino acid sequencing.

6. The application according to claim 5, characterized in that, The sequencing method used is the Sanger sequencing method.

7. The application according to claim 1, characterized in that, The products include reagent kits, test strips, and chips.

8. The application according to claim 7, characterized in that, The kit also includes genomic DNA extraction reagents, PCR reaction system reagents, DHPLC related reagents, or protein extraction reagents.

9. The application according to claim 8, characterized in that, The genomic DNA extraction reagents include phenol, chloroform, isoamyl alcohol, or ethanol.

10. The application according to claim 8, characterized in that, The PCR reaction system reagents include fluorescent dyes, Taq DNA polymerase, and dNTPs.

11. The application according to claim 1, characterized in that, The sample was selected from peripheral blood.

12. The application according to claim 1, characterized in that, The multiple epiphyseal dysplasia mentioned is selected from occult multiple epiphyseal dysplasia.

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