Gck mutants and uses thereof

Abstract

The application discloses a GCK mutant and application thereof. Compared with a wild type GCK gene, the GCK mutant has a c.263T>C mutation, is a new pathogenic mutation of juvenile-onset adult-onset diabetes (MODY), and can effectively detect whether a subject has MODY by detecting whether the gene pathogenic mutation exists in the subject. Discovery of the pathogenic mutation site further expands the understanding and cognition of MODY, provides a new detection site and a treatment idea of a detection method for diagnosis or treatment of MODY, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a GCK mutant and its applications. Background Technology

[0002] Maturity-onset diabetes of the young (MODY) is a special type of monogenic diabetes with genetic heterogeneity. It is an autosomal dominant inherited form of diabetes, and its clinical characteristics overlap to some extent with type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM), leading to frequent misdiagnosis. To date, at least 14 MODY subtypes (MODY1-14) have been identified, each caused by different gene mutations. Among them, the GCK gene, belonging to MODY2, was the first gene confirmed to cause MODY. According to the ClinVar database, over 1200 variants of this gene have been identified.

[0003] The "Expert Consensus on Screening and Treatment of Adult-onset Diabetes Mellitus (MODY) in Adolescents" states that genetic testing is the gold standard for diagnosing MODY, especially suitable for patients with a high clinical suspicion. The consensus recommends appropriately relaxing the screening criteria for genetic testing to help improve the detection rate, reduce the risk of missed diagnoses, and enable more MODY patients to benefit from the precise management of genetic screening. Currently, a large number of MODY cases still fail to achieve molecular diagnosis because pathogenic variants are not detected in the 14 known MODY-related genes; these cases are classified as MODY-X. Existing research speculates that MODY-X may be related to undiscovered pathogenic genes, pathogenic sites, or non-coding region regulatory sites. Therefore, conducting molecular genetic testing on suspected patients is of great significance: by clarifying the type of pathogenic variant, not only can the diagnosis and precise subtyping of MODY be confirmed, but the clinical course can also be predicted to some extent, providing a basis for the development of individualized treatment strategies. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a GCK gene mutant and its application.

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

[0006] The first aspect of the present invention provides a GCK mutant protein, wherein the GCK mutant protein has a p.Leu88Pro mutation compared with the protein encoded by the wild-type GCK gene.

[0007] Furthermore, the accession number of the wild-type GCK protein is NP_000153.3.

[0008] In this invention, the mutations include gene mutations such as point mutations, deletions, and insertions. Point mutations include synonymous mutations, missense mutations, and nonsense mutations. A "missense mutation" refers to a mutation in which a codon encoding a certain amino acid is replaced by a base substitution, thus changing the type and sequence of amino acids in the polypeptide chain. Missense mutations usually result in the polypeptide chain losing its original function, causing protein abnormalities.

[0009] The second aspect of the present invention provides a GCK gene mutant encoding the GCK mutant protein described in the first aspect of the present invention, wherein the GCK gene mutant has a c.263T>C mutation compared with the wild-type GCK gene.

[0010] In some implementations, when applied to nucleic acid molecules, the term "encoding" refers to a nucleic acid that, if in its natural state or when manipulated by methods known to those skilled in the art, can be transcribed and / or translated to produce mRNA containing a polypeptide and / or fragments thereof, is referred to as "encoding" a polypeptide. The antisense strand is the complement of such a nucleic acid, from which the coding sequence can be deduced.

[0011] Furthermore, the accession number of the wild-type GCK gene is NM_000162.5.

[0012] A third aspect of the present invention provides a recombinant vector comprising the GCK gene mutant described in the second aspect of the present invention.

[0013] The vector of this invention refers to an artificial construct capable of delivering and preferably expressing one or more target genes or sequences in a host cell. The vector of this invention is not limited and can be an expression vector, viral vector, etc. In some embodiments, the vector contains the GCK gene mutant, promoter, and terminator described in this invention, or optionally further contains a marker gene. The vector can be a known vector or a self-constructed vector. Known vectors include plasmid vectors, lentiviral vectors, adenovirus vectors, AAV viral vectors, etc.

[0014] In some embodiments, the vector may contain expression regulatory sequences, such as transcription and translation start and stop codons, which are specific to the type of host cell into which the vector is to be introduced (e.g., bacteria, fungi, plants, or animals), depending on whether the vector is DNA-based or RNA-based. Recombinant expression vectors may contain restriction sites to facilitate cloning.

[0015] In some embodiments, the vector may also contain one or more marker genes that allow selection of host cells for transformation or transfection. Marker genes include biocidal resistance (e.g., resistance to antibiotics, heavy metals, etc.); and prototrophic complementation in auxotrophic hosts. Suitable marker genes for the expression vector of this invention include neomycin / G418 resistance genes, hygromycin resistance genes, histidine resistance genes, tetracycline resistance genes, ampicillin resistance genes, kanamycin resistance genes, and puromycin resistance genes.

[0016] In some implementations, the expression vector can be constructed using methods well known to those skilled in the art. These methods include, but are not limited to, recombinant DNA technology, DNA synthesis technology, etc. DNA encoding the antibody or its antigen-binding fragment can be effectively ligated to a multiple cloning site in the vector to guide mRNA synthesis and thereby protein expression, or for homologous recombination.

[0017] A fourth aspect of the present invention provides a recombinant cell comprising the GCK mutant protein described in the first aspect of the present invention, the GCK gene mutant described in the second aspect of the present invention, or the recombinant vector described in the third aspect of the present invention.

[0018] The recombinant cells of this invention refer to any cell type suitable for transformation, transfection, transduction, etc., using a GCK mutant protein as described in the first aspect of this invention, a GCK gene mutant as described in the second aspect of this invention, or a recombinant vector as described in the third aspect of this invention. Recombinant cells include any offspring of the parent cell that differ from the parent cell due to mutations occurring during replication. Preferably, the recombinant cells include prokaryotic cells and eukaryotic cells; more preferably, the prokaryotic cells include bacteria, actinomycetes, cyanobacteria, mycoplasma, chlamydia, and rickettsiae; even more preferably, the eukaryotic cells include mammalian cells, insect cells, plant cells, and yeast cells.

[0019] In some embodiments, the recombinant cells are prepared by introducing the mutant or recombinant vector of the present invention as described above into the cells. The methods of introduction include, but are not limited to, physical, chemical, and biological methods. The physical methods include, but are not limited to, microinjection, electroporation, calcium phosphate precipitation, lipid transfection, and particle bombardment. The chemical methods include, but are not limited to, colloidal dispersion systems and lipid-based systems. The colloidal dispersion systems include, but are not limited to, macromolecular complexes, nanocapsules, microspheres, and beads. The lipid-based systems include, but are not limited to, oil-in-water emulsions, micelles, mixed micelles, and liposomes. The biological methods include, but are not limited to, DNA vectors, lentiviral vectors, poxvirus vectors, herpes simplex virus vectors, adenovirus vectors, and adeno-associated virus vectors.

[0020] The fifth aspect of this invention provides the use of a reagent for detecting the GCK mutant protein described in the first aspect of this invention or the GCK gene mutant described in the second aspect of this invention in the preparation of any one or more of the following products:

[0021] 1) Products for diagnosing adult-onset diabetes in adolescents;

[0022] 2) Products for diagnosing preconception and / or prenatal genetic diseases.

[0023] Furthermore, the reagents include antibodies, probes, primers, or guide RNAs specifically targeting the GCK mutant protein described in the first aspect of the present invention or the GCK gene mutant described in the second aspect of the present invention.

[0024] Furthermore, the product includes at least one of a reagent kit, a chip, a test strip, and a high-throughput sequencing platform.

[0025] The sixth aspect of the present invention provides a product for diagnosing adolescent-onset adult-onset diabetes or preconception and / or prenatal genetic diseases, the product comprising a reagent for detecting the GCK mutant protein described in the first aspect of the present invention or the GCK gene mutant described in the second aspect of the present invention.

[0026] In this invention, diagnosis refers to the discovery, judgment, or recognition of an individual's health status or condition based on one or more symptoms, data, or other information related to the individual. An individual's health status can be diagnosed as healthy / normal (i.e., without disease or ailment), or as unhealthy / abnormal (i.e., with disease or ailment), or as a specific degree of disease severity or progression. The term diagnosis includes the early detection of a disease / symptom associated with a specific disease or ailment; the characteristics or classification of the disease; the discovery of disease progression, cure, or recurrence; and the discovery of an individual's response to the disease after treatment or intervention.

[0027] Furthermore, the adolescent-onset adult-onset diabetes includes adolescent-onset adult-onset type 2 diabetes.

[0028] Furthermore, the reagents include antibodies, probes, primers, or guide RNAs specifically targeting the GCK mutant protein described in the first aspect of the present invention or the GCK gene mutant described in the second aspect of the present invention.

[0029] In this invention, "antibody" refers to any form of antibody that exhibits the desired biological activity (e.g., inhibiting the binding of a ligand to its receptor or inhibiting ligand-induced receptor signal transduction). Therefore, "antibody" is used in its broadest sense and is explicitly included, but not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and multispecific antibodies (e.g., bispecific antibodies), fully human, humanized, primate-derived, chimeric antibodies, single-chain antibodies, etc.

[0030] In this invention, a "probe" refers to a molecule capable of binding to a specific sequence, subsequence, or other portion of another molecule. Unless otherwise specified, a probe generally 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.

[0031] In this invention, the term "primer" refers to an oligonucleotide, whether naturally occurring in purified restriction digest or synthetically produced, which can function as a starting point for synthesis when placed under conditions that induce the synthesis of primer extension products complementary to the nucleic acid chain (e.g., in the presence of nucleotides and inducers such as DNA polymerase and at suitable temperature and pH).

[0032] In this invention, the “guide RNA” refers to a class of short non-coding RNA molecules with sequence guidance function. Through a base complementary pairing mechanism, it precisely locates nucleic acid editing effector proteins to specific DNA or RNA targets. It is a key component for realizing programmable genome editing or RNA editing and typically contains 20-50 nucleotides.

[0033] In some embodiments, the reagents include those suitable for at least one of the following methods: restriction fragment length polymorphism (RFLP), single-strand conformation polymorphism (SCM), polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGE), nucleic acid sequencing, nucleic acid genotyping chip detection, mass spectrometry of flight (MS / MS), denaturing high-performance liquid chromatography (HPLC), snapshot method, in situ hybridization, biomolecular mass spectrometry (BMS), immunoassay, Western blotting, flow cytometry, and HRM.

[0034] In some embodiments, the reagent further includes a detectable marker. The detectable marker refers to a composition capable of generating a detectable signal indicating the presence of a target polynucleotide in the sample being tested. Suitable markers include, but are not limited to, radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent components, magnetic particles, and bioluminescent components. Therefore, the marker is any composition detectable by a device or method, including but not limited to spectroscopic, photochemical, biochemical, immunochemical, electrochemical, optical, chemical detection devices, or any other suitable device. In some embodiments, the marker can be detected visually without the aid of a device.

[0035] Furthermore, the product includes at least one of a reagent kit, a chip, a test strip, and a high-throughput sequencing platform.

[0036] In some embodiments, the kit further includes at least one of a negative control, a positive control, a nucleic acid extraction reagent, a PCR amplification reagent, a sequencing reagent, and an electrophoresis detection reagent. The positive control is a sample of GCK with the aforementioned mutation; the negative control is deionized water; it is understood that in other specific examples, the negative control may be other samples of GCK without the mutation; the PCR amplification reagent includes a PCR premix, and in one optional specific example, the PCR premix includes Mg... 2+ The kit contains reaction buffer, DNA polymerase, and dNTPs. Similarly, the kit may exclude any of the following reagents: positive control, negative control, PCR amplification reagent, sequencing reagent, and electrophoresis detection reagent; the excluded reagents may be reasonably obtained from external sources.

[0037] In some embodiments, the chip includes a gene chip and a protein chip; the gene chip includes a solid support and oligonucleotide probes ordered immobilized on the solid support, the oligonucleotide probes specifically corresponding to part or all of the sequence of the GCK mutant. The protein chip includes a solid support and specific antibodies or ligands, etc., for the protein encoded by the GCK mutant immobilized on the solid support. The solid support can be made of various commonly used materials in the chip field, such as plastic products, microparticles, membrane carriers, etc.

[0038] In some implementations, the solid support may be made of various commonly used materials in the field of gene chips, including but not limited to: nylon membranes, glass or silicon slides modified with active groups (e.g., aldehydes, aminos, etc.), unmodified glass slides, plastic sheets, etc.

[0039] The seventh aspect of the present invention provides a biological model of adolescent-onset adult-onset diabetes, wherein the biological model carries the GCK mutant protein described in the first aspect of the present invention or the GCK gene mutant described in the second aspect of the present invention.

[0040] Furthermore, the biological model includes a cell model or an animal model.

[0041] Furthermore, the cells include, but are not limited to, HEK293 cells, Huh7 cells, INS-1 cells, MIN6 cells, HEK293T cells, CHO cells, COS-7 cells, yeast cells, and Escherichia coli cells.

[0042] In this invention, the animals include non-human vertebrates, more preferably mammals, which refer to all members of the class Mammalia, such as cattle, horses, pigs, sheep, monkeys, pets (e.g., dogs, cats), or rodents. The term "rodent" refers to any and all members of the phylogenetic rodent class (e.g., mice, rats, squirrels, beavers, groundhogs, voles, hamsters, guinea pigs, spiny guinea pigs), including any offspring derived therefrom.

[0043] The eighth aspect of the present invention provides any of the following methods:

[0044] 1) A method for constructing a biological model of adolescent-onset adult-onset diabetes mellitus, the method comprising introducing, in vivo, the GCK mutant protein of the first aspect of the present invention, the GCK gene mutant of the second aspect of the present invention, or the recombinant vector of the third aspect of the present invention.

[0045] 2) A method for screening drugs for treating adolescent-onset adult-onset diabetes, the method comprising applying drug candidates to a biological model constructed by the aforementioned method for constructing a biological model of adolescent-onset adult-onset diabetes, and screening for drugs that have therapeutic effects on adolescent-onset adult-onset diabetes.

[0046] Furthermore, the organism includes cells or non-human animals.

[0047] Furthermore, the biological model includes a cell model or an animal model.

[0048] In this invention, the drug candidates can be obtained from a wide variety of sources. For example, they can be obtained by random and directed synthesis of a variety of organic compounds and biomolecules, or libraries of natural compounds in the form of bacterial, fungal, plant or animal extracts, or libraries and compounds of natural or synthetic compounds modified by conventional chemical, physical or biochemical means, or by directed or random chemical modification of known pharmacological agents, such as acylation, alkylation, esterification, amidation, etc., to generate structural analogs.

[0049] The ninth aspect of the present invention provides any of the following applications:

[0050] 1) The application of the GCK mutant protein described in the first aspect of the present invention, the GCK gene mutant described in the second aspect of the present invention, or the recombinant vector described in the third aspect of the present invention in the preparation of a biological model of adolescent-onset adult-onset diabetes mellitus.

[0051] 2) Application of the biological model constructed using the method for constructing a biological model of adolescent-onset adult-onset diabetes as described in the seventh aspect of the present invention in screening drugs for the treatment of adolescent-onset adult-onset diabetes.

[0052] Furthermore, the biological model includes a cell model or an animal model.

[0053] Advantages and beneficial effects of the present invention:

[0054] The GCK mutant of this invention has a c.263T>C mutation compared to the wild-type GCK gene, which is a novel pathogenic mutation in adolescent-onset adult-onset diabetes mellitus (MODY). By detecting the presence of this pathogenic mutation in subjects, it is possible to effectively detect whether subjects have MODY. The discovery of this pathogenic mutation site provides a reliable basis for the accurate clinical diagnosis of MODY associated with this mutation, helps to improve the accuracy of MODY diagnosis and treatment, and also provides new experimental ideas and data support for gene mutation research in monogenic diabetes. Attached Figure Description

[0055] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the accompanying drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0056] Figure 1 This is a pedigree chart of genetic families with the GCK c.263T>C (p.Leu88Pro) mutation, where "Male with diabetes" indicates males with diabetes, "Female with diabetes" indicates females with diabetes, "Male without diabetes" indicates males without diabetes, "Female without diabetes" indicates females without diabetes, "Dead" indicates deceased, "Unknown phenotype" indicates unknown phenotype, and "Heterozygous GCKc.263T>C carriers" indicates heterozygous carriers of GCK c.263T>C.

[0057] Figure 2This is a Sanger sequencing peak diagram of the proband.

[0058] Figure 3 This is a three-dimensional structure diagram of the GCK protein, where A is the wild-type GCK protein, B is the GCK c.263T>C (p.Leu88Pro) mutant protein, C is a local conformation diagram of site 88 of A, and D is a local conformation diagram of site 88 of B.

[0059] Figure 4 The graph shows the kinetics of glucase in the p.L88P mutant.

[0060] Figure 5 The graph shows the thermal stability results of the p.L88P mutant. Detailed Implementation

[0061] To make the above-mentioned objectives, features, and advantages of this application more apparent and understandable, a detailed description of specific embodiments of this application is provided. Many specific details are set forth in the following description to provide a thorough understanding of this application. However, this application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of this application. Therefore, this application is not limited to the specific embodiments disclosed below.

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Unless otherwise specifically stated, all raw materials, reagents, instruments, and equipment used in this application are commercially available or can be prepared by existing methods.

[0063] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0064] In this application, terms such as "preferred," "better," "more suitable," and "ideal" are merely used to describe implementation methods or embodiments that achieve better results, and should be understood not to limit the scope of protection of this application.

[0065] The terms “having,” “containing,” “comprising,” and “including” as used in this application are synonyms and are inclusive or open-ended, not excluding additional, uncited members or features. Members or features include, for example, materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions under which actions occur, timing, states, etc.

[0066] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0067] In this application, if the unit of a data range is only followed by the right endpoint, it means that the units of the left and right endpoints are the same.

[0068] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0069] In this application, the exemplary descriptions such as "in some implementations (or embodiments)" and "in one implementation (or embodiment)" may cover, but are not limited to, the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0070] In this application, the terms "first aspect," "second aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first aspect," "second aspect," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0071] In this application, when numerical intervals (i.e., numerical ranges) are mentioned, unless otherwise specified, the distribution of selectable numerical values ​​within the numerical interval is considered continuous, and includes the two endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. The term "numerical interval" can be broadly included to include numerical interval types such as percentage intervals, ratio intervals, and proportion intervals.

[0072] In this application, the terms "room temperature" or "normal temperature" generally refer to 4°C to 35°C, for example, 20°C ± 5°C. In some embodiments of this application, "room temperature" or "normal temperature" refers to 10°C to 30°C. In some embodiments of this application, "room temperature" or "normal temperature" refers to 20°C to 30°C.

[0073] The following are some examples.

[0074] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, reference should be made to the guidelines given in this application, or to experimental manuals or conventional conditions in the art, or to the conditions recommended by the manufacturer, or to experimental methods known in the art.

[0075] In the following examples, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0076] Example 1: Study on pathogenic genes and mutation sites

[0077] I. Experimental Methods

[0078] 1. Principles of Case Collection and Organization

[0079] (1) Case screening: Screen families with clinically suspected monogenic diabetes, clarify the composition of family members, and collect detailed information on the proband and family members (age, gender, height, weight, BMI, etc.), chief complaint, present illness, past medical history, personal history and family history of genetic diseases, and focus on recording diabetes-related symptoms (such as polydipsia, polyphagia, polyuria, weight loss, etc.) and age of onset.

[0080] (2) Clinical examination: A comprehensive physical examination was conducted on the proband and his family members, with a focus on measuring height, weight, BMI, blood pressure and other indicators; fasting venous blood was collected to measure fasting blood glucose, 2-hour postprandial blood glucose, glycated hemoglobin (HbA1c), insulin level, C-peptide level and other diabetes-related laboratory indicators to clarify the clinical phenotype.

[0081] (3) Case analysis: All clinical data were analyzed, pedigrees were established, family inheritance patterns were identified, families with typical diabetic phenotypes and family history of diabetes were screened out, probands were identified, and clinical evidence was provided for subsequent sequencing experiments.

[0082] 2. Peripheral blood genomic DNA extraction and whole-exome sequencing

[0083] With the informed consent of the proband's parents, 2 mL of peripheral blood was drawn from the proband and his / her parents and sent to Beijing Maikino Gene Technology Co., Ltd. for DNA extraction. Whole exome sequencing was performed on the proband, and Sanger verification was performed on the parents. Subsequently, peripheral blood was drawn from the proband's family (including maternal grandmother, aunt, uncle, etc.) for DNA extraction and Sanger verification.

[0084] 3. Interpretation of variations in sequencing results

[0085] The pathogenicity of this variant was assessed according to the ACMG / AMP grading system. Sites detected by whole-exome sequencing were searched in population databases such as 1000 Genomes, gnomAD, and ExAC, as well as variant databases such as ClinVar and Varcard. The function of this variant was predicted using multiple prediction software programs, including CADD scoring, REVEL, AlphMissense, and structure-function prediction software such as PolyPhen-2, SIFT, PROVEAN, and MutationTaster.

[0086] 4. GCK protein structure prediction and analysis

[0087] The amino acid sequence (NP_000153.3) of the wild-type GCK protein was obtained from the NCBI database. Three-dimensional structural models of the wild-type and variant (p.Leu88Pro) GCK proteins were constructed using the SWISS-MODEL tool. The crystal structure of the GCK protein in the PDB database (PDB ID: 1V4S) was used as a template, and the modeling parameters were set to default values. The two protein structures were compared and analyzed using the PyMOL tool. The focus was on observing the effects of the variant site on the protein's spatial conformation, active sites (glucose binding site, ATP binding site), and functional domains to clarify the possible mechanism of action of the variant.

[0088] II. Experimental Results

[0089] 1. Clinical data of the proband:

[0090] In February 2023, a 6-year-old girl (the proband) was found to have elevated fasting blood glucose (6.15 mmol / L) during a follow-up examination after surgery for papillary thyroid carcinoma. The child was 130 cm tall, weighed 23 kg (one standard deviation above the mean), and had a BMI of 13.61 kg / m². 2The child was diagnosed with impaired fasting glucose. The child did not exhibit typical symptoms such as polydipsia, polyphagia, polyuria, or weight loss, and was not given medication. During follow-up, fasting blood glucose remained stable at 6.3–6.5 mmol / L, and symptoms did not progress. A follow-up examination in January 2024 showed: fasting blood glucose 7.51 mmol / L, glycated hemoglobin (HbA1c) 6.4%; fasting C-peptide 2.36 ng / mL (reference range 0.80–4.20 ng / mL); fasting insulin 17.9 μIU / mL (reference range 3.0–25.0 μIU / mL). The Homeostasis Model for Insulin Resistance (HOMA-IR) score was 5.97, indicating significant insulin resistance. Pancreatic autoantibody detection: Insulin autoantibody (IAA) 0.71 COI, islet cell antibody (ICA) 0.57 COI, glutamate decarboxylase antibody (GADA) 15.83 IU / mL, tyrosine phosphatase-like protein islet antigen 2 antibody (IA-2A) 9.27 IU / mL. At this time, the child's height was 131.3 cm, weight was 33.5 kg (3 standard deviations above the mean), and BMI was 19.43 kg / m². 2 This exceeds the obesity threshold for the same age and sex (18.9 kg / m²). 2 The child's waist circumference was 70 cm, hip circumference was 74 cm, and waist-to-hip ratio was 0.95. The child appeared obese but did not exhibit Cushing's facies, physical deformities, or acanthosis nigricans. Based on these results, type 1 diabetes was initially suspected.

[0091] The proband was born full-term via cesarean section, weighing 3.5 kg. The mother, aged 28, was diagnosed with gestational diabetes at 20 weeks of gestation and did not take medication. The mother, maternal grandmother, and great-aunt all had mild fasting hyperglycemia, requiring no medication. On the paternal side, the grandfather had type 2 diabetes with persistent fasting blood glucose >10.0 mmol / L and was taking metformin; the father was diagnosed with type 2 diabetes at age 39. Sequencing and pedigree verification confirmed that the proband in this family presented with asymptomatic mild fasting hyperglycemia, belonging to the MODY disease group, with a novel mutation in the GCK gene and a three-generation family history of diabetes. Testing revealed that the proband, his mother, maternal grandmother, and great-aunt all carried the same mutation at this locus. Figure 1 ).

[0092] 2. Whole exome sequencing and Sanger validation results

[0093] By comparing sequencing data, detecting variants, and annotating them, one potential pathogenic variant related to MODY was finally identified—the GCK gene missense variant c.263T>C (p.Leu88Pro). Figure 2Sequencing results showed that the base at position 263 of the GCK gene coding region was replaced by C (c.263T>C), and the corresponding amino acid sequence at position 88 was replaced by proline (Pro), i.e. p.Leu88Pro (p.L88P). This mutation is located in exon 3 of the GCK gene and belongs to the functional region mutation, which may affect the function of GCK protein.

[0094] 3. Explanation of the pathogenicity of this variant

[0095] This variant was initially assessed as a variant of unknown significance based on the ACMG / AMP guidelines (evidence combination: PM2+PP3); subsequent verification by this family pedigree ( Figure 1 Further evidence was provided (evidence combination: PP1+PP4). At the same time, through a rare search of benign variants in the GCK gene, it was found that the constraint Z value of this missense variant in the ExAC database was 4.39, which met the judgment criterion of Z value > 3.09. Therefore, PP2 evidence was added, and the variant was re-evaluated as a suspected pathogenic variant.

[0096] This variant was not found in population databases such as 1000 Genomes, gnomAD, and ExAC, nor in variant databases such as ClinVar and Varcard. The CADD predictive value is 29.5, the REVEL predictive value is harmful, and the AlphaMissense predictive value is 0.986. PolyPhen-2, SIFT, PROVEAN, and MutationTaster all consider this variant to be harmful.

[0097] 4. This mutation resulted in changes in the structure of the GCK protein.

[0098] like Figure 3 As shown, this mutation leads to a local conformational change at site 88 of the GCK protein, and the interaction between this amino acid site and surrounding residues is also altered.

[0099] Example 2: Cellular validation of the GCK gene missense variant c.263T>C (p.Leu88Pro)

[0100] I. Experimental Methods

[0101] 1. Construction of Huh7 cell line carrying GCK c.263T>C heterozygous mutant

[0102] We commissioned Guangzhou Yuanjing Biotechnology Co., Ltd. to construct a Huh7 human hepatocellular carcinoma cell line carrying the GCK c.263T>C heterozygous mutation using gene editing technology. This yielded initial mutant cell material that can be used for subsequent culture, quality control, and enzyme activity assays, ensuring accurate mutation sites and good cell viability, thus providing core experimental subjects for subsequent GCK enzyme activity detection.

[0103] 2. Preparation of complete culture medium for Huh7 cells

[0104] Prepare the complete culture medium in a sterile laminar flow hood according to the following ratio: 89% high glucose DMEM medium + 10% fetal bovine serum + 1% penicillin-streptomycin. Mix thoroughly by inverting and store at 4°C in the dark. Before use, preheat in a 37°C water bath for 30 minutes to ensure no precipitation or turbidity.

[0105] 3. Sequencing verification of GCK c.263T>C heterozygous mutation

[0106] 1) Cell collection: Take mutant cells cultured to 80%-90% confluence and wild-type control cells provided by the same company, discard the culture medium, wash once with 3 mL of pre-warmed PBS buffer, and discard the PBS; add 1 mL of 0.25% trypsin digestion solution, digest until cells detach, add 1 mL of complete culture medium to stop digestion, gently pipette to resuspend the cells, transfer to a 10 mL centrifuge tube; centrifuge at 1200 rpm for 3-5 min at room temperature, discard the supernatant, and retain the cell pellet.

[0107] 2) Cell sequencing: The collected cells were sent to BGI Genomics Co., Ltd. in Shenzhen for mutation site sequencing and STR typing.

[0108] 3) Interpretation of sequencing validation results:

[0109] a) Electrophoresis of PCR products: The PCR products of both mutant strains and wild-type controls showed a single band, approximately 308 bp in size (consistent with the expected amplified fragment length), while the blank control showed no band, indicating that the PCR amplification was uncontaminated and had good specificity.

[0110] b) Sequencing peak diagram: Wild-type Huh7 cells show a single T peak at the GCK c.263 site (homozygous wild-type); mutant Huh7 cells show a clear T / C heterozygous double peak at the GCK c.263 site, with no obvious overlapping peaks and a clean background, indicating that the cells carry the GCK c.263T>C heterozygous mutation, which is stable and has no loss or abnormality.

[0111] 4. Enzyme activity was measured using the GCK enzyme activity assay kit.

[0112] 1) Cell samples: Quality-controlled GCK c.263T>C heterozygous mutant Huh7 cells and wild-type Huh7 cells (3 replicates per group to ensure experimental reproducibility);

[0113] 2) Core reagent: GCK enzyme activity assay kit (abcam, ab273303).

[0114] 3) Cell Collection and Pretreatment: Collect mutant cells cultured to 80%-90% confluence and wild-type control cells (3 replicates per group), discard the culture medium, wash the cells twice with pre-cooled PBS buffer, discarding the PBS after each wash and aspirating as much residual liquid as possible; add 1 mL of 0.25% trypsin digestion solution, digest at 37℃ for 1-2 min until the cells shrink, become rounded, and detach; add 1 mL of complete culture medium to stop digestion, and gently pipette to disperse the cells into a single-cell suspension; take a small amount of the cell suspension, count the cells using a cell counter, and adjust the cell concentration to the recommended concentration in the kit (1×10⁻⁶). 6 (cells / mL) Ensure consistent cell concentration in each group and place in an ice bath for later use; add the cell lysis aid solution provided with the kit to the adjusted cell suspension, gently pipette to mix, incubate on ice for 10 minutes, and set aside for later use.

[0115] 4) GCK enzyme activity assay: Prepare the required reagents according to the kit instructions, add pre-chilled 2.5 mM DTT buffer to the cells for homogenization; after incubating on ice for 10 min, centrifuge at 12000×g for 10 min at 4℃ and collect the supernatant; dilute the supernatant 10–20 times with DTT-free GCK buffer. Prepare and plot the standard curve according to the proportions in Table 1, add samples according to the dilution ratio (2–10 µL sample / positive control diluted to 50 µL), and perform the assay: add 50 µL of the corresponding mixture to each well; room temperature, kinetic mode, 20–30 min, 535 / 587 nm; record the linear interval t1 and t2 readings.

[0116] Table 1. Standard curve sample addition ratio (final volume per well: 50 µL)

[0117]

[0118] 5) GCK activity calculation method:

[0119] A) Subtract 0 from the standard sample reading and plot the slope of the curve.

[0120] B) Sample ΔRFU = Sample (RFU2 - RFU1) - Background (RFU2 - RFU1);

[0121] C) Substitute the values ​​into the curve to obtain the NADPH amount B;

[0122] D) GCK activity = B ÷ (time × loading volume × protein concentration) × dilution factor;

[0123] E) Unit: pmol / min / µg.

[0124] 6) Result Interpretation:

[0125] A) Cell pretreatment qualified: The pretreated cell suspension is uniform, without a large number of cell clumps or obvious cell breakage, and the cell viability is good (trypan blue staining viability ≥90%), indicating that it can be used for enzyme activity determination;

[0126] B) Standard curve is qualified: the standard curve R²≥0.99, the OD value of the standard wells increases linearly with the increase of the standard concentration, and there is no abnormal deviation, indicating that the enzyme reaction system is stable and the test results are reliable;

[0127] II. Experimental Results

[0128] PCR and sequencing results showed that the constructed mutant Huh7 cells carried a GCK c.263T>C heterozygous mutation, which was stable without loss or abnormality. The GCK enzyme activity of the mutant Huh7 cells was significantly lower than that of wild-type cells, indicating that the GCK c.263T>C heterozygous mutation can reduce GCK enzyme activity and is a pathogenic mutation that induces MODY. Treatment with a low concentration of 0.1 μM of the glucokinase activator Dorzagliatin (Hual Medicines, catalog number: HUA 2013011-090-K, purity: 97.9%) only increased the maximum reaction rate Vmax but did not improve glucose sensitivity, and the half-maximum saturation concentration Khalf / S0.5 further increased. Increasing the drug concentration to 0.5 μM reduced Khalf / S0.5 by 36% compared to the untreated mutant, while Vmax significantly increased. High-dose Dorzagliatin can partially repair the substrate response defect caused by p.L88P and enhance catalytic activity. Figure 4 (Table 2).

[0129] Table 2. Glucose kinetics of p.L88P mutant.

[0130]

[0131] Temperature gradient experiments showed that both wild-type and p.L88P proteins maintained high catalytic activity in the 30-35℃ range; at 40℃, the wild-type showed only slight inactivation (activity decreased by about 14%), while the p.L88P mutant showed a significant 70% decrease in activity; at temperatures above 45℃, the activity of both types of proteins continued to decrease, and the residual activity of the mutant was consistently significantly lower than that of the wild-type. This suggests that the p.L88P mutation disrupts the spatial conformation of the GCK protein, significantly impairing its thermal stability and making it more sensitive to temperature stimuli. Figure 5 ).

[0132] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0133] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A GCK mutant protein, characterized in that, The GCK mutant protein, compared to the protein encoded by the wild-type GCK gene, exhibits the p.Leu88Pro mutation.

2. The GCK mutant protein according to claim 1, characterized in that, The accession number for the wild-type GCK protein is NP_000153.

3.

3. A GCK gene mutant encoding the GCK mutant protein of claim 1 or 2, characterized in that, The GCK gene mutant, compared with the wild-type GCK gene, has a c.263T>C mutation; Preferably, the accession number of the wild-type GCK gene is NM_000162.

5.

4. A recombinant vector, characterized in that, The recombinant vector contains the GCK gene mutant as described in claim 3.

5. A recombinant cell, characterized in that, The recombinant cells contain the GCK mutant protein of claim 1 or 2, the GCK gene mutant of claim 3, or the recombinant vector of claim 4.

6. The use of the reagent for detecting the GCK mutant protein of claim 1 or 2 or the GCK gene mutant of claim 3 in the preparation of any one or more of the following products: 1) Products for diagnosing adult-onset diabetes in adolescents; 2) Products for diagnosing preconception and / or prenatal genetic diseases; Preferably, the reagent comprises an antibody, probe, primer, or guide RNA specifically targeting the GCK mutant protein of claim 1 or 2 or the GCK gene mutant of claim 3; Preferably, the product includes at least one of a reagent kit, a chip, a test strip, and a high-throughput sequencing platform.

7. A product for diagnosing adolescent-onset adult-onset diabetes or preconception and / or prenatal genetic diseases, characterized in that, The product includes reagents for detecting the GCK mutant protein of claim 1 or 2 or the GCK gene mutant of claim 3; Preferably, the adolescent-onset adult-onset diabetes includes adolescent-onset adult-onset type 2 diabetes; Preferably, the reagent comprises an antibody, probe, primer, or guide RNA specifically targeting the GCK mutant protein of claim 1 or 2 or the GCK gene mutant of claim 3; Preferably, the product includes at least one of a reagent kit, a chip, a test strip, and a high-throughput sequencing platform.

8. A biological model of adolescent-onset adult-onset diabetes, characterized in that, The biological model carries the GCK mutant protein as described in claim 1 or 2 or the GCK gene mutant as described in claim 3; Preferably, the biological model includes a cell model or an animal model.

9. Any of the following methods: 1) A method for constructing a biological model of adolescent-onset adult-onset diabetes mellitus, the method comprising introducing the GCK mutant protein of claim 1 or 2, the GCK gene mutant of claim 3, or the recombinant vector of claim 4 into an organism; 2) A method for screening drugs for treating adolescent-onset adult-onset diabetes, the method comprising applying drug candidates to the biological model of claim 8 to screen for drugs that have therapeutic effects on adolescent-onset adult-onset diabetes. Preferably, the organism includes cells or non-human animals; Preferably, the biological model includes a cell model or an animal model.

10. Any of the following applications: 1) The application of the GCK mutant protein of claim 1 or 2, the GCK gene mutant of claim 3, or the recombinant vector of claim 4 in the preparation of a biological model of adolescent-onset adult-onset diabetes mellitus; 2) The use of the biological model described in claim 8 in screening drugs for the treatment of adolescent-onset adult-onset diabetes; Preferably, the biological model includes a cell model or an animal model.

Images