Primer, gene mutant, mutant protein and reagent for detecting McCone-Alblight syndrome and application of primer, gene mutant, mutant protein and reagent

Abstract

The invention provides a virulence gene capable of causing McCone-Alblight syndrome as well as detection and application of the virulence gene capable of causing McCone-Alblight syndrome. Compared with the wild type GNAS gene, the virulence gene has the CAT deletion from the first base to the second base of the first exon of the wild type GNAS gene. The mutation of the pathogenic gene disclosed by the invention can be used as a biomarker for diagnosing the McCone-Alblight syndrome. According to the present invention, whether the subject carries the mutation can be detected so as to screen or diagnose the genetic diagnosis of the McCone-Alblight syndrome, such that the treatment can be guided. The detection kit provided by the invention can be used for rapidly and effectively predicting or diagnosing the McCin-Alblight syndrome.

Description

Technical Field

[0001] The present invention relates to the field of detection reagents, and in particular to a primer, a gene mutant, a mutant protein, a reagent and an application thereof for detecting McCune-Albright syndrome. Background Art

[0002] McCune-Albright syndrome (MAS; MIM 133780) is a rare disease. MAS was first discovered and reported by American doctors McCune and Albright in 1937. The incidence rate is between 1 / 1,000,000 and 1 / 100,000. The main clinical manifestations are dysplastic bone fiber structure, café-au-lait spots, and hypersecretion of hormones. The main symptoms of dysplastic bone fiber structure are bone pain and bone deformity. Long bones are very prone to fracture under the gravity of the limbs. The incidence rate of males and females is roughly the same. The main cause of MAS is the mutation or abnormal development of the GNAS gene during the embryonic process, which leads to the accumulation of cAMP in cells, activates G protein-cAMP dependent receptors, promotes the secretion of hormones in target organs, causes hyperfunction of endocrine glands, and then triggers a series of disease reactions.

[0003] The McCune-Albright syndrome causative gene GNAS (MIM 139320) is located on chromosome 20q13.32. The gene is 19.9 kb in length, contains 13 exons and 12 introns, and encodes a stimulatory guanine nucleotide binding protein (G protein) α subunit (Gsα) composed of 394 amino acid residues, which transduces signals from G protein-coupled receptors (GPCRs) to adenylate cyclase (AC). Mutations in the GNAS gene lead to the loss of Gsα's inherent GTPase activity, resulting in constitutive receptor activation and incorrect cAMP production. Gsα includes two domains, the first of which is the guanosine triphosphatase (GTPase) domain, which is formed after folding of 39 to 394 amino acid residues. The GTPase domain serves as the guanosine binding site for receptors and effectors. GTPase has four GTP / GDP binding sites and two magnesium ion binding sites. Two of the four GTP / GDP binding sites are highly conserved regions, Arg201 and Gln227. Arg201 is the most common pathogenic mutation site in MAS. More than 95% of missense mutations in MAS cases occur at positions R201H and R201C; rare mutations occur at Q227 and other positions. The symptoms of MAS depend largely on the tissue range and location of the mutation.

[0004] Gene mutation is an important genetic basis for the development of McCune-Albright syndrome, and genetic diagnosis is an important criterion for the diagnosis of McCune-Albright syndrome. Clinically, it is necessary to establish corresponding detection technologies for different mutations and use them to clarify the cause and diagnose the disease, develop detection methods, determine mutation sites, and assist in the screening and diagnosis of McCune-Albright syndrome gene mutations, as well as for drug screening, efficacy evaluation and targeted treatment, which is of great significance. Summary of the invention

[0005] The main purpose of the present invention is to provide a primer, a gene mutant, a mutant protein, a reagent and an application for detecting McCune-Albright syndrome, so as to solve the technical problems of screening and diagnosis of McCune-Albright syndrome.

[0006] To achieve the above object, the present invention provides a GNAS mutant protein, wherein compared with the wild-type GNAS protein, the GNAS mutant protein lacks amino acids 2 to 60.

[0007] The present invention provides a GNAS gene mutant. Compared with a wild-type GNAS gene, the GNAS gene mutant lacks CAT from the -1st base to the 2nd base in exon 1 of the wild-type GNAS gene.

[0008] The present invention provides a use of the GNAS gene mutant as described above as a detection target in the preparation of a reagent or a kit, wherein the reagent includes a reagent for detecting McCune-Albright syndrome;

[0009] The kit includes one or more of a kit for preventing McCune-Albright syndrome, a kit for diagnosing McCune-Albright syndrome, a kit for screening genetic diseases before pregnancy, a kit for screening genetic diseases before birth, a kit for diagnosing genetic diseases before pregnancy, a kit for diagnosing genetic diseases before birth, and a kit for assisting in treating McCune-Albright syndrome.

[0010] The present invention provides an amplification primer for detecting McCune-Albright syndrome, wherein the amplification primer comprises an upstream primer GNAS-1F and a downstream primer GNAS-1R; the upstream primer GNAS-1F comprises a nucleotide sequence as shown in SEQ ID NO.1, and the downstream primer GNAS-1R comprises a nucleotide sequence as shown in SEQ ID NO.2.

[0011] The present invention provides a sequencing primer for detecting McCune-Albright syndrome. The sequencing primer comprises an upstream primer GNAS-Seq1F and a downstream primer GNAS-Seq1R; the upstream primer GNAS-Seq1F comprises a nucleotide sequence as shown in SEQ ID NO.3; the downstream primer GNAS-Seq1R comprises a nucleotide sequence as shown in SEQ ID NO.4.

[0012] The present invention provides a primer combination for detecting McCune-Albright syndrome, comprising the amplification primers and / or the sequencing primers as described above.

[0013] The present invention provides an application of the primer combination as described above in preparing a reagent for detecting McCune-Albright syndrome.

[0014] Furthermore, the detection target of McCune-Albright syndrome includes a GNAS gene mutant, and compared with the wild-type GNAS gene, the GNAS gene mutant has a CAT deletion from the -1 base to the 2 base in exon 1 of the wild-type GNAS gene.

[0015] The present invention provides a reagent for detecting McCune-Albright syndrome, wherein the reagent comprises the primer combination as described above.

[0016] The present invention provides an application of the above-mentioned reagent in a kit and its preparation, wherein the kit comprises one or more of a kit for preventing McCune-Albright syndrome, a kit for diagnosing McCune-Albright syndrome, a kit for screening pre-pregnancy genetic diseases, a kit for screening prenatal genetic diseases, a kit for diagnosing pre-pregnancy genetic diseases, a kit for diagnosing prenatal genetic diseases, and a kit for assisting the treatment of McCune-Albright syndrome.

[0017] This application proposes a pathogenic gene that causes McCune-Albright syndrome, which can effectively distinguish McCune-Albright syndrome patients from normal people. Therefore, the pathogenic gene mutation of the present invention can be used as a biomarker for diagnosing McCune-Albright syndrome. The present invention can be used for genetic diagnosis, screening and eugenics guidance of McCune-Albright syndrome by detecting whether the subject carries the above mutation. The detection kit provided by the present invention can be used to quickly and effectively predict or diagnose McCune-Albright syndrome. The present invention lays a new foundation and approach for the study of the pathogenesis of McCune-Albright syndrome, provides a new theoretical basis for the treatment of McCune-Albright syndrome patients, and provides possible drug targets for the treatment of McCune-Albright syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0019] Figure 1 This is the genetic map of McCune-Albright syndrome family No. 1; □ represents a normal male individual, ○ represents a normal female individual, ■ represents a male patient, and ↗ represents a proband;

[0020] Figure 2 This is the result of using Sanger sequencing to detect the genotype of the GNAS: NM_000516.7: exon1: c.-1_2delCAT: p.Gly2_Met60del site in Family 1, where layer B: heterozygous mutants in Family 1; layers A and C: wild-type genotypes in Family 1 (the arrows in the sequencing diagram indicate the location of the mutation);

[0021] Figure 3 This is the genetic map of McCune-Albright syndrome family No. 2; □ represents a normal male individual, ● represents a female patient, ○ represents a normal female individual, and ↗ represents a proband;

[0022] Figure 4This is the result of using the kit to detect the genotype of the proband of Family No. 2 at GNAS: NM_000516.7: exon1: c.-1_2delCAT site; among them, layer B: heterozygous mutants in Family No. 2; layers A and C: the genotype in Family No. 2 is wild type (the arrow in the sequencing image indicates the location of the mutation).

[0023] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with the implementation methods and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0024] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] In the present invention, the term "autosomal dominant inheritance" means that as long as one parent has a disease-causing gene on the autosome and passes it on to the child, the disease may occur regardless of whether the other gene of the child is normal, regardless of gender. Those with a family history of genetic diseases need to undergo prenatal examinations to prevent genetic diseases from affecting the next generation.

[0026] In the present invention, the term "heterozygous mutation" means that the mutation exists in only one gene in a pair of alleles.

[0027] In the present invention, the term "homozygous mutation" means that both alleles have the same mutation, that is, a biallelic mutation, and each chromosome has a mutation.

[0028] In the present invention, the term "deletion mutation" refers to the situation where a codon encoding a certain amino acid is changed to a codon encoding another amino acid after the base is deleted, thereby changing the type and sequence of amino acids in the polypeptide chain.

[0029] In the present invention, the term "frameshift mutation" refers to the deletion or insertion of a base at a certain site in a DNA molecule, which causes a change in the reading frame, resulting in a series of downstream code changes, so that the gene originally encoding a certain peptide chain becomes another completely different peptide chain sequence.

[0030] The term "diagnosis" as used herein includes prediction of disease risk, diagnosis of whether a disease has occurred, and assessment of disease prognosis.

[0031] The term "prenatal diagnosis" in this article refers to the clear diagnosis of high-risk fetuses based on genetic counseling, mainly through genetic testing and imaging examinations, and the purpose of fetal selection is achieved through selective abortion of sick fetuses, thereby reducing the birth defect rate and improving the quality of eugenics and population quality.

[0032] Furthermore, the technical solutions between the various embodiments of the present invention may be combined with each other, but this must be based on the fact that they can be implemented by ordinary technicians in the field. When the combination of technical solutions is mutually contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0033] The present invention provides a GNAS mutant protein. Compared with a wild-type GNAS protein, the GNAS mutant protein lacks amino acids 2 to 60.

[0034] The present invention provides a GNAS gene mutant. Compared with a wild-type GNAS gene, the GNAS gene mutant lacks CAT from the -1st base to the 2nd base in exon 1 of the wild-type GNAS gene.

[0035] The GNAS gene mutant includes c.-1_2delCAT, and the accession number of the GNAS gene of the present invention is NM_000516.7.

[0036] In the present invention, the c.-1_2delCAT indicates that the bases CAT at positions -1 to 2 of exon 1 of the GNAS gene are deleted, resulting in a nucleotide fragment shown in SEQ ID NO.5 (5'-GCCGC***GGGCT-3') (the asterisk indicates the location where the deletion mutation occurs); the c.-1_2delCAT causes the encoded GNAS protein to be missing from amino acids 2 to 60 compared with the protein encoded by the wild-type GNAS gene, i.e., a deletion mutation, recorded as GNAS: NM_000516.7: exon1: c.-1_2delCAT: p.Gly2_Met60del, wherein the sequence of the core amino acids generated by the mutation is shown in SEQ ID NO.6 (M R ILHVNGFN) (the underlined letter is the first amino acid after the deletion mutation part), which affects the normal function of GNAS, causes McCune-Albright syndrome, and is pathogenic.

[0037] The GNAS gene mutant provided by the present invention can distinguish McCune-Albright syndrome patients from normal people, is a biomarker for diagnosing McCune-Albright syndrome, is helpful for the screening and diagnosis of McCune-Albright syndrome gene mutations, and provides new technical support for its drug screening, efficacy evaluation and targeted treatment.

[0038] According to the outstanding role of the above-mentioned GNAS gene mutant in McCune-Albright syndrome of the present invention, the following contents all belong to the protection scope of the present invention: the GNAS gene mutant is used as a detection target to prepare a reagent for detecting McCune-Albright syndrome; the GNAS gene mutant is used as a detection target to prepare a kit for diagnosing McCune-Albright syndrome; the GNAS gene mutant is used as a detection target to prepare a kit for preventing McCune-Albright syndrome; the GNAS gene mutant is used as a detection target to prepare a kit for auxiliary treatment of McCune-Albright syndrome; the GNAS gene mutant is used as a detection target to prepare a pre-pregnancy and / or prenatal genetic disease screening kit; the GNAS gene mutant is used as a detection target to prepare a pre-pregnancy and / or prenatal genetic disease diagnosis kit.

[0039] The present invention provides a use of the GNAS gene mutant as described above as a detection target in the preparation of a reagent or a kit, wherein the reagent includes a reagent for detecting McCune-Albright syndrome;

[0040] The kit includes one or more of a kit for preventing McCune-Albright syndrome, a kit for diagnosing McCune-Albright syndrome, a kit for screening genetic diseases before pregnancy, a kit for screening genetic diseases before birth, a kit for diagnosing genetic diseases before pregnancy, a kit for diagnosing genetic diseases before birth, and a kit for assisting in treating McCune-Albright syndrome.

[0041] The present invention provides an amplification primer for detecting McCune-Albright syndrome, wherein the amplification primer comprises an upstream primer GNAS-1F and a downstream primer GNAS-1R; the upstream primer GNAS-1F comprises a nucleotide sequence as shown in SEQ ID NO.1, and the downstream primer GNAS-1R comprises a nucleotide sequence as shown in SEQ ID NO.2.

[0042] The nucleotide sequences of SEQ ID NO.1-2 of the present invention are as follows:

[0043] SEQ ID NO.1: 5'-CCGAGGCAATAAGAGCG-3';

[0044] SEQ ID NO.2: 5'-ACCCCTGGCGTCGTGTA-3';

[0045] The present invention provides a sequencing primer for detecting McCune-Albright syndrome. The sequencing primer comprises an upstream primer GNAS-Seq1F and a downstream primer GNAS-Seq1R; the upstream primer GNAS-Seq1F comprises a nucleotide sequence as shown in SEQ ID NO.3; the downstream primer GNAS-Seq1R comprises a nucleotide sequence as shown in SEQ ID NO.4.

[0046] The nucleotide sequences of SEQ ID NO.3-4 of the present invention are as follows:

[0047] SEQ ID NO.3: 5'-AGCTCCTGCTCTGGTCC-3';

[0048] SEQ ID NO.4: 5'-CGGTAGACCTGCTTGTCC-3';

[0049] The sequencing primers of the present invention sequence the amplification products of the amplification primers to distinguish between GNAS gene mutants and wild-type GNAS genes; the present invention utilizes the sequencing primer 1 to sequence the fragments amplified by the amplification primers, which can quickly and accurately diagnose McCune-Albright syndrome.

[0050] The present invention provides a primer combination for detecting McCune-Albright syndrome, comprising the amplification primers and / or the sequencing primers as described above.

[0051] The primer combination of the present invention can detect whether there is a c.-1_2delCAT mutation site on the GNAS gene, and specifically amplify the GNAS gene mutant. Specifically, the amplification primer 1 specifically amplifies the GNAS gene mutant fragment or the wild-type GNAS gene fragment containing the c.-1_2delCAT mutation site, and the GNAS gene mutant and the wild-type GNAS gene can be distinguished after sequencing by the sequencing primer; the GNAS gene mutant and the wild-type GNAS gene can be distinguished after sequencing by the sequencing primer.

[0052] The present invention provides an application of the primer combination as described above in preparing a reagent for detecting McCune-Albright syndrome.

[0053] Exemplarily, the reagents further include one or more of dNTPs, PCR buffer, magnesium ions and Tap polymerase.

[0054] Furthermore, the detection target of McCune-Albright syndrome includes a GNAS gene mutant, and compared with the wild-type GNAS gene, the GNAS gene mutant has a CAT deletion from the -1 base to the 2 base in exon 1 of the wild-type GNAS gene.

[0055] The present invention provides a reagent for detecting McCune-Albright syndrome, wherein the reagent comprises the primer combination as described above.

[0056] The present invention utilizes exon sequencing to screen for pathogenic gene mutations that are highly associated with McCune-Albright syndrome. In order to avoid false positive results, verification is performed through Sanger sequencing. It is first discovered that when c.-1_2delCAT is present in the GNAS gene, it is associated with McCune-Albright syndrome. Therefore, by detecting whether c.-1_2delCAT is present in the GNAS gene, McCune-Albright syndrome can be detected.

[0057] The present invention provides an application of the above-mentioned reagent in a kit and its preparation, wherein the kit comprises one or more of a kit for preventing McCune-Albright syndrome, a kit for diagnosing McCune-Albright syndrome, a kit for screening pre-pregnancy genetic diseases, a kit for screening prenatal genetic diseases, a kit for diagnosing pre-pregnancy genetic diseases, a kit for diagnosing prenatal genetic diseases, and a kit for assisting the treatment of McCune-Albright syndrome.

[0058] The present invention also provides a kit for diagnosing McCune-Albright syndrome, comprising the reagents described in the above technical solution. The kit of the present invention diagnoses whether an individual suffers from McCune-Albright syndrome by the genotype of the GNAS gene mutant in the test sample of the male individual and / or the female individual; the test sample preferably includes blood and lesion site tissue. The genotype diagnosis standard of whether an individual suffers from McCune-Albright syndrome is specifically:

[0059] When the genotype of the individual c.-1_2delCAT locus is "c.-1_2delCAT heterozygous mutation", the individual is a patient;

[0060] When the genotype of the individual c.-1_2delCAT locus is "c.-1_2delCAT homozygous mutation", the individual is a patient;

[0061] When the genotype of the individual c.-1_2delCAT locus is "wild type", the individual is normal;

[0062] In the present invention, the term "heterozygous mutation" means that the mutation exists in only one gene in a pair of alleles; the term "homozygous mutation" means that the mutation exists in both genes in a pair of alleles, that is, biallelic mutation, and each chromosome is mutated.

[0063] To further illustrate the present invention, the primer combination, reagents and kit, GNAS gene mutant and application for detecting McCune-Albright syndrome provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0064] The experimental methods in the following examples where specific conditions are not specified are generally performed according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 2014), or according to the conditions recommended by the manufacturer.

[0065] Example 1

[0066] 1. Diagnostic criteria:

[0067] Please refer to the 2010 edition of "Single Gene Inherited Disorders in Humans" and the fifth edition of "WHO Classification of Bone and Soft Tissue Tumors";

[0068] MAS is very rare. Due to endocrine dysfunction, patients experience a variety of symptoms.

[0069] The disease characteristics of the manifestations are also different. The recommended clinical diagnostic criteria for McCune-Albrigh syndrome are that it should have two or more of the following characteristics:

[0070] 1) Characteristic cafe au lait spots: located in the midline of the skin, with jagged edges

[0071] 2) Single or multiple bone fibrous dysplasia

[0072] 3) Having any of the following endocrine diseases

[0073] Gonadotropin-independent precocious puberty (peripheral precocious puberty)

[0074] Testicular lesions with or without precocious puberty

[0075] Hyperthyroidism

[0076] Elevated growth hormone / mixed elevation of growth hormone and prolactin

[0077] Renal phosphate wasting, with or without hypophosphatemia

[0078] Neonatal Cushing's syndrome

[0079] Note: Monofibrous dysplasia should be diagnosed by testing for GNAS mutation gene.

[0080] 2. Detection object

[0081] The McCune-Albright syndrome family (referred to as Family 1) was used as the test subject for testing. The clinical information of some members of Family 1 is shown in Table 1, and the family pedigree is shown in Figure 1 .

[0082] Table 1 Clinical information of the members of McCune-Albright syndrome family 1

[0083]

[0084]

[0085] Note: Ⅰ and Ⅱ represent the first and second generations respectively. Peripheral blood DNA of Ⅰ:1 and Ⅰ:2 and DNA of surgical tissue samples of family members No. 1 were used for sequencing.

[0086] Example 2

[0087] Exome sequencing

[0088] 1. Instruments and equipment are shown in Table 2.

[0089] Table 2 Instruments and equipment

[0090]

[0091] 2. Reagents and consumables

[0092] Human whole exome sequencing kit (Agilent), DNA 1000 kit (Agilent), 96-well plate (Axygen), different types of pipette tips (Axygen), 200 μL centrifuge tubes (Eppendorf), 1.5 mL centrifuge tubes (Eppendorf), capillary electrophoresis buffer (Thermo), sequencing standards (Thermo), anhydrous ethanol (Thermo), BigDyeTerminator V3.1 (Thermo), peripheral blood gDNA extraction kit (TIANGEN), agarose (TIANGEN) and EB stain (Amresco).

[0093] 3. Reagent Formulation

[0094] 1) Prepare 5×TBE electrophoresis buffer stock solution according to Table 3.

[0095] Table 3 5×TBE electrophoresis buffer formula

[0096]

[0097] 2) 0.5×TBE electrophoresis buffer working solution: dilute the 5×TBE electrophoresis buffer stock solution in Table 3 10 times with ddH2O.

[0098] 3) Prepare 10× red blood cell lysis buffer according to Table 4.

[0099] Table 4 10× red blood cell lysis buffer formula

[0100]

[0101]

[0102] 4) The formula of 1× cell nuclear lysis buffer was prepared according to Table 5.

[0103] Table 5 1× Cell Nuclear Lysis Solution Formula

[0104] Reagents 2M Tris-HCl, pH 8.2 4M NaCl 2mM EDTA Volume / Weight 0.5mL 10mL 0.4mL

[0105] 4. Experimental Procedure

[0106] After signing the informed consent form, 3-5 mL of peripheral blood and 3-8 g of surgical tissue from members Ⅰ:1 and Ⅰ:2 of family No. 1 and Ⅱ:1 were collected as research samples.

[0107] 4.1 Sample DNA extraction

[0108] 1) Place 3-5 mL of the sample into a 15 mL centrifuge tube, add 2-3 times the volume of 1× red blood cell lysis buffer, mix well, and place on ice for 30 minutes until the solution becomes transparent; if the sample is amniotic fluid, proceed directly to step 2).

[0109] 2) Centrifuge at 3000 rpm for 10 minutes at 4°C and carefully remove the supernatant. Add 1 mL of 1× nuclear lysis buffer to the precipitate and mix well. Then add 2 mL of 1× nuclear lysis buffer and 150 μL of 20% SDS and shake well until it becomes viscous and transparent. Add 10 μL of 20 mg / mL proteinase K and shake well. Digest at 37°C for more than 6 hours or overnight.

[0110] 3) Add an equal volume of saturated phenol, shake gently to mix, and centrifuge at 3000 rpm for 10 minutes at room temperature.

[0111] 4) Carefully transfer the supernatant to another centrifuge tube, add an equal volume of phenol and chloroform mixture (the volume ratio of phenol to chloroform is 1:1), mix well, and centrifuge at 3000 rpm for 10 minutes at room temperature.

[0112] 5) Carefully transfer the supernatant. If the supernatant is not clear and transparent, extract again with an equal volume of chloroform.

[0113] 6) Transfer the supernatant to another centrifuge tube, add two times the volume of anhydrous ethanol, shake well, and white flocculent DNA will be seen. Use a flame-sterilized glass hook to hook out the DNA, wash twice with 70% ethanol, dry at room temperature for 5 minutes, and then dissolve the DNA in 200 μL 1×TE and dissolve overnight in a drum. Measure the OD value under UV.

[0114] 7) DNA dissolved in TE can be stored at 4°C for one year. If long-term storage is required, add 2 times the volume of anhydrous ethanol and store at -70°C.

[0115] 4.2 Exome sequencing

[0116] 1) Take 2 μg DNA, mechanically shear it to a fragment size of about 200 bp, and cut the gel to recover 150-250 bp fragments;

[0117] 2) DNA fragments are end-repaired and 3' end A is added;

[0118] 3) Connect the sequencing adapter, purify the connection product, perform PCR amplification, and purify the amplified product;

[0119] 4) The probe of the Agilent kit is added to the purified amplified product for hybridization capture. The hybridized product is eluted and recovered for PCR amplification. The final product is then recovered and a small sample is taken for agarose gel electrophoresis for quality control analysis.

[0120] 5) NextSeq500 sequencer sequencing and data analysis.

[0121] 4.3 Results

[0122] Finally, the pathogenic gene mutation GNAS was obtained: NM_000516.7: exon1: c.-1_2delCAT: p.Gly2_Met60del; this mutation is the deletion of bases CAT from -1 to 2 located in exon 1, resulting in the deletion of amino acids from positions 2 to 60, that is, a deletion mutation.

[0123] In the patient individuals of Family No. 1, the genotype of the GNAS:NM_000516.7:exon1:c.-1_2delCAT:p.Gly2_Met60del site is "c.-1_2delCAT heterozygous mutation", while in the normal individuals of Family No. 1, the genotype of this site is "wild type".

[0124] Example 3

[0125] Sanger sequencing validation

[0126] The exome sequencing results of the two families were further verified by Sanger sequencing for the GNAS: NM_000516.7: exon1: c.-1_2delCAT: p.Gly2_Met60del site. The genotype of the GNAS: NM_000516.7: exon1: c.-1_2delCAT: p.Gly2_Met60del site was tested for three people (the proband, the proband's father, and the proband's mother) in family No. 1 in Example 1 and 100 normal people outside the family.

[0127] The specific steps are as follows:

[0128] 1. DNA Extraction

[0129] The genomic DNA was extracted according to the method of Example 2.

[0130] 2. Design, verification and optimization of candidate primers

[0131] 2.1 Candidate primer design refers to the human genome sequence database hg19 / build36.3 (https: / / www.ncbi.nlm.nih.gov / genome, or http: / / genome.ucsc.edu / cgi-bin / hgGateway?redirect=manual&source=genome.ucsc.edu).

[0132] 2.2 Design 19 pairs of candidate primers for the mutation site c.-1_2delCAT (see Table 6), and use PCR experiments to verify and evaluate the advantages and disadvantages of each pair of candidate primers

[0133] Table 6 Basic information and verification experiment results of candidate primers for c.-1_2delCAT site

[0134]

[0135]

[0136]

[0137] Note: Normal PCR amplification results will only have one specific band after electrophoresis. If primer dimer bands or non-specific product bands appear, they are the result of abnormal primer reactions. Avoid using such primers as much as possible as target primers.

[0138] 2.3 Candidate Primer PCR Verification Reaction

[0139] Carry out PCR according to the reaction system in Table 7 and keep the reaction system on ice; set up 8 reaction test tubes for each pair of primers (sequence numbers 1 to 8 in Table 7).

[0140] Table 7 Primer detection PCR reaction system

[0141]

[0142]

[0143] Reaction conditions: Place the above test reaction tube into the PCR instrument and perform the following reaction program:

[0144] Step 1: pre-denaturation at 95°C for 5 min;

[0145] Step 2: 30 cycles (95°C denaturation for 30 sec→Tm annealing for 30 sec→72°C extension for 60 sec); (PCR amplification parameters are set according to the Tm values ​​of each primer in Table 6).

[0146] Step 3: 72°C extension for 7 min;

[0147] Step 4: 4°C until sampling.

[0148] 2.4 The PCR results of candidate primers were tested by agarose gel electrophoresis to evaluate the effectiveness and specificity of the primer reaction:

[0149] 1) Seal both ends of the cleaned and dried gel sampler with tape, place it on a flat surface, and place a comb about 1 cm from one end of the sampler.

[0150] 2) Weigh 2 g of agar powder into a conical flask, add 100 mL of 0.5×TBE electrophoresis buffer, shake well, and heat in a microwave oven or electric stove (with asbestos mesh). After boiling, take out and shake well, and heat again until the gel is completely melted, then take out and cool at room temperature.

[0151] 3) When the gel cools to about 50°C, pour it into the sealed gel sampler to make the thickness about 5mm.

[0152] 4) After the gel solidifies, remove the tape and place the gel and sampler into the electrophoresis tank.

[0153] 5) Add electrophoresis buffer so that the liquid level is 1 to 2 mm above the gel surface, and pull out the comb upwards; use a micropipette to mix the sample and DNA size standard with the loading solution respectively, and add them to each well. Since the specific gravity of sucrose in the loading solution is relatively large, the DNA sinks to the bottom of the well.

[0154] 6) Cover the electrophoresis tank, turn on the power, adjust to the appropriate voltage, and start electrophoresis. According to the indication of bromophenol blue in the sample loading solution, determine the approximate position of the sample and decide whether to terminate the electrophoresis.

[0155] 7) Turn off the power, take out the gel, and place it in a 0.5 g / mL EB aqueous solution for staining for 10 to 15 minutes.

[0156] 8) Place the gel under a transmission UV irradiator to observe the results at a wavelength of 254 nm, and take photos with a camera equipped with a red filter or use a gel scanning system to record the electrophoresis results.

[0157] 2.5 Result evaluation:

[0158] 1) If only one bright target band appears in tube 7 and no other bands are detected, it is judged that the primer pair and reaction system have good effectiveness and strong specificity;

[0159] 2) If no target band appears in tube 7, the primer pair and reaction system are considered invalid;

[0160] 3) If a primer dimer band other than the target band appears in tube 7, and also appears in some tubes 2, 3, 4, 5, and 6, it is judged that the effectiveness of the primer pair and reaction system is poor;

[0161] 4) If a non-specific band other than the target band appears in tube 7, and non-specific bands also appear in some tubes 5 and 6, it is judged that the primer pair and reaction system have poor specificity;

[0162] 5) If primer dimers and non-specific bands outside the target band appear in tube 7, and primer dimers and non-specific bands also appear in some tubes 2, 3, 4, 5, and 6, it is judged that the effectiveness and specificity of this pair of primers and reaction system are poor.

[0163] 2.6 According to the statistical results after the verification test in Table 7, SEQ ID NO.1 and SEQ ID NO.2 in Table 6 were selected as amplification primers for the GNAS:NM_000516.7:exon1:c.-1_2delCAT:p.Gly2_Met60del site.

[0164] GNAS-1F: 5'-CCGAGGCAATAAGAGCG-3'(SEQ ID NO.1)

[0165] GNAS-1R: 5'-ACCCCTGGCGTCGTGTA-3'(SEQ ID NO.2)

[0166] 3. PCR amplification of mutation sites in family members No. 1 and 100 non-family members

[0167] Carry out PCR according to the reaction system in Table 8 and keep the reaction system on ice.

[0168] Table 8 Mutation point PCR reaction system

[0169]

[0170] Reaction conditions: Place the reaction system into a PCR instrument and perform the following reaction procedure for the c.-1_2delCAT site:

[0171] Step 1: pre-denaturation at 95°C for 5 min;

[0172] Step 2: 30 cycles (95°C denaturation for 30 sec → 55°C annealing for 30 sec → 72°C extension for 60 sec);

[0173] Step 3: 72°C extension for 7 min;

[0174] Step 4: 4°C until sampling.

[0175] 4. Agarose gel electrophoresis detection

[0176] Refer to step 2.4 above.

[0177] 5. Enzymatic purification of PCR products: Add 0.5 μL of exonuclease I (Exo I) and 1 μL of alkaline phosphatase (AIP) to 5 μL of PCR product, digest at 37°C for 15 min, and inactivate the enzyme at 85°C for 15 min.

[0178] 6. BigDye reaction

[0179] The BigDye reaction system is shown in Table 9.

[0180] Table 9 BigDye reaction system

[0181]

[0182] Sequencing PCR cycling conditions:

[0183] Step 1: pre-denaturation at 96°C for 1 min;

[0184] Step 2: 33 cycles (96°C denaturation for 30 sec → 55°C annealing for 15 sec → 60°C extension for 4 min);

[0185] Step 3: 4°C until sampling.

[0186] 7. Purification of BigDye reaction products:

[0187] 1) Add 1 μL 125 mM EDTA (pH 8.0) to each tube until it reaches the bottom of the tube, then add 1 μL 3 mol / L NaAc (pH 5.2);

[0188] 2) Add 70 μL of 70% alcohol, shake and mix 4 times, and leave at room temperature for 15 min;

[0189] 3) Centrifuge at 3000g, 4°C for 30 min; immediately invert the 96-well plate and centrifuge at 185g for 1 min;

[0190] 4) Leave at room temperature for 5 minutes to allow the residual alcohol to evaporate, add 10 μL Hi-Di formamide to dissolve the DNA, denature at 96°C for 4 minutes, quickly place on ice for 4 minutes, and sequence on the machine.

[0191] 8. Sequencing

[0192] The purified BigDye reaction product was subjected to DNA sequencing, and the sequencing primers were designed as nested primers based on the above SEQ ID NO.1 and SEQ ID NO.2 (the second set of primers was designed within the product sequence range amplified by the first set of primers) as sequencing primers. The primer sequences are shown below.

[0193] The sequencing primer sequences for the GNAS:NM_000516.7:exon1:c.-1_2delCAT:p.Gly2_Met60del site are as follows:

[0194] GNAS-Seq1F: 5'-AGCTCCTGCTCTGGTCC-3' (SEQ ID NO.3)

[0195] GNAS-Seq1R: 5'-CGGTAGACCTGCTTGTCC-3' (SEQ ID NO.4);

[0196] 9. Results Analysis

[0197] The sequencing results for the GNAS:NM_000516.7:exon1:c.-1_2delCAT:p.Gly2_Met60del site are as follows Figure 2 As shown. Figure 2 It can be seen that the GNAS: NM_000516.7: exon1: c.-1_2delCAT site genotype of one patient in Family No. 1 is a "c.-1_2delCAT" heterozygous mutation; the GNAS: NM_000516.7: exon1: c.-1_2delCAT site genotype of two normal individuals in Family No. 1 and 100 unrelated normal controls is "wild type".

[0198] Example 4

[0199] McCune-Albright syndrome diagnostic kit and its application

[0200] 1. Kit composition:

[0201] Kit 1: 1) Amplification primers: SEQ ID NO.1 and SEQ ID NO.2 in Example 3; 2) PCR buffer (10×PCR buffer, composed of KCl 500mmol / L, Tris-HCl (pH8.3) 100mmol / L and MgCl2 15mmol / L); 3) Taq enzyme (20U); 4) dNTPs (4mM each of four dNTPs); 5) c.-1_2delCAT positive reference DNA, which is a double-stranded DNA. The specific sequence of the c.-1_2delCAT mutation site positive reference is shown in SEQ ID NO.7, specifically:

[0202] The single underlined bases are the positions of the upstream and downstream primers for PCR amplification, the boxes are the deletion mutation sites, and the double underlined bases are the positions of the upstream and downstream sequencing primers; 6) Sequencing primers:

[0203] As shown in SEQ ID NO.3 and SEQ ID NO.4.

[0204] 2. Usage:

[0205] A total of 522 individuals from 139 bone development families were screened and tested, and 20 patients from 18 families that were consistent with the present invention were found again. The application of the gene mutation detection kit is now described using family No. 2 as an example. The clinical information of family No. 2 is shown in Table 11, and the pedigree of family No. 2 is shown in Table 12. Figure 3 .

[0206] Table 10 Summary of McCune-Albright syndrome screening

[0207]

[0208]

[0209] Table 11 Clinical information of the members of McCune-Albright syndrome family 2

[0210]

[0211]

[0212] Note: Ⅰ and Ⅱ represent the first and second generations respectively.

[0213] The peripheral blood DNA of members Ⅰ:1 and Ⅱ:1 of family No. 2 and the surgical resection specimens of Ⅰ:2 were used for detection by the kit 1; the steps are as follows:

[0214] 1) Genomic DNA extraction: extract sample genomic DNA according to the steps in Example 2;

[0215] 2) First, a PCR amplification reaction is performed using the PCR amplification primers, Taq enzyme, buffer, dNTPs, sample genomic DNA, etc. in the kit, as described in step 3 of Example 3;

[0216] 3) Purifying the PCR amplification product, as described in step 5 of Example 3;

[0217] 4) using the sequencing primers in the above kit to perform BigDye reaction on the purified PCR product, as described in step 6 of Example 3;

[0218] 5) Purifying the BiyDye reaction product, as described in step 7 of Example 3;

[0219] 6) Sequencing the BiyDye reaction product and comparing the sequence with the normal sequence, as described in step 8 of Example 3.

[0220] The test results of the No. 2 family members using the test kit are as follows Figure 4 As shown, the position indicated by the arrow in layer B shows that the genotype of the proband in the family GNAS: NM_000516.7: exon1: c.-1_2delCAT site is a "c.-1_2delCAT" heterozygous mutation; the husband of the proband in layer A and the daughter of the proband in layer C do not have mutations; the test results confirmed that the proband is a patient with McCune-Albright syndrome, and the genotypes of the proband's husband and daughter are wild type, and they are normal individuals.

[0221] Example 5

[0222] Gene mutation rating and interpretation (pathogenicity of mutations)

[0223] Mutation interpretation is based on our current understanding of McCune-Albright syndrome and the causative gene GNAS (https: / / www.omim.org / entry / 139320) and the clinical phenotype association of the test results. Mutations follow the HGVS guidelines for mutation nomenclature (http: / / www.hgvs.org / ) and are named according to GenBank accession numbers (https: / / www.ncbi.nlm.nih.gov / genbank / ). The rules for interpreting genetic variant data refer to the relevant guidelines of the American College of Medical Genetics and Genomics (ACMG): Richards, S, et al., Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med, advance online publication 5March 2015. doi:10.1038 / gim.2015.30; and China's "Standards and Guidelines for the Classification of Genetic Variation": Wang Qiuju, Shen Yiping, Wu Lingqian, et al. Standards and Guidelines for the Classification of Genetic Variation. Science China: Life Sciences, 2017, 47:668–688.

[0224] The classification of genetic variation in the "Standards and Guidelines for the Classification of Genetic Variation" is based on typical data types (such as population data, computational data, functional data, and co-segregation data) to classify variations into five levels, namely: "pathogenic (P)", "likely pathogenic (LP)", "variant of uncertain significance (VUS)", "likely benign (LB)" and "benign (B)". The five-level classification is based on the comprehensive score after interpreting and analyzing each aspect / sub-item of the variation (Table 12).

[0225] Table 12 Criteria for determining pathogenicity of variants

[0226]

[0227]

[0228] Before the five-level assessment, each aspect / sub-item of the mutation / variation needs to be analyzed / interpreted. Among them, the pathogenic variation criteria can be divided into: very strong (PVS1), strong (PS1~4), moderate (PM1~6), or supporting evidence (PP1~5; benign variation evidence can be divided into: stand-alone (BA1), strong (BS1~4), or supporting evidence (BP1~6) (see Table 13 below). For a given mutation / variation, it is first necessary to select the criteria in Table 13 based on the observed evidence to determine whether the various aspects / sub-items of the mutation / variation can meet the criteria. Which of the items in Table 13 is each rated as PVS1 / PS1~4 / PM1~6 / PP1~5 / BA1 / BS1~4 / BP1~6? Finally, the sub-item standards that can be met are combined according to the scoring rules in Table 12, and then a classification is selected from the five-level system according to the combination standards in Table 12; for example, if the aspects / sub-items of the mutation have PVS1, PM1, and PM2 after analysis, the mutation is rated as "pathogenic" [that is, it meets the criteria (i) + (b) in "Pathogenic (P)" in Table 12].

[0229] Table 13. Variant interpretation criteria and variant pathogenicity determination criteria

[0230]

[0231]

[0232]

[0233] The analysis / interpretation of each aspect / sub-item of mutation / variation needs to be based on the corresponding bioinformatics analysis tools (see Table 15) and a large number of available data (libraries) (see Table 16), including data obtained from existing cases and published data, such as public databases (such as ClinVar or site-specific databases) and laboratory-owned databases. When using various data (libraries) to analyze mutations / variations, the degree judgment evaluation criteria used are shown in Table 14.

[0234] Table 14 Evaluation criteria for degree judgment

[0235]

[0236]

[0237] Table 15 Bioinformatics analysis tools

[0238]

[0239]

[0240] Table 16 Population databases, disease-specific databases and sequence databases

[0241]

[0242]

[0243] According to the above standards or criteria, the GNAS gene c.-1_2delCAT mutation in the present invention is evaluated as "pathogenic", and the judgment criteria and specific evidence are as shown in Table 17:

[0244] Table 17 Interpretation of pathogenicity of GNAS gene c.-1_2delCAT mutation

[0245]

[0246] AD: autosomal dominant inheritance

[0247] GNAS:NM_000516.7:Exon1:c.-1_2delCAT:p.Gly2_Met60del Variant rating evidence is as follows:

[0248] 1. PVS1: The c.-1_2delCAT mutation in the GNAS gene causes a frameshift mutation, resulting in a truncated protein. This mutation affects the protein function.

[0249] 2. PS4: Based on the literature and this case, this variant was detected in 21 patients;

[0250] 3. PM2: The c.-1_2delCAT variant of the GNAS gene was not found in the 1000 Genomes (1000G) of the reference population, the Exome Database (ExAC), and the GnomAD database;

[0251] 4. PP3: Multiple computer software predicts that the variant will have a deleterious effect on the gene or gene product;

[0252] Therefore, the comprehensive evidence of this mutation / variation (PVS1+PS4+PM2+PP3) meets (a) or (c) in the "pathogenic (P)" judgment criterion (i) in Table 12, and the GNAS gene c.-1_2delCAT variation is comprehensively judged to be "pathogenic".

[0253] Example 6

[0254] Follow-up and diagnostic kit performance analysis

[0255] All family members were followed up, and the GNAS gene targeted capture chip method was resequencing analysis and validation for all individuals (see Table 18).

[0256] Table 18c.-1_2delCAT site detection performance analysis results

[0257] index formula result True positive variant sites (TP) / 21 True negative variant site (TN) / 42 False positive variant sites (FP) / 0 False negative variant site (FN) / 0 Sensitivity (N=21) TP / (TP+FN) 100% (95%CI:99.03%~100%) Specificity (N=42) TN / (TN+FP) 100% (95%CI:99.03%~100%) Positive predictive value TP / (TP+FP) 100% (95%CI:99.03%~100%) Negative predictive value TN / (TN+FN) 100% (95%CI:99.03%~100%)

[0258] Note: This table contains follow-up data of family 1.

[0259] As can be seen from Table 10, positive patients (21 cases) were found when testing 19 families. The above positive site detection results were verified by the GNAS gene targeted capture chip method. According to the follow-up and verification results, a total of 21 true positive cases, 42 true negative cases, 0 false negative cases and 0 false positive cases were found. The sensitivity of the c.-1_2delCAT variant site marker for detection was 100.00%, and its 95% CI was 99.03% to 100%, and the specificity was 100%, and its 95% CI was 99.03% to 100%. The above results show that this kit has good detection performance in clinical application.

[0260] It can be seen from the above examples that the GNAS gene mutants of the present invention can be used as biomarkers for diagnosing McCune-Albright syndrome and provide possible drug targets for treating McCune-Albright syndrome.

[0261] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A GNAS mutant protein, characterized in that Compared with the wild-type GNAS protein, the GNAS mutant protein lacks amino acids 2 to 60.

2. A GNAS gene mutant, characterized in that: Compared with the wild-type GNAS gene, the GNAS gene mutant lacks CAT from the -1st base to the 2nd base in exon 1 of the wild-type GNAS gene.

3. A use of the GNAS gene mutant as claimed in claim 2 as a detection target in the preparation of a reagent or a kit, characterized in that: The reagents include reagents for detecting McCune-Albright syndrome; The kit includes one or more of a kit for preventing McCune-Albright syndrome, a kit for diagnosing McCune-Albright syndrome, a kit for screening genetic diseases before pregnancy, a kit for screening genetic diseases before birth, a kit for diagnosing genetic diseases before pregnancy, a kit for diagnosing genetic diseases before birth, and a kit for assisting in treating McCune-Albright syndrome.

4. An amplification primer for detecting McCune-Albright syndrome, characterized in that: The amplification primers include an upstream primer GNAS-1F and a downstream primer GNAS-1R; the upstream primer GNAS-1F includes a nucleotide sequence as shown in SEQ ID NO.1, and the downstream primer GNAS-1R includes a nucleotide sequence as shown in SEQ ID NO.

2.

5. A sequencing primer for detecting McCune-Albright syndrome, characterized in that: The sequencing primers include an upstream primer GNAS-Seq1F and a downstream primer GNAS-Seq1R; the upstream primer GNAS-Seq1F includes a nucleotide sequence as shown in SEQ ID NO.3; the downstream primer GNAS-Seq1R includes a nucleotide sequence as shown in SEQ ID NO.

4.

6. A primer combination for detecting McCune-Albright syndrome, characterized in that: It comprises the amplification primer as claimed in claim 4 and / or the sequencing primer as claimed in claim 5.

7. Use of the primer combination according to claim 6 in preparing a reagent for detecting McCune-Albright syndrome.

8. The use according to claim 7, characterized in that: The detection target of McCune-Albright syndrome includes a GNAS gene mutant, and compared with the wild-type GNAS gene, the GNAS gene mutant has a CAT deletion from the -1 base to the 2 base in exon 1 of the wild-type GNAS gene.

9. A reagent for detecting McCune-Albright syndrome, characterized in that: The reagent comprises the primer combination as claimed in claim 6.

10. Use of the reagent according to claim 9 in a kit and its preparation, characterized in that: The kit includes one or more of a kit for preventing McCune-Albright syndrome, a kit for diagnosing McCune-Albright syndrome, a kit for screening genetic diseases before pregnancy, a kit for screening genetic diseases before birth, a kit for diagnosing genetic diseases before pregnancy, a kit for diagnosing genetic diseases before birth, and a kit for assisting in treating McCune-Albright syndrome.

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