CN115925869A

CN115925869A - AXIN2 mutants and uses thereof

Info

Publication number: CN115925869A
Application number: CN202211589650.5A
Authority: CN
Inventors: 廖灿; 符芳; 李东至; 李茹; 杨昕; 潘敏; 甄理; 韩瑾; 党孝; 王丹; 喻秋霞; 邓琼
Original assignee: Guangzhou Women and Childrens Medical Center
Current assignee: Guangzhou Women and Childrens Medical Center
Priority date: 2022-12-12
Filing date: 2022-12-12
Publication date: 2023-04-07

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an AXIN2 mutant and application thereof. The invention discloses for the first time that compared with wild type AXIN2 protein, at least one of the following mutations is present: the protein and/or the nucleic acid coding the protein are pathogenic factors of embryonic development deformity, the embryonic development deformity can be diagnosed by detecting the protein and/or the nucleic acid coding the protein, and the effect of preventing and/or treating the embryonic development deformity can be achieved by specifically changing the protein and/or the substance coding the protein.

Description

AXIN2 mutants and uses thereof

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to an AXIN2 mutant and application thereof.

Background

Birth defects are a global public health problem affecting millions of newborns worldwide, with structural birth defects accounting for approximately 60-70% of birth defects, meaning structural changes in the fetus that occur in utero during pregnancy, with clinical manifestations ranging from single minute structural defects to severe and fatal multiple system structural abnormalities, with a morbidity rate of 3% in live-born infants and a mortality rate of approximately 25% in perinatal. Depending on the system involved, it can be classified into central nervous system, facial, thoracic, cardiovascular, abdominal, digestive, urogenital and skeletal malformations, and others including Nuchal Translucency (NT) thickening and lymphocystoma, fetal edema and intrauterine growth restriction, etc. When two or more malformations of the fetus occur simultaneously, the fetus is called multiple malformations, and related organ systems can randomly appear and also appear in the forms of various syndromes, sequence characteristics, associations and malformation spectrums according to a certain rule.

Most fetal abnormalities occur in the early stages of major organ system development, most of which can be found by routine prenatal ultrasound examination. Additional prenatal assessment is required to discover fetal abnormalities, and the clear cause is a prerequisite for the assessment of pregnancy outcome, fetal prognosis, and perinatal treatment and for genetic counseling. The etiology of fetal abnormalities is complex and is known to include primarily genetic etiology, pregnancy-associated infections, medication, history of exposure to teratogens or other toxic or harmful substances, and maternal factors (e.g., fetal alcohol syndrome, gestational diabetes, etc.). The genetic etiology can be related to various types of genetic diseases, including chromosomal abnormalities, copy Number Variations (CNVs), and monogenic diseases. The remaining cases of unknown etiology are defined as fetal abnormalities of unknown cause. In general, about 30% of fetuses with abnormal fetal karyotype detected by ultrasound and other imaging examinations during pregnancy, and another 6% of fetuses can detect pathogenic copy number variation by Chromosome Microarray Analysis (CMA), which means that more than 60% of fetuses cannot be clearly diagnosed by conventional prenatal genetic diagnosis techniques.

The discovery and detection of the pathogenic gene causing the fetal deformity can strengthen the understanding of the embryonic development and provide guidance for prenatal diagnosis. The newly developed Next Generation high throughput Sequencing (NGS) technology can directly and accurately detect the mutation site of a gene within 30 hundred million bases of the whole genome, and provides technical convenience and feasibility for exploring and discovering the pathogenic gene of a genetic disease. Among them, whole Exome Sequencing (WES) has been widely used in the research of genetic diseases and complex diseases due to its economical and effective advantages. With the development of WES, more genes related to fetal malformation can be found, so that the genetic cause of the disease can be further understood.

Disclosure of Invention

The first aspect of the present invention is directed to a protein.

It is an object of the second aspect of the invention to provide a nucleic acid encoding the first aspect of the invention.

The third aspect of the present invention is directed to a substance for detecting the protein of the first aspect of the present invention and/or a substance for detecting the second aspect of the present invention for use in the manufacture of a product for diagnosing an embryonic development deformity.

The fourth aspect of the present invention is directed to a method for constructing a biological model.

The fifth aspect of the present invention is directed to the use of a biological model for screening drugs.

The sixth aspect of the present invention is directed to the use of a substance which specifically alters a protein of the first aspect of the present invention and/or a nucleic acid of the second aspect of the present invention in the preparation of a medicament for the prevention and/or treatment of an embryo development deformity.

An object of a seventh aspect of the present invention is to provide a medicament.

An eighth aspect of the invention is directed to a product.

An object of the ninth aspect of the present invention is to provide a biological material related to the nucleic acid of the second aspect of the present invention.

A tenth aspect of the present invention is directed to an application of AXIN 2.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

in a first aspect of the invention, there is provided a protein having at least one of the following mutations compared to the wild-type AXIN2 protein: p.G288A and p.R714W.

Preferably, the protein has any one of the following mutations compared to the wild-type AXIN2 protein: p.G288A and p.R714W.

Preferably, the protein has the following mutations compared to the wild-type AXIN2 protein: p.G288A and p.R714W.

Preferably, the amino acid sequence of the wild-type AXIN2 protein is shown as SEQ ID No. 2.

In a second aspect of the invention there is provided a nucleic acid encoding a protein according to the first aspect of the invention.

Preferably, the nucleic acid has at least one of the following mutations compared to the wild-type AXIN2 gene: c.863G > C, c.2140C > T.

Preferably, the nucleic acid has a mutation compared to the wild-type AXIN2 gene of any one of the following: c.863G > C, c.2140C > T.

Preferably, the nucleic acid has the following mutations compared to the wild-type AXIN2 gene: c.863G > C and c.2140C > T.

Preferably, the nucleotide sequence of the wild-type AXIN2 gene is shown in SEQ ID NO. 1.

In a third aspect of the present invention, there is provided a use of at least one of (e 1) to (e 3) in the preparation of a product for diagnosing an embryo development abnormality;

(e1) Detecting a substance of the protein of the first aspect of the invention;

(e2) A substance that detects the nucleic acid of the second aspect of the invention;

(e3) Detecting a substance of wild-type AXIN 2.

Preferably, the substance for detecting the protein of the first aspect of the present invention is a substance for quantitatively detecting the protein of the first aspect of the present invention.

Preferably, the substance for detecting the protein of the first aspect of the present invention is a substance in one or more detection methods selected from the group consisting of: immunohistochemistry, western blotting, and biochip.

Preferably, the immunohistochemistry is selected from the group consisting of: immunofluorescence, immunoenzyme linked immunosorbent assay (ELISA) and immunocolloidal gold.

Preferably, the substance for detecting a protein of the first aspect of the invention comprises: a substance specific for a protein of the first aspect of the invention.

Preferably, the substance specific to the protein of the first aspect of the present invention is any one of (a 1) to (a 3):

(a1) An antibody that specifically binds to a protein of the first aspect of the invention;

(a2) A ligand protein or polypeptide that specifically binds to a protein of the first aspect of the invention;

(a3) A non-proteinaceous compound which specifically recognizes the protein of the first aspect of the invention.

Preferably, the antibody comprises at least one of polyclonal antibody, monoclonal antibody, single-chain antibody, functional antibody fragment, antibody Fab region, nanobody, chimeric antibody and multispecific antibody.

Preferably, the substance for detecting a nucleic acid of the second aspect of the present invention is a substance for quantitatively detecting a nucleic acid of the second aspect of the present invention.

Preferably, the means for detecting a nucleic acid of the second aspect of the invention is selected from one or more of the following detection techniques or methods: northern blotting, PCR, and biochip methods.

Preferably, the substance for detecting the nucleic acid of the second aspect of the present invention comprises a probe, a gene chip, a PCR primer, etc. of the nucleic acid of the second aspect of the present invention.

Preferably, the means for detecting a nucleic acid according to the second aspect of the invention comprises AXIN2-863G >.

Preferably, the sequence of the AXIN2-863G > -C primer is shown as SEQ ID NO.3 and SEQ ID NO. 4.

Preferably, the sequence of the AXIN2-2140C > T primer is shown as SEQ ID NO.5 and SEQ ID NO. 6.

Preferably, the substance that detects wild-type AXIN2 comprises a substance that quantitatively detects wild-type AXIN 2.

Preferably, the substance that detects wild-type AXIN2 comprises a substance that detects wild-type AXIN2 at the gene level and/or protein level.

Preferably, the substance comprises a substance for use in one or more detection techniques or methods selected from the group consisting of: immunohistochemistry, western blotting, northern blotting, PCR, and biochip methods.

Preferably, the immunohistochemistry is at least one selected from the group consisting of: immunofluorescence analysis, reverse enzyme-linked immunosorbent assay and immunocolloidal gold method.

Preferably, the substance for detecting wild-type AXIN2 is at least one selected from the group consisting of: substances specific to wild-type AXIN2, such as antibodies (preferably monoclonal antibodies) thereto; wild type AXIN2 specific probes, gene chips, PCR primers, and the like.

Preferably, the product comprises at least one of a reagent, a kit, a strip, a chip, a system.

Preferably, the test sample of the product is selected from at least one of blood, amniotic fluid, villi, cord blood, tissue, cell sample.

The inventors have found that the aforementioned gene mutation is present in an exon of a patient having an embryonic development abnormality, and is a heterozygous mutation and/or a homozygous mutation; and/or a reduced expression level of wild-type AXIN 2; and/or a reduced activity of wild type AXIN2 protein. The product prepared from the substance for detecting the nucleic acid and/or the substance for detecting the protein and/or the substance for detecting the wild AXIN2 can be used for diagnosing the embryo development deformity.

In a fourth aspect of the present invention, a method for constructing a biological model is provided, which includes the following steps: allowing the biological model to carry at least one of (b 1) to (b 4):

(b1) A protein of the first aspect of the invention;

(b2) A nucleic acid according to the second aspect of the invention;

(b3) Reduced expression of wild-type AXIN 2;

(b4) The activity of the wild type AXIN2 protein is reduced.

Preferably, said wild type AXIN2 in (b 3) comprises a protein and a nucleic acid.

Preferably, the amount of wild-type AXIN2 expression is reduced by degrading substances of wild-type AXIN2 and/or substances that reduce the level of expression of wild-type AXIN 2.

Preferably, the activity of a wild type AXIN2 protein is reduced by degrading substances of the wild type AXIN2 protein and/or substances inhibiting the activity of the wild type AXIN2 protein.

Preferably, the biological model comprises at least one of an animal model, a cellular model; further preferably, the biological model comprises an animal model.

Preferably, the animal model comprises a zebrafish model.

Preferably, the biological model is an embryonic development deformity model.

In a fifth aspect of the present invention, there is provided a use of a biological model for screening a drug, the biological model carrying at least one of (b 1) to (b 4):

(b1) A protein of the first aspect of the invention;

(b2) A nucleic acid according to the second aspect of the invention;

(b3) Reduced expression of wild-type AXIN 2;

(b4) The activity of the wild type AXIN2 protein is reduced.

Preferably, the biological model comprises at least one of an animal model, a cellular model; further preferably, the biological model comprises a cell model.

Preferably, the animal model comprises a zebrafish model.

The model carrying the nucleic acid and the protein, with the reduced expression level of wild-type AXIN2 and the reduced activity of wild-type AXIN2 protein can be used as a model of c.863G > C and/or c.2140C > T related diseases, and the disease model can be used for scientific research, such as screening of drugs for treating c.863G > C and/or c.2140C > T related diseases.

Preferably, the biological model is an embryonic development deformity model.

Preferably, the medicament is for the prevention and/or treatment of an embryonic development deformity.

In a sixth aspect, the present invention provides a use of at least one of (f 1) to (f 3) in the preparation of a medicament for preventing and/or treating an embryo development deformity;

(f1) A substance which increases the expression level of wild-type AXIN 2;

(f2) A substance that increases the activity of a wild-type AXIN2 protein;

(f3) A substance which specifically alters a protein of the first aspect of the invention and/or a nucleic acid of the second aspect of the invention.

As mentioned above, patients with embryonic development deformities carry the gene mutations described above, and are heterozygous and/or homozygous; and/or a reduced expression level of wild-type AXIN 2; and/or reduced activity of wild-type AXIN2 protein; drugs that restore the aforementioned nucleic acids or proteins to wild-type or non-pathogenic, and/or that increase the expression of wild-type AXIN2, and/or that increase the activity of wild-type AXIN2 protein, would have the potential to treat and/or prevent malformations in embryonic development.

Preferably, the change in specificity is such as to restore the protein of the first aspect of the invention and/or the nucleic acid of the second aspect of the invention to wild type.

Preferably, the substance that specifically alters the protein of the first aspect of the invention and/or the nucleic acid of the second aspect of the invention is a substance based on at least one gene editing method including single base gene editing, zinc Finger Nuclease (ZFN), transcription activator-like effector nuclease (TALEN), CRISPR/Cas9 (which can be combined with iPSC and AAV vector technologies) and the like.

In a seventh aspect of the present invention, there is provided a medicament comprising: at least one of (f 1) to (f 3):

(f1) A substance which increases the expression level of wild-type AXIN 2;

(f2) A substance that increases the activity of a wild-type AXIN2 protein;

In an eighth aspect of the present invention, there is provided a product comprising at least one of (g 1) to (g 3):

(g1) Detecting a substance of the protein of the first aspect of the invention;

(g2) A substance that detects the nucleic acid of the second aspect of the invention;

(g3) Detecting a substance of wild-type AXIN 2.

Preferably, the product comprises (g 1) or (g 2).

Preferably, the product comprises at least two of (g 1) to (g 3).

Preferably, the product comprises: at least one of a substance that detects the protein of the first aspect of the invention, a substance that detects the nucleic acid of the second aspect of the invention, and a substance that detects wild-type AXIN 2.

Preferably, the substance for detecting a protein of the first aspect of the invention comprises: a substance specific for the protein of the first aspect of the invention.

(a3) A non-proteinaceous compound which specifically recognizes a protein according to the first aspect of the invention.

Preferably, the sequence of the AXIN2-2140C >.

Preferably, the product is used for diagnosing embryo development deformity.

Preferably, the product further comprises other substances for diagnosing malformations of embryonic development.

Preferably, the above-mentioned embryonic development deformities comprise: cleft lip and palate, fallo tetrad, leukoplakia, hypospadias, ventricular septal defect, diaphragmatic hernia, NT thickening, and bipedal varus.

In a ninth aspect of the present invention, there is provided a biological material related to the nucleic acid of the second aspect of the present invention, the biological material comprising at least one of (c 1) to (c 7):

(c1) An expression cassette comprising a nucleic acid of the second aspect of the invention;

(c2) A vector comprising a nucleic acid of the second aspect of the invention;

(c3) A vector comprising the expression cassette of (c 1);

(c4) A transgenic cell line comprising a nucleic acid of the second aspect of the invention;

(c5) A transgenic cell line comprising the expression cassette of (c 1);

(c6) A transgenic cell line comprising the vector of (c 2);

(c7) A transgenic cell line comprising the vector of (c 3).

Preferably, the transgenic cell line does not comprise propagation material.

It is an object of a tenth aspect of the present invention to provide an application of AXIN 2.

Use of AXIN2 in any one of (d 1) to (d 6):

(d1) Regulating the proliferation activity of the embryonic stem cells;

(d2) Regulating late death of embryonic stem cells;

(d3) Regulating the expression of an anti-apoptosis factor Bcl-2 of the embryonic stem cells;

(d4) Regulating the expression of apoptosis factor clear caspase-3 of embryonic stem cell;

(d5) Key factors for regulating Wnt/beta-catenin signal channels of embryonic stem cells;

(d6) Regulating and controlling downstream molecules of a Wnt/beta-catenin signal channel of the embryonic stem cells;

the key factors of the Wnt/beta-catenin signal channel comprise beta-catenin and TCF4;

the downstream molecules of the Wnt/beta-catenin signal path comprise c-jun, c-myc and cyclin D1.

Preferably, the AXIN2 is over-expressed to reduce the proliferation activity of the embryonic stem cells, induce the late death of the embryonic stem cells, reduce the expression of Bcl-2, promote the expression of clear Caspase-3, reduce the expression of key factors of the Wnt/beta-catenin signaling pathway of the embryonic stem cells and reduce the expression of downstream molecules of the Wnt/beta-catenin signaling pathway of the embryonic stem cells; by inhibiting the expression of AXIN2, the proliferation activity of the embryonic stem cells can be improved, the late death of the embryonic stem cells can be reduced, the expression of Bcl-2 can be promoted, the expression of clear Caspase-3 can be reduced, the expression of key factors of the Wnt/beta-catenin signal path of the embryonic stem cells can be promoted, and the expression of downstream molecules of the Wnt/beta-catenin signal path of the embryonic stem cells can be promoted.

The invention has the beneficial effects that:

the invention discloses for the first time that compared to the wild-type AXIN2 protein, there is at least one of the following mutations: the protein and/or the nucleic acid coding the protein are pathogenic factors of embryonic development deformity, the embryonic development deformity can be diagnosed by detecting the protein and/or the nucleic acid coding the protein, and the effect of preventing and/or treating the embryonic development deformity can be achieved by specifically changing the protein and/or the substance coding the protein.

Drawings

FIG. 1 is a validation diagram of the mutation site sanger of AXIN2 gene; wherein, A picture is AXIN2 gene mutation site c.863G > Cseger verification picture (antisense chain); panel B shows validation of AXIN2 gene mutation site c.2140C > T sanger (antisense strand).

FIG. 2 is a schematic diagram of the experimental technique of AXIN2 gene effect on cell development in vitro.

FIG. 3 is a graph showing the effect of AXIN2 gene on embryonic stem cells: wherein A is the result of mRNA expression level after AXIN2 gene interference and overexpression in embryonic stem cells (P <0.05 compared to LV 3-vector); b is a graph of the results of AXIN2 gene interference and cell proliferation activity following overexpression in embryonic stem cells (P <0.05 compared to LV 3-vector); c is a graph of statistics of early and late apoptosis following AXIN2 gene interference and overexpression in embryonic stem cells (P <0.05 compared to LV 3-vector); d is a graph of the results of the expression of cleared caspase-3 following AXIN2 gene interference and overexpression in embryonic stem cells (P <0.05, P <0.01 compared to LV 3-vector).

FIG. 4 is the result of electrophoretic identification of the genotype of zebra fish AXIN2 gene mutant embryos: wherein A is an electrophoresis identification result picture of the homozygous mutant embryo genotype of the zebra fish AXIN2 gene; b is a gene type electrophoresis identification result picture of the zebra fish AXIN2 gene heterozygous mutant embryo.

FIG. 5 is a diagram showing examples of the development of zebrafish homozygous mutant embryos at various stages: wherein, A is a development example diagram of the zebrafish homozygous mutant embryo at 8 hpf; b is a diagram of a development example of a zebrafish homozygous mutant embryo at 24 hpf; c is a diagram of an example of the development of zebrafish homozygous mutant embryos at 48 hpf; d is a diagram of an example of the development of a zebrafish homozygous mutant embryo at 72 hpf; e is a diagram of a development example of a zebrafish homozygous mutant embryo at 96hpf, axin2+/+ is a wild type zebrafish control group, and axin 2-/-is a zebrafish homozygous mutant experimental group.

FIG. 6 is a statistical chart of the observed phenotype of the zebra fish homozygous mutant embryo in microscopic morphology at each stage.

FIG. 7 is a representation of zebrafish heterozygous mutant and wild type TU zebrafish embryos at various stages.

FIG. 8 is a graph showing the results of comparing heart rate data of zebra fish heterozygous mutants with wild type TU zebra fish embryos at each period: * P < 0.001; * P < 0.0001.

Detailed Description

The present invention will be described in further detail with reference to specific examples.

The materials, reagents and the like used in the present examples are commercially available reagents and materials unless otherwise specified.

Interpretation of terms:

gnomad: https:// gnomad. Broadedition. Org/, population database, data for 15,708 whole genomes and 125,748 exomes have been collected so far;

2.1000Genomes: https:// www.internationalgenome.org/, thousand human genome, containing 2,504 sequencing samples;

OMIM: https:// www.omim.org/, the human mendelian genetic online database;

4, BWA: http:// bio-bw. Sourceform. Net/, software that aligns the original sequencing data onto a reference genome;

5.GATK: https// gatk. Broadinsulation. Org/hc/en-us, an abbreviation of Genome Analysis ToolKit, is a piece of software that analyzes variant information from high throughput sequencing data;

6.VEP: (ii) a Variant Effect Predictor for annotating different types of variation generated by a second generation test, https:// asia.ensemblel.org/info/docs/tools/vep/index.html;

SIFT, polyphen 2. U HDIV, REVEL, mutationTaster, metaSVM: various hazard prediction software for predicting the hazard caused by the mutation;

ClinVar: https:// www.ncbi.nlm.nih.gov/clinvar/, stores the relationship between human mutation sites and phenotypes, and provides supporting evidence;

9.HGMD: http:// www.hgmd.cf.ac.uk/ac/gene. Php? Human Gene Mutation Database (The Human Gene Mutation Database), collecting mutations associated with genetic diseases of humans;

10.Orphanet: https:// www.orpha.net/connector/cgi-bin/index. Php, rare human disease database;

DDG2P: https:// decipher.sanger.ac.uk/ddd/ddgenes, developmental disorder disease-associated genotype and phenotype databases;

12.PCR: polymerase chain reaction, a molecular biology technique for amplifying specific DNA fragments;

crispr-Cas9: is a genome editing technology developed by a specific acquired immune system in microorganisms such as bacteria and archaea;

QPCR: quantitative Real-time PCR, real-time fluorescent Quantitative PCR, and Quantitative analysis of a specific DNA sequence in a sample to be detected by using PCR;

hpf.

Example 1 screening and verification of causative genes causing fetal abnormalities

1. Sample collection

The inventor collects 56 families from 2016, 1 month to 2018, 12 months to child medical center of Guangzhou city, wherein the family is subjected to repeated fetal malformation (more than or equal to 2 times) due to unknown reasons. None of these families have previously found a cause of fetal abnormalities by conventional genetic testing methods such as karyotyping or CMA techniques. The enrolled pedigree retains at least one sample of the abnormal fetus. The average puncture gestation week of the fetuses in the family of the group is 24 weeks (12-32 weeks), and the average age of the pregnant women is 29 years (22-39 years). Proband sample sources include villi (n = 7), amniotic fluid (n = 19), cord blood (n = 33), induced fetal tissue (n = 1) and fetal postnatal peripheral blood (n = 2), with parental peripheral blood collected for testing. Both partners of the married couple are not close but have been given detailed genetic counseling before and after exon sequencing. Meanwhile, 123 women who once born the congenital development defect offspring and 100 women who have no bad birth history are collected as candidate disease-causing gene row extension screening verification screened by WES. The invention is approved by ethical committee of child medical center of Guangzhou city women, and adheres to the standard established in Helsinki declaration in 1964 and the revised contents thereof. All participating individuals have signed informed consent by themselves or their guardians. Clinical information including age, gender and clinical symptoms was collected.

2. Whole exon sequencing

The inventors hybridized All DNA samples with a SureSelect XT Human All Exon 50Mb V5 Kit (Agilent) capture chip to obtain the target region sequence, and performed double-ended 150bp sequencing on an Illumina HiSeq 2500 sequencer. Performing original data quality control on original data obtained after sequencing by using fastp (V0.20), removing reads containing joints or low quality, comparing clean data after quality control to a human reference genome GRCH37 (hg 19) by using BWA (V0.7.17), sequencing the compared data by using Samtools (V1.9) and Picard software, marking a repeated sequence, and finally detecting variation information of a sample by using a HaplotpypeCaller module of GATK (V3.8); filtering the obtained initial variation result to leave high-quality information, and removing the variation meeting one of the following conditions: total mutation depth is less than 10, genotype quality value is less than 50 and alignment quality value is less than 30. VEP (Variant Effect Predictor) is used for annotating the variation, and information such as a disease database, a crowd frequency database, a hazard or conservative prediction software, a variation site database and the like is annotated; population databases include the thousand human genomes (1000 genomics), gnomAD and the local frequency database of the laboratory; the dangerous or conservative prediction software comprises SIFT, polyphen2_ HDIV, REVEL, mutationTaster, metaSVM and the like, mutation site databases ClinVar and HGMD and disease databases OMIM, orphanet, medGen, DDG2P and the like.

By data analysis of all the above samples, the coding regions and splicing regions that may affect protein function were preferentially focused on variations with population data frequencies less than 0.05. The inventor uses WES technology to detect the families of 56 repeatedly born abnormal fetuses with unknown reasons, and the results show that the known pathogenicity or possible pathogenicity variation related to the abnormal phenotype of the fetuses is detected in 14 families, the molecular diagnosis rate of the total Mendelian monogenic disease is 25%, and BBS7, L1CAM, NOTCH3, CEP290, ARX, AMPD2, EPHB4, COL1A1, TMEM231, ABCA12, DCHS1, ASPM, FGFR 3 and EIF2B3 genes are involved, wherein 7 cases are autosomal recessive inheritance, 2 cases are X staining recessive inheritance, and 5 cases are autosomal dominant inheritance (2 cases are inherited from father or mother with new phenotype, and 3 cases are inherited from 3). The clinical significance of the association with fetal phenotype detected in the other 6 families was unknown (VUS), involving the ZIC3, PKD1, ABCA1, TSC2, ARHGAP29 and CCDC22 genes. No clinically significant variation was detected in the remaining 36 families, and these 36 families were further analyzed to identify and screen 6 oocytes maturation, cell cycle regulation, meiosis and embryonic development-associated genes and their mutation sites, including AXNI2 gene (2), POLG, PTPN13, NINL, SH3PXD2B, KIAA1109, in 7 women.

3. Candidate pathogenic gene expansion screening verification

The 6 genes are screened and verified by adopting a Sanger sequencing technology, and the screening is expanded in another 123 cases of women who have born congenital malformed fetuses and 100 cases of women without adverse birth history, and the Sanger sequencing result indicates that AXIN2 gene mutation with uncertain clinical significance is found in another 4 cases of women who have born congenital malformed fetuses, and similar mutation is not detected in a normal control group without adverse birth history. The remaining 5 candidate gene mutation sites were eliminated due to polymorphic variation in the control population. In conclusion, two AXIN2 gene missense heterozygous mutations, c.863G > C (p.G288A) and c.2140C > T (p.R714W), were detected in 2+4 women with poor fertility history (see Table 1), and were not detected in 100 women with no poor fertility history. Wherein rs number of c.863C > G variation site is 754388559, and the minimum allele frequency in GnomAD database is 0.000013; c.2140C > T mutation site rs number 148765149, the minimum allele frequency is 0.000159, and bioinformatics software predicts that the two sites have harmful variation and can damage the structure and the function of the wild-type protein. Prenatal ultrasound and MRI suggested that in these 6 women carrying the AXIN2 gene mutation, the organ malformation in their offspring fetal cases involved cardiovascular, facial and cervical, skeletal and thoracic abnormalities (table 2, table 3). It can be seen that this mutation in the AXIN2 gene is the causative site of fetal malformation. Wherein, the nucleic acid sequence of the wild AXIN2 gene is as follows: ATGAGTAGCGCTATGTTGGTGACTTGCCTCCCGGACCCCA GCAGCAGCTTCCGTGAGGATGCCCCGCGGCCCCCAGTGCCAGGGGAAGAAGGGGAGACCCCACCGTGTCAGCCAGGGGTGGGCAAGGGCCAGGTCACCAAACCCATGCCTGTCTCTTCCAACACCAGGCGGAACGAAGATGGGTTGGGGGAGCCGGAGGGGCGGGCATCTCCGGATTCCCCTCTGACCCGGTGGACCAAGTCCTTACACTCCTTATTGGGCGATCAAGACGGTGCTTACCTGTTCCGAACTTTCCTGGAGAGGGAGAAATGCGTGGATACCTTAGACTTCTGGTTTGCCTGCAATGGATTCAGGCAGATGAACCTGAAGGATACCAAAACTTTACGAGTAGCCAAAGCGATCTACAAAAGGTACATTGAGAACAACAGCATTGTCTCCAAGCAGCTGAAGCCTGCCACCAAGACCTACATAAGAGATGGCATCAAGAAGCAGCAGATTGATTCCATCATGTTTGACCAGGCGCAGACCGAGATCCAGTCGGTGATGGAGGAAAATGCCTACCAGATGTTTTTGACTTCTGATATATACCTCGAATATGTGAGGAGTGGGGGAGAAAACACAGCTTACATGAGTAATGGGGGACTCGGGAGCCTAAAGGTCGTGTGTGGCTATCTCCCCACCTTGAATGAAGAAGAGGAGTGGACTTGTGCCGACTTCAAGTGCAAACTTTCGCCAACCGTGGTTGGCTTGTCCAGCAAAACTCTGAGGGCCACGGCGAGTGTGAGGTCCACGGAAACTGTTGACAGTGGATACAGGTCCTTCAAGAGGAGCGATCCTGTTAATCCTTATCACATAGGTTCTGGCTATGTCTTTGCACCAGCCACCAGCGCCAACGACAGTGAGATATCCAGTGATGCGCTGACGGATGATTCCATGTCCATGACGGACAGCAGTGTAGATGGAATTCCTCCTTATCGTGTGGGCAGTAAGAAACAGCTCCAGAGAGAAATGCATCGCAGTGTGAAGGCCAATGGCCAAGTGTCTCTACCTCATTTCCCGAGAACCCACCGCCTGCCCAAGGAGATGACCCCCGTGGAACCCGCCACCTTTGCAGCTGAGCTGATCTCGAGGCTGGAAAAGCTGAAGCTGGAGTTGGAGAGCCGCCACAGCCTGGAGGAGCGCCTGCAGCAGATCCGAGAGGATGAAGAGAGAGAGGGCTCCGAGCTCACACTCAATTCGCGGGAGGGGGCGCCCACGCAGCACCCCCTCTCCCTACTGCCCTCCGGCAGCTACGAGGAAGACCCGCAGACGATACTGGACGATCACCTGTCCAGGGTCCTCAAGACCCCTGGCTGCCAGTCTCCAGGCGTAGGCCGCTATAGCCCCCGCTCCCGCTCCCCGGACCACCACCACCACCACCATTCGCAGTACCACTCCCTGCTCCCGCCCGGTGGCAAGCTGCCTCCCGCGGCCGCCTCGCCGGGCGCCTGCCCCCTCCTCGGGGGCAAAGGCTTTGTGACCAAGCAGACGACGAAGCATGTCCACCACCACTACATCCACCACCATGCCGTCCCCAAGACCAAGGAGGAGATCGAGGCGGAGGCCACGCAGCGGGTGCACTGCTTCTGCCCTGGGGGCAGCGAGTATTACTGCTACTCGAAATGCAAAAGCCACTCCAAGGCTCCGGAAACCATGCCCAGCGAGCAGTTTGGCGGCAGCAGAGGCAGTACCTTGCCCAAACGCAATGGGAAAGGCACGGAGCCGGGCCTGGCCCTGCCCGCCAGGGAAGGAGGGGCCCCCGGCGGAGCTGGGGCCCTGCAGCTTCCCCGGGAGGAAGGAGACAGGTCGCAGGATGTCTGGCAGTGGATGCTGGAGAGTGAGCGGCAGAGCAAGCCCAAGCCCCATAGTGCCCAAAGCACAAAAAAGGCCTACCCCTTGGAGTCTGCCCGCTCGTCTCCAGGCGAACGAGCCAGCCGGCACCATCTGTGGGGGGGCAACAGCGGGCACCCCCGCACCACCCCCCGTGCCCACCTGTTCACCCAGGACCCTGCGATGCCTCCCCTGACCCCACCCAACACGCTGGCTCAGCTGGAGGAGGCCTGTCGCAGGCTAGCTGAGGTGTCGAAGCCCCCAAAGCAGCGGTGCTGTGTGGCCAGTCAGCAGAGGGACAGGAATCATTCGGCCACTGTTCAGACGGGAGCCACACCCTTCTCCAATCCAAGCCTGGCTCCAGAAGATCACAAAGAGCCAAAGAAACTGGCAGGTGTCCACGCGCTCCAGGCCAGTGAGTTGGTTGTCACTTACTTTTTCTGTGGGGAAGAAATTCCATACCGGAGGATGCTGAAGGCTCAGAGCTTGACCCTGGGCCACTTTAAAGAGCAGCTCAGCAAAAAGGGAAATTATAGGTATTACTTCAAAAAAGCAAGCGATGAGTTTGCCTGTGGAGCGGTGTTTGAGGAGATCTGGGAGGATGAGACGGTGCTCCCGATGTATGAAGGCCGGATTCTGGGCAAAGTGGAGCGGATCGATTGA (SEQ ID NO. 1); the amino acid sequence of the wild-type NOTCH3 protein is as follows: MSSAMLVTCLPDPSSSFREDAPRPPVPGEEGETPPCQPGVGKGQVT KPMPVSSNTRRNEDGLGEPEGRASPDSPLTRWTKSLHSLLGDQDGAYLFRTFLEREKCVDTLDFWFACNGFRQMNLKDTKTLRVAKAIYKRYIENNSIVSKQLKPATKTYIRDGIKKQQIDSIMFDQAQTEIQSVMEENAYQMFLTSDIYLEYVRSGGENTAYMSNGGLGSLKVVCGYLPTLNEEEEWTCADFKCKLSPTVVGLSSKTLRATASVRSTETVDSGYRSFKRSDPVNPYHIGSGYVFAPATSANDSEISSDALTDDSMSMTDSSVDGIPPYRVGSKKQLQREMHRSVKANGQVSLPHFPRTHRLPKEMTPVEPATFAAELISRLEKLKLELESRHSLEERLQQIREDEEREGSELTLNSREGAPTQHPLSLLPSGSYEEDPQTILDDHLSRVLKTPGCQSPGVGRYSPRSRSPDHHHHHHSQYHSLLPPGGKLPPAAASPGACPLLGGKGFVTKQTTKHVHHHYIHHHAVPKTKEEIEAEATQRVHCFCPGGSEYYCYSKCKSHSKAPETMPSEQFGGSRGSTLPKRNGKGTEPGLALPAREGGAPGGAGALQLPREEGDRSQDVWQWMLESERQSKPKPHSAQSTKKAYPLESARSSPGERASRHHLWGGNSGHPRTTPRAHLFTQDPAMPPLTPPNTLAQLEEACRRLAEVSKPPKQRCCVASQQRDRNHSATVQTGATPFSNPSLAPEDHKEPKKLAGVHALQASELVVTYFFCGEEIPYRRMLKAQSLTLGHFKEQLSKKGNYRYYFKKASDEFACGAVFEEIWEDETVLPMYEGRILGKVERID (SEQ ID NO. 2).

TABLE 1AXIN2 Gene mutation sites

TABLE 2 Main fetal abnormal phenotype of offspring of women carrying AXIN2 Gene mutation

TABLE 3 sample information carrying AXIN2 gene mutation

Example 2 sequencing validation by Sanger method

1, DNA extraction: all sample DNAs were extracted using QIAamp DNA Blood Mini Kit (methods see instructions).

2. Primer design and PCR reaction

a) Designing a primer: with reference to the human genome reference sequence Hg19/GRCH37, mutation site-specific primers were designed, as shown in Table 4.

TABLE 4 specific primers for AXIN2-863G >

b) Reaction system: the method comprises the following specific steps: mgCl ₂ 3. Mu.L (25 mM), 3. Mu.L GC Buffer I15. Mu. L, dNTP (2.5 mM), 2. Mu.L each of the forward primer/reverse primer (10. Mu. Mol/L), 0.1. Mu.L of Promega Taq DNA polymerase, and 1. Mu. L, ddH as template DNA ₂ 0 7μL。

c) Reaction procedures are as follows: the method comprises the following specific steps: 3min at 94 ℃; 30 cycles of 94 ℃ for 30s, 59 ℃ for 30s and 72 ℃ for 100s; 5min at 72 ℃; infinity at 10 ℃.

Sanger sequencing

The PCR amplification products obtained in step 2 from the above 123 women who had born an congenital malformed fetus and 100 women who had no history of poor fertility were directly subjected to DNA sequencing (ABI 3730 DNAanalyzer) to obtain sequencing results. Based on the sequencing results, AXIN2 gene sequence alignment was performed on the samples, and the results are shown in table 2: missense mutations c.863G > C and/or c.2140C > T are present in the AXIN2 gene of 6 women who had born congenital malformed fetuses (as shown in FIG. 1), while missense mutations c.863G > C and/or c.2140C > T are not present in the AXIN2 gene of 100 women who had no history of poor birth. It can be seen that missense mutation c.863G > C and/or c.2140C > T of AXIN2 gene is a pathogenic mutation causing fetal abnormality.

Example 3 the AXIN2 Gene on the developmental mechanism of embryonic Stem cells

The inventor utilizes human embryonic stem cells (H9 cells purchased from cell banks of Shanghai national academy of sciences) to construct an AXIN2 gene interference and over-expression lentiviral vector to silence and over-express the AXIN2 gene in the embryonic stem cells (experimental groups: normal control group, negative plasmid group (transfection blank plasmid), AXIN2 gene interference group (interference sequence information of AXIN2 gene: AXIN 2-F5'-ATCCAGTCGGTGATGGAGGA-3' (SEQ ID NO. 7), AXIN 2-R5'-GTTTCCGTGGACCTCACACT-3' (SEQ ID NO. 8), target sequence: GCGATCCTGTTAATCCTTATC (SEQ ID NO. 9)) and AXIN2 gene over-expression group), and experiments are carried out in three groups respectively. The proliferation and apoptosis conditions of cells are respectively detected by applying a tetramethyltetrazolium blue (MTT) method and a flow cytometer, the changes of anti-apoptosis factors and apoptosis factors of each group of cells and the expression of key factors and downstream molecules of a Wnt signal channel are detected by using real-time fluorescent quantitative PCR (qPCR) and Western Blotting Assay (WB) technologies, and the regulation mechanism of AXIN2 gene defects on the development and differentiation of embryonic stem cells is evaluated, wherein the specific technical route is shown in figure 2. The QPCR detection result showed that, compared to the control blank vector group, the expression level of embryonic stem cell AXIN2 mRNA was significantly decreased in the AXIN2 gene interference group (LV 3-AXIN2 RNAi), and the expression level of embryonic stem cell AXIN2 mRNA was significantly increased in the AXIN2 gene overexpression group (LV 5-AXIN 2) (a in fig. 3). The growth curves were plotted from the MTT results and found that at 48 hours and 72 hours after transfection, the AXIN2 gene interference group (LV 3-AXIN2 RNAi) had an increased cell proliferation activity and the AXIN2 gene overexpression group (LV 5-AXIN 2) had a significantly decreased cell proliferation activity as compared to the negative plasmid group (L3-vector) (B in FIG. 3). The regulation effect of the AXIN2 gene on apoptosis is researched by a flow cytometer, and the result shows that compared with other groups, the AXIN2 gene overexpression group (LV 5-AXIN 2) remarkably induces the late apoptosis of embryonic stem cells (C in figure 3). The expression conditions of anti-apoptotic factors Bcl-2 and apoptotic factors cleaned caspase-3 in embryonic stem cells after the AXIN2 gene is interfered and over-expressed are detected by a western blot experiment (WB). WB results show that the expression of Bcl-2 in an AXIN2 gene interference group (LV 3-AXIN2 RNAi) is obviously enhanced, and the expression of Bcl-2 in an AXIN2 gene overexpression group (LV 5-AXIN 2) is obviously reduced. The expression of the AXIN2 gene interference group (LV 3-AXIN2 RNAi) cleared Caspase-3 was significantly inhibited, while the expression of the AXIN2 gene overexpression group (LV 5-AXIN 2) cleared Caspase-3 was significantly up-regulated (D in FIG. 3). The key composition factors of the Wnt/beta-catenin signal channel are respectively detected by using a qPCR technology and a WB technology, and the mRNA and protein expression of beta-catenin and TCF4 is obviously increased in an AXIN2 gene interference group (LV 3-AXIN2 RNAi) and is obviously reduced in an AXIN2 gene overexpression group (LV 5-AXIN 2). Meanwhile, the levels of mRNA and protein of downstream molecules c-jun, c-myc and Cyclin D1 of WNT/beta-catenin signal channel in the AXIN2 gene interference group (LV 3-AXIN2 RNAi) are obviously increased, and the levels of c-jun, c-myc, cyclin D1 mRNA and protein of the AXIN2 gene overexpression group (LV 5-AXIN 2) are obviously reduced.

Example 4 Effect of maternal AXIN2 Gene deficiency on the development of offspring embryos

This example used zebrafish of TU strain. The results of the whole in situ hybridization of the embryos show that the AXIN2 gene has obvious expression in the embryos at the 2-cell stage, the 64-cell stage, the 512-cell stage and the 75% of the outer wrapping stage (8 hpf), and the existence of maternal expression of the AXIN2 gene of the zebra fish is confirmed. The inventor successfully constructs an AXIN2 gene double-mutation zebra fish model by a CRISPR Cas9 technology. Respectively selfing the zebra fish axin2 homozygous mutant (-/-) to generate an axin 2-/-homozygous mutant genotype offspring, hybridizing the zebra fish axin2 homozygous mutant female fish (-/-) with a wild type male fish (+/+) to generate an axin2 +/-heterozygous mutant genotype offspring, respectively collecting embryos from the two groups to be an experimental group, simultaneously collecting TU wild type (+/+) embryos from the two groups to be a control group, observing different indexes according to different development periods of the zebra fish, and respectively observing the microscopic morphology of the zebra fish embryos under a stereoscopic microscope at 5 development periods of 8, 24, 48, 72, 96hpf and the like, wherein the observation positions are head, heart, trunk and tail. The development condition of the zebra fish axin 2-/-homozygous mutant lacking the expression product of the maternal axin2 gene and the existence of an axin2 +/-heterozygous mutant embryo is verified. Specifically, 6F 1 adult fishes are selected, partial tail fin tissues are cut out, genomic DNA is extracted, PCR reaction is carried out, PCR products and vectors are recombined, and then single clone sequencing is selected to confirm the genotype of the alleles of the fishes. And screening out 3F 1-generation zebra fishes carrying axin2 biallelic gene mutation. A group of embryos collected by selfing the zebra fish axin 2F 3 homozygous mutant is an experimental group, and a group of TU wild-type embryos is collected as a control group. The 16-tailed 96hpf fry of the experimental group was randomly drawn for genotype identification, and the genotype was confirmed to be axin 2-/-homozygous mutation, as shown in fig. 4. According to different indexes observed in different development stages of the zebra fish, the microscopic morphology of the zebra fish axin 2-/-homozygous mutant embryo is observed under a stereoscopic microscope at 5 development stages of 8hpf, 24hpf, 48hpf, 72hpf, 96hpf and the like. 1) At 8hpf, the homozygous mutant embryos were not significantly different from the wild type. When the embryos were developed to 24hpf (the tunica vaginalis had been torn off), the embryos of the homozygous mutant group were observed to develop with retardation compared with the embryos of the control group. In the experiment, 192 embryos are counted in the experimental group, 91 embryos are delayed in development in the 24hpf period, 101 embryos are normal, and the development delay ratio is 47.4%. Morphological observation shows that at the moment, the eye development of the zebra fish embryo of the control group is obvious, the eye development delay of the embryo of the homozygous mutant group is obvious, and the obvious lens and retina structures in the wild embryo are not seen; the control group had normal trunk development, and the homozygous mutant group had slower trunk development and had not straightened. 2) At 48hpf, 30 embryos of 30/192 homozygous mutant showed slight pericardial edema, 62 (62/192) had delayed development, the remaining 100 embryos were normal (100/192), and the control wild-type embryos developed normally. 3) By 72hpf, the experimental group of 65 homozygous mutant fish showed pericardial edema (65/192), 30 fish 30/192 had a significantly delayed development, and the remaining 97 fish were normal (97/192). 4) By 96hpf, the experimental group had 39 homozygous mutant fish exhibiting pericardial edema (39/192), 27 fish with pericardial edema (27/192) dead, 30 fish with developmental delay (15.6%, 30/192), and the remaining 96 fish with normal (50.0%, 96/192). That is, through preliminary microscopic observation of the zebra fish axin2 homozygous mutant embryo lacking the maternal axin2 gene expression product, the lack of the maternal axin2 gene has an effect on the development of the zebra fish early embryo, which is shown in the aspects of delayed development, pericardial edema of the heart, heart rate reduction and the subsequent death of a part of the larva fish with pericardial edema (fig. 5 and 6). Hybridizing the zebra fish axin2 homozygous mutant female fish (-/-) with a wild type male fish (+/-) to generate an axin2 +/-heterozygous mutant genotype offspring, wherein the morphological observation result of the zebra fish heterozygous mutant embryo development lacking a maternal axin2 gene expression product shows that 99 heterozygous mutant embryos, 97 viable embryos and the survival rate of 98 percent are counted in the experimental group in the 24hpf period; the wild type embryos of the control group account for 185 embryos in total, the survival rate is 182, and the survival rate is 98%. 1) At 24hpf, 42 embryos in the experimental group (42/97) have development delay, the development period is basically 20hpf, the development period of the control group is basically 22hpf, and the development period between the two groups is about 2hpf different; 2) At 48hpf, the experimental group and the control group have no obvious difference; 3) At 72hpf, the experimental group and the control group have no obvious difference; 4) At 96hpf, the experimental group had no significant difference from the control group. Namely, the zebra fish heterozygous mutant which lacks the expression product of the maternal axin2 gene has transient dysplasia in the early embryonic stage, and has the possibility of development delay (24 hpf) and arrhythmia (72 hpf) in the early developmental stage, which is specifically shown in the followings: when the hybrid embryo is developed to 24hpf, the development delay of the hybrid embryo lacking axin2 gene maternal expression products exists, and the development delay phenomenon of the hybrid embryo when the embryo is developed to 48hpf is recovered; at 48hpf and 72hpf, the heart rate of heterozygous embryos lacking the maternal expression product of the axin2 gene was abnormal, but by 96hpf the heart rate of the heterozygotes no longer differed significantly from the heart rate of the wild type (FIGS. 7, 8). By combining the above observation results, it is speculated that the deletion of the expression product of the maternal gene of the heterozygous mutant does not cause abnormal phenotypes such as developmental deformity similar to the emergence of the embryo of the axin2 homozygous mutant on the premise that the axin2 zygote type gene can be normally expressed. However, in the early embryonic development stage (before 24 hpf), the deletion of axin2 maternal expression products will affect the development of zebrafish embryos, resulting in a qualitative stunted phenotype and abnormal heart rate, and after 48hpf development, the morphologies of the heterozygote embryos and heart rate gradually return to normal due to the normal expression of axin2 heterozygote genes. It is further indicated that the AXIN2 protein is associated with embryonic development deformity, mutation or deletion of the AXIN2 protein may cause embryonic development deformity, and embryonic development deformity may be diagnosed by detecting the expression amount of the AXIN2 protein and/or nucleic acid encoding the AXIN2 protein (p.G288A and/or p.R714W) and/or AXIN2 gene (c.863G > C and/or c.2140C > T) having the above point mutation, and accordingly, the effect of preventing and/or treating embryonic development may be achieved by increasing the expression amount of the AXIN2 protein and/or nucleic acid encoding the AXIN2 protein, and/or restoring the AXIN2 protein (p.G288A and/or p.R714W) and/or AXIN2 gene (c.863G > C and/or c.2140C > T) having the above point mutation to wild type.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A protein having at least one of the following mutations compared to a wild-type AXIN2 protein: p.G288A and p.R714W.

2. A nucleic acid encoding the protein of claim 1.

3. (e1) The application of at least one of the components (a) to (e 3) in the preparation of products for diagnosing embryo development deformity;

(e1) A substance that detects the protein of claim 1;

(e2) A substance that detects the nucleic acid according to claim 2;

(e3) Detecting a substance of wild-type AXIN 2.

4. A method for constructing a biological model comprises the following steps: allowing the biological model to carry at least one of (b 1) to (b 4):

(b1) The protein of claim 1;

(b2) The nucleic acid of claim 2;

(b3) A reduced expression level of wild-type AXIN 2;

(b4) Activity of wild-type AXIN2 protein is reduced.

5. Use of a biological model for screening a drug, the biological model carrying at least one of (b 1) to (b 4):

(b1) The protein of claim 1;

(b2) The nucleic acid of claim 2;

(b3) A reduced expression level of wild-type AXIN 2;

(b4) The activity of the wild type AXIN2 protein is reduced.

6. (f1) The application of at least one of (a) to (f 3) in the preparation of medicines for preventing and/or treating embryo development deformity;

(f1) A substance which increases the expression level of wild-type AXIN 2;

(f2) A substance that increases the activity of a wild-type AXIN2 protein;

(f3) A substance which specifically alters the protein of claim 1 and/or the nucleic acid of claim 2.

7. A medicament, comprising: at least one of (f 1) to (f 3):

(f1) A substance which increases the expression level of wild-type AXIN 2;

(f2) A substance that increases the activity of a wild-type AXIN2 protein;

8. The medicament of claim 7, wherein:

the change in specificity is to restore the protein of claim 1 and/or the nucleic acid of claim 2 to wild type; preferably, the agent is an agent based on a gene editing method comprising at least one selected from the group consisting of single base gene editing, zinc finger nucleases, transcription activator-like effector nucleases, CRISPR/Cas9 in combination with ipscs and AAV vector technologies.

9. A product, comprising: at least one of (g 1) to (g 3):

(g1) A substance that detects the protein of claim 1;

(g2) A substance that detects the nucleic acid according to claim 2;

(g3) Detecting a substance of wild-type AXIN 2.

10. A biomaterial related to the nucleic acid according to claim 2, comprising at least one of (c 1) to (c 7):

(c1) An expression cassette comprising the nucleic acid of claim 2;

(c2) A vector comprising the nucleic acid of claim 2;

(c3) A vector comprising the expression cassette of (c 1);

(c4) A transgenic cell line comprising the nucleic acid of claim 2;

(c5) A transgenic cell line comprising the expression cassette of (c 1);

(c6) A transgenic cell line comprising the vector of (c 2);

(c7) A transgenic cell line comprising the vector of (c 3).