CN113981071A - CSF1R related gene mutation as marker for diagnosing CVM and application thereof - Google Patents

Abstract

The invention discloses CSF1R -related gene mutation as a marker for diagnosing CVM and its application. The invention finds the mutation site c. 2749_2758delGACAGGAGAG, c.2797G>T, c.2906_2909dupATCA, the in vitro and in vivo functional experiments have verified that the above mutation sites are pathogenic mutations of CVM, and the genomic DNA of the subjects is detected whether there is one or more of the above mutation sites , which can be used to assist the rapid diagnosis of CVM, and the invention lays a foundation for controlling the incidence of CVM from etiology, and realizing early diagnosis and early intervention of CVM.

Description

CSF1R-related gene mutation as a marker for diagnosis of CVM and its application

technical field

The invention belongs to the technical field of medical detection, and in particular, relates to CSF1R -related gene mutation as a marker for diagnosing CVM and its application.

Background technique

Congenital vertebral malformation (CVM) is a serious congenital abnormality of spine development and one of the main diseases causing disability in adolescents. CVM is a group of diseases mainly characterized by abnormal vertebral structure of the spine caused by abnormal development of somites during embryonic development. It can be manifested as congenital scoliosis (CS), kyphosis and other spinal deformities. This leads to serious physical problems such as neck and back pain, impaired cardiopulmonary function, decreased labor capacity, and even disability (Giampietro P F, Raggio C L, Blank R D, etal. Clinical, genetic and environmental factors associated with congenitalvertebral malformations[J]. Molecular syndromology , 2013, 4(1-2): 94-105.). Epidemiological surveys show that the incidence of CVM in neonates is between 0.5-1‰, but because some patients have no obvious symptoms or mild symptoms and are not found, therefore, the actual incidence of CVM in neonates higher. The teratogenic factors mainly exist before the peak of growth and development. When CVM patients enter the peak of spine development, the deformity usually progresses rapidly. Deformities can easily induce serious psychological problems, and orthopedic surgery is complicated and expensive, and it also brings a great burden to the family and society.

The pathogenesis of CVM is not yet fully understood. CVM is attributed to abnormal development of paraaxial mesoderm, somites or axial bones during embryonic development. The effects of sine may lead to CVM (McMaster M J. Congenital scoliosis caused by a unilateral failureof vertebral segmentation with contralateral hemivertebrae[J]. Spine, 1998,23(9): 998-1005.). Therefore, the occurrence of CVM is the result of a combination of factors, mainly due to the combined effect of genetic factors and environmental factors. Related studies have reported that CVM has a genetic predisposition. Compared with environmental factors, genetic factors are considered to be involved in the occurrence and development of CVM. occupies a dominant position, and it is currently believed that the mutation or abnormal regulation of somite formation-related genes during embryonic development is an important mechanism leading to CVM. With the rapid development of genomics and sequencing technology, studies have reported that the polymorphisms and rare variants of PAX1 , TBX6 , WNT3A , LMX1A , DLL3 and other genes are closely related to CVM; chromosomes 17p11.2, 16p11.2, 10q24.31 , 21p11, 22q11.2 and other regions copy number variation is closely related to CVM. With the deepening of research on CVM, more and more research results indicate that rare mutations may play a more important role in the occurrence and development of genetic disease CVM.

Currently, research on rare mutations associated with CVM has been slow. Aiming at the above-mentioned defects, the present invention finds the mutation sites c.2749_2758delGACAGGAGAG, c.2797G>T, c.2906_2909dupATCA on the CSF1R gene for the first time by performing exome sequencing and bioinformatics analysis on 538 CVM patients. In vivo functional experiments have verified that the above mutation sites are pathogenic mutations of CVM. Therefore, the peripheral blood of subjects can be collected and genomic DNA can be extracted to detect whether one or more of the above mutation sites exist in the CSF1R gene. That is, it can be used to assist the rapid diagnosis of CVM, and the present invention lays a foundation for controlling the incidence of CVM from etiology, and realizing early diagnosis and early intervention of CVM.

SUMMARY OF THE INVENTION

In view of the current technical deficiencies in the field, the purpose of the present invention is to provide CSF1R -related gene mutations as markers for diagnosing CVM and applications thereof, and the CSF1R -related gene mutations include c.2749_2758delGACAGGAGAG, c.2797G>T, c.2906_2909dupATCA, The in vitro and in vivo functional experiments confirmed that the above mutation site is a pathogenic mutation of CVM, indicating that the above pathogenic mutation can be used for early diagnosis of CVM.

The above-mentioned purpose of the present invention is achieved through the following technical solutions:

A first aspect of the present invention provides a gene mutation for diagnosing CVM.

Further, the gene mutation is a mutation in the CSF1R gene.

Further, the mutations are c.2749_2758delGACAGGAGAG, c.2797G>T, and c.2906_2909dupATCA.

As an optional embodiment, the mutation is any one of c.2749_2758delGACAGGAGAG, c.2797G>T, and c.2906_2909dupATCA.

As an optional embodiment, the mutation is any two of c.2749_2758delGACAGGAGAG, c.2797G>T, and c.2906_2909dupATCA.

As an alternative embodiment, the mutations are c.2749_2758delGACAGGAGAG, c.2797G>T, and c.2906_2909dupATCA.

The specific information of the mutation sites on the CSF1R gene described in the present invention are as follows:

Specific information of c.2749_2758delGACAGGAGAG: NM_005211.3: c.2749_2758delGACAGGAGAG(p.Asp917SerfsTer32);

The specific information of c.2797G>T: NM_005211.3: c.2797G>T(p.Gly933Cys);

Specific information of c.2906_2909dupATCA: c.2906_2909dupATCA (p.Phe971SerfsTer7).

A second aspect of the present invention provides a detection reagent.

Further, the detection reagent is the gene mutation detection reagent described in the first aspect of the present invention.

Further, the detection reagent includes specific amplification primers and/or specific recognition probes for the gene mutation;

Preferably, the gene mutation is c.2749_2758delGACAGGAGAG, c.2797G>T, c.2906_2909dupATCA.

Further, the sequences of the specific amplification primers of the gene mutations c.2749_2758delGACAGGAGAG, c.2797G>T, and c.2906_2909dupATCA are respectively as SEQ ID NO.1-SEQ ID NO.2, SEQ ID NO.3-SEQ ID NO 4. Shown in SEQ ID NO.5-SEQ ID NO.6.

Further, the reagents also include dNTPs, Taq enzyme, Mg ²⁺ and PCR reaction buffer.

The genetic mutation can be detected using certain methods known to those of skill in the art, including but not limited to: DNA sequencing; primer extension assays, including cellular mutation-specific nucleotide incorporation assays; and Cell mutation-specific primer extension assays (e.g., cell mutation-specific PCR, cell mutation-specific ligation chain reaction (LCR), and nick-LCR); mutation-specific oligonucleotide hybridization assays (e.g., oligonucleotides) nucleotide ligation assay); cleavage protection assay in which protection from cleavage agents is used to detect mismatched bases in nucleic acid duplexes; analysis of MutS protein binding; comparison of variant and wild-type nucleic acid molecule migration electrophoretic analysis of rates; denaturing gradient gel electrophoresis (DGGE, as described, for example, in Myers et al. (1985) Nature 313:495); analysis of RNase cleavage at mismatched base pairs; analysis of heterologous Chemical or enzymatic cleavage of double-stranded DNA; mass spectrometry (eg: MALDI-TOF-MS); genetic bit analysis (GBA); 5' nuclease assays (eg: TaqMan ^™ ); assays using molecular beacons Law.

Further, the primers or probes described in the present invention can be chemically synthesized using phosphorimide solid phase support method or other well-known methods. The nucleic acid sequence can also be modified using a number of means known in the art. Non-limiting examples of such modifications are methylation, capping, substitution with one or more analogs of natural nucleotides, and modifications between nucleotides, eg, modification of uncharged linkers (eg, methyl phosphates, phosphotriesters, phosphorimides, carbamates, etc.), or modified charged linkers (eg, phosphorothioates, phosphorodithioates, etc.).

A third aspect of the present invention provides a diagnostic product for CVM.

Further, the diagnostic product includes the detection reagent described in the second aspect of the present invention;

Preferably, the diagnostic product includes a kit, a chip, and a test strip.

Further, the kit also includes instructions for use or labels, positive controls, negative controls, buffers, auxiliary agents or solvents; the instructions or labels describe in detail how to use the kit for detection and the kit Used to diagnose CVM. In certain embodiments, the kit further comprises a washing solution. In certain embodiments, the kit further comprises reagents for performing hybridization assays, mRNA isolation or purification tools, detection tools, and positive and negative controls. In certain embodiments, the kit further comprises instructions for using the kit. In certain embodiments, provided herein are kits for detecting protein levels of one or more biomarkers. In certain embodiments, the kits comprise test strips coated with antibodies that recognize protein biomarkers, washing solutions, reagents for performing the assay, protein isolation or purification tools, detection tools, and positive and negative controls. In certain embodiments, the kit further comprises instructions for using the kit. The kits can be customized for home use, clinical use, or scientific research.

Further, the kit of the present invention can include a variety of different reagents suitable for practical use (eg, for different detection methods), and is not limited to the reagents listed in the present invention, as long as it is based on the mutation c on the CSF1R gene Detection of .2749_2758delGACAGGAGAG, c.2797G>T, and c.2906_2909dupATCA to diagnose CVM are all included within the scope of the present invention.

Further, the kit can employ, for example, test strips, membranes, chips, disks, test strips, filters, microspheres, slides, multiwell plates or optical fibers. The solid support of the kit can be, for example, plastics, silicon wafers, metals, resins, glass, membranes, particles, precipitates, gels, polymers, flakes, spheres, polysaccharides, capillaries, films, plates or slides .

Further, the preparation of the chip can adopt the conventional preparation methods of biochips known to those skilled in the art, including (but not limited to): using a solid-phase carrier of modified glass slide or silicon wafer, and the 5' end of the probe contains an amino group The modified poly-dT string, the oligonucleotide probe is prepared into a solution, and then it is spotted on the modified glass slide or silicon wafer by a spotter, arranged in a predetermined sequence or array, and then fixed by leaving it overnight. The chip of the present invention can be obtained; if the nucleic acid does not contain amino modification, its preparation method can also refer to: "Gene Diagnosis Technology-Non-radioactive Operation Manual" edited by Wang Shenwu; J.L.erisi, V.R.Iyer, P.O.BROWN. Exploring the metabolic and genetic control of gene expression on a genomic scale. Science, 1997;278:680 and Ma Liren, edited by Jiang Zhonghua. Biochip. Beijing: Chemical Industry Press, 2000, 1-130.

A fourth aspect of the present invention provides a method for constructing a CVM zebrafish model.

Further, the method includes the following steps:

(1) Cultivate zebrafish embryos;

(2) Construct a mutated CSF1R plasmid containing the mutated gene sequence;

(3) After the plasmid is linearized, transcribe to obtain mRNA;

(4) injecting the mRNA into cells from the single-cell stage to the two-cell stage of the zebrafish embryo;

Preferably, the mutant gene in step (2) is c.2749_2758delGACAGGAGAG on the CSF1R gene;

Preferably, the mRNA in step (4) is 50 pg.

Further, the zebrafish described in step (1) is preferably Tg(Ola.Sp7:nlsGFP) transgenic zebrafish.

The fifth aspect of the present invention provides the application of the gene mutation described in the first aspect of the present invention in preparing the detection reagent described in the second aspect of the present invention.

Further, the gene mutation sites are c.2749_2758delGACAGGAGAG, c.2797G>T, and c.2906_2909dupATCA.

Those skilled in the art design specific amplification primers or specific recognition probes according to the upstream and downstream sequences of the gene mutation site, and the design methods of primers and probes are conventional techniques known in the art.

The sixth aspect of the present invention provides the application of the gene mutation described in the first aspect of the present invention or the detection reagent described in the second aspect of the present invention in the preparation of a diagnostic product for CVM;

Preferably, the diagnostic product for CVM is the diagnostic product for CVM according to the third aspect of the present invention;

More preferably, the diagnostic product diagnoses whether the subject suffers from CVM by detecting the gene mutation described in the first aspect of the present invention in the sample;

Most preferably, the source of the sample is the blood of the subject.

Further, the diagnostic products include kits, chips, and test strips.

The seventh aspect of the present invention provides the application of the CVM zebrafish model prepared by the method described in the fourth aspect of the present invention in screening candidate drugs for the treatment of CVM.

Further, the method for screening a candidate drug for the treatment of CVM comprises the steps of:

(1) Apply the substance to be screened to the CVM zebrafish model;

(2) Detect the relief of CVM symptoms in zebrafish;

(3) If the CVM symptoms of zebrafish are alleviated after administration of the substance to be screened, the substance to be screened is a candidate drug for the treatment of CVM.

The gene mutation described in the present invention includes its corresponding mutant protein. Therefore, after the gene mutation site described in the claims is changed to a mutant protein, it is still included in the protection scope of the present invention. Improvements and modifications of proteins will also fall within the protection scope of the claims of the present invention.

The present invention can utilize any method known in the art to detect genes and their encoded proteins. It will be understood by those skilled in the art that the means of detecting the gene is not an important aspect of the present invention. The genes in the present invention are detected using various detection techniques known to those of ordinary skill in the art, including (but not limited to): nucleic acid sequencing, nucleic acid hybridization, nucleic acid amplification technology, and immunodetection technology.

Further, methods for detecting gene mutations or protein mutations include (but are not limited to): Taqman method, mass spectrometry, DNA microarray method, sequencing method, microsequencing, hybridization, restriction fragment analysis, oligonucleotide ligation detection, allelic Gene-specific PCR-HRM or a combination of the above methods.

Unless otherwise defined, all technical and scientific terms used in the context of the present invention have the same meaning as understood by one of ordinary skill in the art. The terms used in the specification of the present invention are only used to describe specific embodiments, and are not intended to limit the present invention. In addition, some terms are explained as follows.

The term "primer," as used in the present invention, refers to a single-stranded polynucleotide capable of hybridizing to a nucleic acid and permitting polymerization of a complementary nucleic acid (generally by providing a free 3'-OH group).

The term "probe", as used in the present invention, refers to a molecule capable of binding to a specific sequence or subsequence or other portion of another molecule. Unless otherwise indicated, a "probe" generally refers to a polynucleotide probe capable of binding to another polynucleotide by complementary base pairing. Depending on the stringency of the hybridization conditions, a probe can bind to a target polynucleotide that lacks complete sequence complementarity to the probe. Probes can be labeled, either directly or indirectly, to include primers. Hybridization means, including but not limited to: solution phase, solid phase, mixed phase or in situ hybridization assays.

The term "amplification," as used herein, refers to the process of generating one or more copies of a reference nucleic acid sequence or its complement. Amplification can be linear or exponential (eg, polymerase chain reaction (PCR)). "Copy" does not necessarily mean complete sequence complementarity or identity with respect to the template sequence. For example, copies may contain nucleotide analogs such as deoxyinosine, intentional sequence changes (such as those introduced via primers containing sequences that are hybridizable but not fully complementary to the template), and/or sequence errors that occur during amplification .

The term "exon" used in the present invention refers to the portion that is retained in the mature mRNA, that is, the portion of the mature mRNA corresponding to the gene. Introns are parts that are spliced out during mRNA processing and are not present in mature mRNAs. Exons and introns are both for genes, the coding part is exon, the non-coding part is intron, and intron has no genetic effect.

The term "diagnosis", as used herein, refers to the identification or classification of a molecular or pathological state, disease or condition. It can also refer to the classification of a particular subtype of disease, eg by molecular characteristics (eg, a subset of patients characterized by nucleotide variations in a particular gene or nucleic acid region).

The term "treatment", as used herein, refers to treatment involving humans or animals (eg, as applied by a veterinarian) in which some desired therapeutic effect is achieved, eg, inhibiting the progression of a disorder (including reducing the rate of progression, enabling development cessation), amelioration of the condition, and cure of the condition. Treatment as a preventive measure (eg, prophylaxis) is also included. Use in patients who have not yet developed the disorder but are at risk of developing the disorder is also included in the term "treatment".

Advantages and beneficial effects of the present invention:

(1) Through exome sequencing and bioinformatics analysis of 538 CVM patients, the mutation sites c.2749_2758delGACAGGAGAG, c.2797G>T, c.2906_2909dupATCA were found in the CSF1R gene for the first time. The functional experiments confirmed that the above mutation site is a pathogenic mutation of CVM.

(2) The present invention provides a method for constructing a CVM zebrafish model. The zebrafish model constructed by the method shows the vertebral body phenotype of CVM patients, which provides an important study for further research on the pathogenic mechanism of human CVM. Model.

(3) The CSF1R -related gene mutation provided by the present invention is used as a marker for diagnosing CVM, laying a foundation for controlling the incidence of CVM from etiology and realizing early diagnosis, early intervention and early treatment of CVM.

(4) The marker provided by the present invention can be used to diagnose CVM, by detecting whether the mutation site (c.2749_2758delGACAGGAGAG, c.2797G>T, c.2906_2909dupATCA exists on the CSF1R gene on the blood genomic DNA of the subject) ), it can be determined whether the subject suffers from CVM, and the diagnosis method is a non-invasive and non-invasive method, which can quickly and effectively achieve early diagnosis and strive for the earliest intervention time.

Description of drawings

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, wherein:

Figure 1 shows the mutational information and clinical phenotype of CSF1R carboxy-terminal mutation sites in 3 patients with CVM, in which, Panel A: Sanger sequencing results of rare CSF1R carboxy-terminal mutation sites, Panel B: spine plain radiographs and computed tomography ( CT) result map;

Figure 2 shows nonsense-mediated decay (NMD) prediction and distribution of rare mutation sites at the carboxy terminus of CSF1R in CVM patients, where, Panel A: NMD prediction of two CSF1R truncating variants identified in the CVM cohort, Panel B: Simplified diagram of CSF1R protein;

Figure 3 shows in vivo and in vitro functional studies of CSF1R carboxy-terminal mutation sites, where, Panel A: CSF1R protein levels in total cell lysates made from Cos-7 cells transfected with wild-type or mutant CSF1R plasmids, Panel B: Quantitative analysis of CSF1R protein expression levels in total cell lysates prepared from wild-type and mutant plasmid-transfected Cos-7 cells, Panel ^C : Tg (Ola .Sp7: nlsGFP) fluorescence imaging of zebrafish; D: Statistical results of the zebrafish in the experimental group ( CSF1R mRNA, CSF1R ^ΔC mRNA) and the control group showing the CVM phenotype, * p<0.05, **p< 0.01, ***p<0.001, ****p<0.0001.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, which are only used to explain the present invention, and should not be construed as a limitation of the present invention. Those of ordinary skill in the art can understand that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the claims and their equivalents . In the following examples, the experimental methods without specific conditions are usually detected according to conventional conditions or according to the conditions suggested by the manufacturer.

Example 1 Screening of gene mutations related to CVM

1. Sample collection

A total of 583 patients diagnosed with congenital vertebral malformation (CVM) at Peking Union Medical College Hospital (PUMCH) between 2009 and 2018 were collected and included as part of the DISCO Spinal Deformity Research Platform (Deciphering disorders involving scoliosis and comorbidities, DISCO) (http://www.disco.org/ http://www.discostudy.org/), which records detailed clinical phenotype information of patients, including relevant imaging data, such as X-ray, computed tomography (CT) and three-dimensional reconstruction of the spine, and whole-spine magnetic resonance imaging (MRI) , All subjects or their parents signed the informed consent, and this study was approved by the Ethics Committee of Peking Union Medical College Hospital.

2. Exome sequencing and data analysis

All collected CVM patients underwent exome sequencing. DNA was extracted from peripheral blood of patients and exome sequencing was performed for all subjects, Illumina paired-end libraries were prepared from DNA samples and exome capture was performed, and then sequenced on the Illumina HiSeq 4000 platform. Variants were called and filtered using an in-house developed analysis pipeline (Peking Union Medical College Pipeline, PUMP).

In this study, all rare variants in CSF1R were extracted from the exome data pool for further analysis studies.

3. Variant Interpretation and Prioritization

Variant interpretation was performed based on the Genome Aggregation Database (gnomAD, http://gnomad.broadinstitute.org/). Among them, rare variants in CSF1R were extracted and filtered according to the following criteria: (1) truncation (nonsense, frameshift, splice acceptor/donor) and minor allele frequency (MAF) ≤ 0.001; (2) ) missense variants missing from public databases in the general population.

4. Experimental results

The experimental results showed that from 3 CVM patients, 3 novel deleterious heterozygous variants in CSF1R were identified, including 2 truncating variants and 1 missense variant (see Table 1).

Subject DISCO-CSS170368. The subject, a 14-year-old girl undergoing spinal surgery, showed a right curvature of the spine with a coronal Cobb angle (T2-L1) of the main curvature of 114°. CT and 3D reconstruction of the spine showed congenital fusion of T5-T9, dysplasia T3-9, hyperplasia of the L4 hemivertebra, and abnormalities of the left 6th and 7th ribs (see Figure 1B). In addition, a whole-spine MRI was performed, which showed mild redness and syringomyelia, and the girl had no symptoms of dyspnea, numbness or weakness in the extremities, backache, or pain in the extremities as the disease progressed. The subject's clinical diagnosis was severe congenital scoliosis, pulmonary insufficiency, double syringomyelia, and syringomyelia. By exome sequencing analysis, a novel truncating variant in CSF1R (NM_005211.3: c.2749_2758delGACAGGAGAG) was identified in this subject at positions 2749 and 2758 on exon 21 of the patient's CSF1R gene 10 nucleotides were deleted in between, resulting in a frameshift mutation in CSF1R . The present patentee investigated the potential role of nonsense-mediated mRNA decay (NMD) of the NMD Esc predictor (see Figure 2A), and the results suggest that mRNAs with this mutation may escape NMD and, therefore, this frameshift mutation Abnormal protein product (p.Asp917SerfsTer32) may be produced.

Subject DISCO-CSS180319. The subject was a 10-year-old girl with confirmed CVM whose imaging studies showed a Cobb angle of 56° for the main curve (T3-T7). In addition, the structural disorder of T3-T7 was also observed in the results of CT and 3D reconstruction of the spine (see Figure 1B). A missense variant in CSF1R (NM_005211.3: c.2797G>T) was identified in this subject by exome sequencing analysis, resulting in an amino acid change (p.Gly933Cys), with glycine replaced by cystine After acid substitution, cystines tend to be oxidized and form disulfide bonds with each other, possibly affecting the formation of CSF1R homodimers and their biological functions.

Subject DISCO-CSS170278. The subject, a 16-year-old boy with thoracic and lumbar scoliosis, showed the presence of T10 hemivertebrae on plain radiographs of the subject's spine (see Figure 1B), as analyzed by exome sequencing, in this A missense variant in CSF1R (NM_005211.3:c.2906_2909dupATCA) was identified in subjects, this deleterious variant can cause a reading frame shift, i.e. the last two amino acids of CSF1R are deleted, and several additional Amino acids are added to the carboxy terminus of the protein.

In addition, the three rare CSF1R variants identified in the study cohort were all located in or near the carboxy-terminal region downstream of the PTK domain (see Figure 2B), whereas the carboxy-terminal region of CSF1R contained multiple autophosphates involved in protein metabolism chemical site.

Table 1 Detrimental and rare CSF1R mutations screened in CVM patients

Example 2 Validation of pathogenic gene mutations by Sanger sequencing

Sanger sequencing was performed on genomic DNA from all subjects to correctly confirm all the variants identified in Example 1.

1. Experimental method

PCR was used to amplify variant-encoding amplicons from genomic DNA obtained from CVM patients;

Table 2 Amplification system

Table 3 Amplification conditions

The amplification system is shown in Table 2, and the amplification conditions are shown in Table 3; the primers used for PCR amplification and Sanger sequencing are as follows:

(1) Mutation 1 (c.2749_2758delGACAGGAGAG)

Forward primer: GCCGAGCTGTTGAGTGAAAT (SEQ ID NO.1)

Reverse primer: TCTAGTGAGCACCTGACCTG (SEQ ID NO. 2)

(2) Mutation 2 (c.2797G>T)

Forward primer: TGGTACTCCCTGTCGTCAAC (SEQ ID NO.3)

Reverse primer: AGGTCTCTCTAGGGGTGTGTG (SEQ ID NO.4)

(3) Mutation 3 (c.2906_2909dupATCA)

Forward primer: TGGTACTCCCTGTCGTCAAC (SEQ ID NO.5)

Reverse primer: AGGTCTCTCTAGGGGTGTGTG (SEQ ID NO.6)

Amplification products were purified using the Axygen AP-GX-50 kit (Lot: 05915KE1). Sanger sequencing was then performed on an ABI3730XL instrument.

2. Experimental results

The experimental results are shown in Figure 1A. The Sanger sequencing results confirmed the existence of CSF1R variants c.2749_2758delGACAGGAGAG, c.2797G>T, and c.2906_2909dupATCA in the patient, confirming that the above mutations are true mutations.

Example 3 In vitro functional verification of pathogenic gene mutations

In order to further study the pathogenesis of variants of CSF1R , in this example, the pEGFP-C1 vector plasmid was used to transfect the variants of CSF1R c.2749_2758delGACAGGAGAG, c.2797G>T, and c.2906_2909dupATCA into Cos-7 cells, respectively. All plasmids were verified by sequencing, and expression of CSF1R in transfected cells was analyzed by Western blotting.

1. Plasmid construction and site-directed mutagenesis

The constructed plasmids include wild-type CSF1R plasmids and mutant CSF1R plasmids;

(1) Design primers respectively to amplify the mutant sequences of wild-type CSF1R and mutant CSF1R (c.2749_2758delGACAGGAGAG, c.2797G>T, c.2906_2909dupATCA), and add homologous recombination sequences with the vector before and after the target gene according to the principle of seamless cloning Design synthetic primers, the primers are as follows:

(2) PCR amplification

The reaction system and reaction conditions of PCR amplification are shown in Table 4 and Table 5;

Table 4 Reaction system

Table 5 Reaction conditions

(3) Agarose cutting gel to recover the synthesized PCR fragments;

(4) EcoRI/BamHI double digestion vector pCS2+, the digestion system is shown in Table 6;

Table 6 Enzyme digestion system

The digestion product was recovered after digestion at 37°C for 0.5 h;

(5) recombination reaction and conversion, the recombination reaction system is shown in Table 7;

Table 7 Recombination reaction system

a) After mixing, reconstitute at 37°C for 30 min, and immediately after the reaction, ice-bath the reconstituted reaction solution for 5 min;

b) Transformation: Add 100 μL of ToP10 competent cells to the reconstituted product, mix well, heat shock at 42°C for 60 s, ice-water bath for 120 s, and spread the transformed mixture evenly on LB plates containing ampicillin resistance Incubate overnight at 37°C.

(6) Screening and identification of positive clones

Pick a single clone for PCR identification reaction, identification primers are as follows:

CMV-F: CGCAAATGGGGCGGTAGGCGTG (SEQ ID NO. 15)

CSF1R-CX-1R: CACTGGTGTGAAGAGGAACT (SEQ ID NO. 16)

2. Cell Culture and Plasmid Transfection

Cos-7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) in 6-well plates supplemented with fetal bovine serum (Gibco), penicillin (50 U/mL) and streptomycin (50 U/mL). μg/mL). The wild-type and mutant CSF1R plasmids (pEGFP-C1 plasmid 1 μg) were transfected into the above cells, respectively, and the cells were harvested 48 hours later.

3. Western blot to verify the expression of related proteins in cells

Take part of the cells collected after 48 hours, extract the total protein and perform Western blot to verify the expression of CSF1R protein in the cells, and use the following antibody to evaluate the protein expression in the cell lysate: mouse anti-GFP monoclonal antibody (1:1000, ZSJQB Co., Ltd.) and mouse anti-GAPDH monoclonal antibody (1:1000, ZSJQB Co., Ltd.).

4. Statistical analysis

Statistical differences between the different experimental and control groups were assessed using the chi-square test, all statistical procedures were performed using GraphPad Prism8, and P≤0.05 was considered statistically significant. Among them, *, **, *** and **** represent p<0.05, p<0.01, p<0.001 and <0.0001, respectively.

5. Experimental results

The experimental results are shown in Figure 3A and Figure 3B, the results show that compared with the cells transfected with the wild-type CSF1R plasmid, the expression level of CSF1R protein in the cells transfected with the mutant CSF1R plasmid was significantly increased (c.2797G>T, p=0.00259 ; c.2906_2909dupATCA, p=0.00005; c.2749_2758delGACAGGAGAG, p=0.00004), indicating that the mutation sites c.2749_2758delGACAGGAGAG, c.2797G>T, and c.2906_2909dupATCA on the CSF1R gene can improve the stability of the CSF1R protein.

Example 4 In vivo functional verification of pathogenic gene mutations

In order to further verify the pathogenic gene mutation, this example uses transgenic zebrafish to carry out the verification experiment.

1. Breeding and breeding of zebrafish

Using Tg (Ola.Sp7:nlsGFP) transgenic zebrafish, in osteoblasts, the Sp7 promoter drives GFP expression for the construction of animal models. Zebrafish were reared at 28°C and fed with brine shrimp twice a day. Embryos were incubated in methylene blue from the cell stage of the two- to four-cell stage until 3 days after fertilization to avoid fungal infection.

2. In vitro mRNA transcription

The DNA sequence of human wild-type CSF1R was cloned into PCS2+ plasmid to construct human wild-type CSF1R plasmid. Then, an insertion variant (c.2749_2758delGACAGGAGAG) was constructed to generate a human mutant CSF1R plasmid containing the mutant gene sequence. After plasmid linearization, transcription was performed with the mMESSAGE mMACHINE™ SP6 Ultra Transcription Kit (Ambion) to obtain the corresponding mRNA.

3. Embryo injection

Wild-type CSF1R mRNA and mutated CSF1R mRNA (50 pg each) were dissolved in distilled water (2 nL each) and injected into embryos at the cell stage from one-cell to two-cell stages, respectively, with an equal amount of water. Injected into embryos as a control group. All individuals in the experimental and control groups were retained for phenotypic evaluation.

The present invention overexpressed a mutated CSF1R allele (NM_005211.3: c.2749_2758delGACAGGAGAG), which depleted all carboxy-terminal regions downstream of the PTK domain, and the carboxy-terminal region depleted of the mutated mRNA was referred to as CSF1R ^ΔC mRNA.

4. Fluorescence Imaging and Phenotypic Assessment

Fluorescence images of zebrafish larvae at 14 dpf (14 days post-fertilization) were collected by fluorescence microscopy. Vertebral morphology was observed and recorded, and phenotypic evaluation and statistical analysis were performed.

5. Statistical analysis

Statistical differences between the different experimental and control groups were assessed using the chi-square test, all statistical procedures were performed using GraphPad Prism8, and P≤0.05 was considered statistically significant. Among them, *, **, *** and **** represent p<0.05, p<0.01, p<0.001 and <0.0001, respectively.

6. Experimental results

The results showed that fluorescence images of spines were collected by fluorescence microscopy at 21 dpf (21 days after fertilization). Zebrafish in the CSF1R mRNA overexpression group exhibited vertebral deformities, including vertebral fusions, hemivertebrae, and fused pedicles (see Figure 3C), overlaying the phenotypes of human CVMs, and these vertebral-related phenotypes suggest that CSF1R expression The dose-effect of vertebral deformity may be the cause. There was a significant difference (chi-square) in the percentage of zebrafish showing the CVM phenotype between the experimental group ( CSF1R mRNA: 24%, 7/29; CSF1R ^ΔC mRNA: 52%, 13/25) and the control group (0%) test, CSF1R mRNA: p=0.00635; CSF1R ^ΔC mRNA: p=0.00002), zebrafish injected with CSF1R ^ΔC mRNA had a significantly higher percentage of the vertebral malformation phenotype compared to zebrafish injected with CSF1R mRNA at the embryonic stage (Ca. Fang test p=0.03452) (see Figure 3D), indicating that injection of human CSF1R ^ΔC mRNA resulted in a higher proportion of zebrafish vertebral deformities than wild-type mRNA, further demonstrating the pathogenicity of variants in the carboxy-terminal region of CSF1R .

Through the above-mentioned in vitro and in vivo functional verification experiments, this study determined that the mutation sites c.2749_2758delGACAGGAGAG, c.2797G>T, and c.2906_2909dupATCA on the CSF1R gene inhibit the degradation of CSF1R by interfering with the binding of ubiquitin protein ligase c-Cbl. , thereby increasing the expression level of CSF1R protein, enhancing the biological function of CSF1R , further affecting bone metabolism and vertebral development, leading to the occurrence of CVM.

The description of the above embodiment is only for understanding the method and the core idea of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can also be made to the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

sequence listing

<110> Peking Union Medical College Hospital, Chinese Academy of Medical Sciences

<120> CSF1R-related gene mutation as a marker for diagnosis of CVM and its application

<141> 2021-11-17

<150> 2021102659988

<151> 2021-03-11

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Claims (10)

1. a kind of gene mutation for diagnosing CVM, is characterized in that, described gene mutation is the mutation on CSF1R gene; The mutations are c.2749_2758delGACAGGAGAG, c.2797G>T, and c.2906_2909dupATCA on the CSF1R gene. 2 . A detection reagent, wherein the detection reagent is the detection reagent for gene mutation according to claim 1 . 3 . 3. The detection reagent according to claim 2, wherein the detection reagent comprises a specific amplification primer and/or a specific identification probe of the gene mutation; The gene mutations are c.2749_2758delGACAGGAGAG, c.2797G>T, c.2906_2909dupATCA. 4. detection reagent according to claim 3 is characterized in that, the sequence of the specific amplification primer of described gene mutation c.2749_2758delGACAGGAGAG, c.2797G>T, c.2906_2909dupATCA is respectively as SEQID NO.1-SEQ ID NO.2, SEQ ID NO.3-SEQ ID NO.4, SEQ ID NO.5-SEQ ID NO.6. 5. The detection reagent according to claim 2, wherein the reagent further comprises dNTPs, Taq enzyme, Mg ²⁺ and PCR reaction buffer. 6. A diagnostic product for CVM, wherein the diagnostic product comprises the detection reagent according to any one of claims 2-5; The diagnostic products include kits, chips, and test strips. 7. a method for building CVM zebrafish model, is characterized in that, described method comprises the steps: (1) Cultivate zebrafish embryos; (2) Construct a mutated CSF1R plasmid containing the mutated gene sequence; (3) After the plasmid is linearized, mRNA is obtained by transcription; (4) injecting the mRNA into cells from the one-cell stage to the two-cell stage of the zebrafish embryo; The mutation described in step (2) is the gene mutation described in claim 1. 8. The application of the gene mutation of claim 1 in the preparation of the detection reagent of any one of claims 2-5. 9. The gene mutation of claim 1 or the detection reagent of any one of claims 2-5 are used in the preparation of a diagnostic product for CVM; The diagnostic product of described CVM is the diagnostic product of CVM as claimed in claim 6; The diagnostic product diagnoses whether the subject suffers from CVM by detecting the gene mutation of claim 1 in the sample. 10. The application of the CVM zebrafish model prepared by the method according to claim 7 in screening candidate drugs for the treatment of CVM.

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