CN117248004A - Application of RUNX2 polypeptide mutant in preparation of kit for evaluating patient suffering from craniocaudal dysplasia - Google Patents

Application of RUNX2 polypeptide mutant in preparation of kit for evaluating patient suffering from craniocaudal dysplasia Download PDF

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CN117248004A
CN117248004A CN202310549073.5A CN202310549073A CN117248004A CN 117248004 A CN117248004 A CN 117248004A CN 202310549073 A CN202310549073 A CN 202310549073A CN 117248004 A CN117248004 A CN 117248004A
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runx2
dysplasia
craniocaudal
mutation
detecting
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陈丽
任建民
侯新国
龚蕾
刘福强
孙玉静
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Qilu Hospital of Shandong University
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Abstract

The present disclosure provides an application of a reagent for detecting a RUNX2 polypeptide mutant in a sample in preparing a kit for assessing susceptibility to craniocaudal dysplasia, wherein the polypeptide mutant is RUNX2p.w518gfs, and RUNX2p.w518gfs is located in a PST domain. The disclosure also provides an application of a reagent for detecting RUNX2 polypeptide mutant in a sample in preparation of a product for detecting single nucleotide polymorphism or genotype easily related to craniocaudal dysplasia, wherein the polypeptide mutant is RUNX2p.W518Gfs, and the RUNX2p.W518Gfs is located in a PST domain. According to the present disclosure, a kit for detecting a polypeptide mutant of craniocaulous dysplasia can be provided.

Description

Application of RUNX2 polypeptide mutant in preparation of kit for evaluating patient suffering from craniocaudal dysplasia
The application is filing date2022, 06, 20 daysThe application number is 202210697983.3 and the invention name isRUNX2 Application of gene mutation in preparation of kit for screening patients with craniocaudal dysplasiaIs a divisional application of the patent application of (2).
Technical Field
The invention relates to the technical field of molecular biology, in particular to application of RUNX2 polypeptide mutants in a detection sample in preparation of a kit for evaluating patients suffering from craniocaudal dysplasia.
Background
Bone formation and remodeling are mainly differentiation, proliferation and extracellular matrix formation of osteoprogenitor cells and chondrocyte progenitor cells, which are independent of activation and inhibition of several related genes, and transcription factors play an important regulatory role on these genes. The dynamic balance between osteoblasts and osteoclasts is the basis for ensuring normal sprouting of teeth, and if the balance is out of balance, the sprouting of teeth can be blocked. The craniocaulous dysplasia (Cleidocranial Dysplasia, CCD) is a typical tooth eruption disorder disease, belongs to autosomal dominant hereditary diseases involving teeth and bones, has high exon rate and different phenotypes, has obvious family aggregation, and has no obvious difference in incidence rate of men and women. Typical clinical symptoms of craniocerebral dysplasia include delayed closure of fontanel, widening of the suture of the skull, poor calcification of the collarbone, loss of the skull, widening of the pubic symphysis, resulting in poor pelvic development, short stature, insufficient development of the maxilla, multiple teeth, retention of deciduous teeth, delayed eruption of permanent teeth, etc.
The RUNX2 gene (RUNX family transcription factor 2), also known as core tuberculosis factor α1, polyomavirus enhancer binding protein or acute myeloid leukemia factor, is located in human autosome 6p21, approximately 220kb long. RUNX2 belongs to the RUNXx related factor (RUNt-related gene) family, which is a generic term for a class of transcription factors, which are heterodimers composed of alpha and beta subunits, and the RUNXx family mainly comprises the RUNX1 gene, the RUNX2 gene and the RUNX3 gene, with RUNX2 having RUNT domains similar to other family members. RUNX2 is the most critical transcription factor in regulating differentiation and maturation of bone marrow mesenchymal stem cells into osteoblasts during bone development, and expression of RUNX2 is a marker for the onset of differentiation of osteoblasts, so it is the earliest and most specific marker gene in bone formation, RUNX2 comprises an N-terminal polyglutamine/alanine repeat domain (Q/a), a central RUNT domain, and a C-terminal proline, serine and threonine rich PST domain. Missense mutation, nonsense mutation, gene insertion/deletion mutation or frameshift mutation of RUNX2 gene are all important causes of craniocaudal dysplasia.
Although there are many studies on the pathogenesis of the craniocaudal dysplasia and the RUNX2 gene, the screening and verification analysis of the disease pathogenic gene mutation are far from sufficient, and unknown pathogenic gene loci still exist. The identification of new pathogenic gene sites of craniocaudal dysplasia is of great significance for early diagnosis, management and guidance of prenatal and postnatal care. Thus, there is an urgent need in the art to develop new pathogenic genes for craniocaudal dysplasia and their associated pathogenic mutation sites.
Disclosure of Invention
The present disclosure has been made in view of the above-described state of the art, and an object thereof is to provide a novel craniocaudal dysplasia-related pathogenic gene mutation site.
To this end, a first aspect of the present disclosure provides the use of a reagent for detecting a RUNX2 gene mutation in a sample, said gene mutation being RUNX2c.1550delt, i.e. said gene mutation being a deletion of base T1550 th of the nucleotide sequence of the wild-type RUNX2 gene, in the preparation of a kit for screening patients with craniocaudal dysplasia. In the disclosure, through the gene mutation of RUNX2c.1550delT as a marker, a patient with craniocaudal dysplasia can be screened, and further the application of a reagent for detecting the RUNX2 gene mutation in a sample in preparing a kit for screening the patient with craniocaudal dysplasia is provided.
In the application to which the first aspect of the present disclosure relates, optionally, the kit may be applied to at least one of the following techniques: DNA sequencing, restriction enzyme fragment length polymorphism, single-stranded conformational polymorphism, denaturing high performance liquid chromatography, SNP chip, microfluidic chip technology, taqMan probe technology and Sequenom MassArray technology. Thus, the RUNX2 gene mutation site can be detected by using these techniques.
In the application to which the first aspect of the present disclosure relates, optionally, the kit includes a primer pair for amplifying the RUNX2 gene and/or a probe for detecting RUNX2c.1550 delt. Thus, the RUNX2 gene can be detected using the primer set and/or the probe.
In the application related to the first aspect of the present disclosure, optionally, the probe for detecting RUNX2c.1550delt is designed according to the sequence of 1550 th base of RUNX2 gene coding region in human genome. Thus, the mutation of RUNX2c.1550delt can be detected.
In the application to which the first aspect of the present disclosure relates, optionally, the kit further comprises dNTPs, a DNA polymerase and a PCR reaction buffer. Thus, the RUNX2 gene can be amplified.
In the application related to the first aspect of the disclosure, optionally, the kit includes a real-time fluorescent quantitative detection reagent, where the real-time fluorescent quantitative detection reagent includes a primer for real-time fluorescent quantitative detection of gene expression of the RUNX2 new mutation site, the sequence of the upstream primer includes 5'-CTCTTCCCAAAGCCAGAGTG-3', and the sequence of the downstream primer includes 5'-GCAGACAGCTCACAAAACCAG-3'.
In the application related to the first aspect of the present disclosure, optionally, the sample is at least one of peripheral blood, saliva, and tissue samples from a subject, and the RUNX2 gene mutation refers to a germ line mutation of the RUNX2 gene. Thus, by collecting a sample of the peripheral blood, saliva, or tissue of the subject, the germ line mutation of the RUNX2 gene of the subject can be detected.
The second aspect of the present disclosure provides an application of a reagent for detecting a RUNX2 gene mutation in a sample in preparing a kit for detecting susceptibility to craniocaudal dysplasia, wherein the gene mutation is a deletion of base T1550 th in a nucleotide sequence of a wild-type RUNX2 gene.
In a third aspect, the disclosure provides an application of a reagent for detecting RUNX2 gene mutation in a sample in preparing a product for detecting a single nucleotide polymorphism or genotype easily associated with craniocaudal dysplasia, wherein the gene mutation is deletion of base T1550 of a nucleotide sequence of a wild-type RUNX2 gene.
The fourth aspect of the present disclosure provides an application of a reagent for detecting a RUNX2 polypeptide mutant in a sample in preparing a kit for screening a patient suffering from craniocaudal dysplasia, wherein the polypeptide mutant is RUNX2p.w518 gfs.
According to the present disclosure, a novel craniocaudal dysplasia-related pathogenic gene mutation can be provided.
Drawings
Fig. 1 is an X-ray image of the oral cavity of a prover in accordance with an embodiment of the present disclosure.
Fig. 2 is a chest X-ray image showing a prover in accordance with an embodiment of the present disclosure.
FIG. 3 is a graph showing the peak sequencing of genes according to embodiments of the present disclosure.
Fig. 4 is a diagram illustrating RUNX2 domains according to embodiments of the present disclosure.
Fig. 5 is a schematic diagram showing structural models of a wild-type RUNX2 protein and a W518Gfs-RUNX2 protein established in accordance with an embodiment of the present disclosure.
Fig. 6 is a sequence alignment diagram illustrating RUNX2 in different species according to an embodiment of the present disclosure.
Fig. 7 is a schematic diagram showing results of detection of the expression level of RUNX2 gene of a prover according to an embodiment of the present disclosure.
Fig. 8 is a schematic diagram showing subcellular localization results of observation of RUNX2 wild-type and RUNX 2W 518Gfs mutations by an in situ immunofluorescence microscope according to an embodiment of the present disclosure.
Fig. 9 is a graph showing the results of detection of osteocalcin promoter activity induced by RUNX2p.w518gfs variation according to the examples of the present disclosure.
Detailed Description
Hereinafter, preferred embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. In the following description, the same members are denoted by the same reference numerals, and overlapping description thereof is omitted. In addition, the drawings are schematic, and the ratio of the sizes of the components to each other, the shapes of the components, and the like may be different from actual ones.
It should be noted that the terms "comprises" and "comprising," and any variations thereof, in this disclosure, such as a process, method, system, article, or apparatus that comprises or has a list of steps or elements is not necessarily limited to those steps or elements expressly listed or inherent to such process, method, article, or apparatus, but may include or have other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
In addition, headings and the like referred to in the following description of the disclosure are not intended to limit the disclosure or scope thereof, but rather are merely indicative of reading. Such subtitles are not to be understood as being used for segmenting the content of the article, nor should the content under the subtitle be limited only to the scope of the subtitle.
It will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the prior art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
As used herein, the singular forms "a", "an", "the" and "the" are intended to include the plural forms as well, unless expressly stated otherwise, as understood by those skilled in the art. It will be further understood that the terms "comprises" and/or "comprising," when used in this specification of this disclosure, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, and/or groups thereof.
To facilitate an understanding of the disclosure, the disclosure is further illustrated in the accompanying drawings and the specific examples, which are not to be construed as limiting the embodiments of the disclosure. Those skilled in the art will appreciate that the drawings are merely schematic representations of the embodiments, and that the components in the drawings are not necessarily required to practice the present disclosure.
The present disclosure relates to any one of the following applications:
application of a reagent for detecting RUNX2 gene mutation in a sample in preparing a kit for screening patients suffering from craniocaudal dysplasia;
application of a reagent for detecting RUNX2 gene mutation in a sample in preparing a kit for detecting susceptibility of craniocaudal dysplasia;
application of a reagent for detecting RUNX2 gene mutation in a sample in preparing a product for detecting single nucleotide polymorphism or genotype easily related to craniocaudal dysplasia;
use of a RUNX2 gene mutation in a test sample for the manufacture of a product for identifying or aiding in the identification of a single nucleotide polymorphism associated with craniocaudal dysplasia;
an application of a reagent for detecting RUNX2 polypeptide mutant in a sample in preparing a kit for screening patients suffering from craniocaudal dysplasia.
According to the application of the present disclosure, a pathogenic mutation site of craniocaulon dysplasia can be provided, which is helpful for assisting in screening patients with craniocaulon dysplasia so as to facilitate prenatal and postnatal care guidance, and early discovery, prevention and treatment of diseases.
In some examples, in the above-described applications to which the present disclosure relates, the RUNX2 gene may be detected by detecting at least one sample of peripheral blood, saliva, tissue samples of a subject. In some examples, the subject may be a general population, an individual suspected of having a craniocaudal dysplasia, or a high risk population of having a craniocaudal dysplasia.
In some examples, germ line mutations of the RUNX2 gene can be detected. Germ line mutations, also called germ cell mutations, are mutations carried by germ cells, such as sperm or ovum.
In some examples, the germ line mutation result of the RUNX2 gene may be obtained by extracting gDNA (genomic DNA) of a subject and detecting the genomic DNA.
In some examples, the mutation at base 1550 of the RUNX2 gene can be detected. Further, the RUNX2c.1550delt mutation may be detected. RUNX2c.1550delT, i.e.deletion of base T1550 of the nucleotide sequence of the wild-type RUNX2 gene.
In the embodiment, the gene mutation of RUNX2c.1550delT is used as a marker, so that a patient with craniocaudal dysplasia can be screened, and further the application of a reagent for detecting the RUNX2 gene mutation in a sample in preparing a kit for screening the patient with craniocaudal dysplasia is provided.
In some examples, the presence of only one RUNX2c.1550delt mutation in the RUNX2 gene in the subject can aid in diagnosing the subject as a patient with craniocaudal dysplasia. In other words, when the RUNX2c.1550delt of the subject is detected as heterozygous mutation, diagnosis of the subject as a patient suffering from craniocerebral dysplasia can be assisted. Of course, when the RUNX2c.1550delt of the subject is detected as a homozygous mutation, diagnosis of the subject as a patient suffering from craniocerebral dysplasia may be assisted.
In some examples, the mutation of the gene RUNX2c.1550delt results in a mutation of the amino acid sequence of RUNX2, which results in an amino acid mutation (which may also be referred to as an amino acid mutation or polypeptide mutant) of RUNX2p.w518fs, accordingly. Specifically, RUNX2p.w518fs means that frame shift mutation occurs at 518 th position of an amino acid sequence encoded by a wild-type RUNX2 gene, and tryptophan, arginine, proline and tyrosine at 518, 519, 520 and 521 positions of the amino acid sequence are replaced. In the embodiment, the amino acid mutation of RUNX2p.W518fs is used as a marker, so that the screening of patients suffering from craniocaudal dysplasia can be performed, and further the application of the reagent for detecting the RUNX2 amino acid mutation in the sample in preparing a kit for screening patients suffering from craniocaudal dysplasia is provided.
In some examples, mutation sites other than RUNX2c.1550delt mutation may also be detected. In some examples, the other mutations may include all pathogenic mutations and suspected pathogenic mutations currently known for craniocaudal dysplasia. Therefore, the detection of the relevant sites of the craniocaudal dysplasia is realized, and the more comprehensive screening of the craniocaudal dysplasia is facilitated.
In some examples, in the above-described applications to which the present disclosure relates, the kit may be in the form of a reagent or kit of reagents. In some examples, the kit may further comprise a system of instruments. For example, in some examples, the kit may be a system consisting of primers and a DNA sequencer; a system consisting of a PCR reagent, a DNA sequencing reagent and a DNA sequencer; the system consists of a TaqMan probe, a PCR primer pair, a quantitative PCR instrument and a module for genotyping, and other reagents required by the TaqMan probe technology; a system consisting of probes, PCR primer pairs, and other reagents and instrumentation required for the Ligase Detection Reaction (LDR); a system consisting of PCR primer pairs, single base extension primers, chips, PCR instruments, modules for genotyping and/or other reagents and instruments required by the Sequenom MassArray technique.
In one embodiment of the present disclosure, a Taqman (Thermo Fisher) genotyping platform may be used to perform a genotyping assay on RUNX2 c.155delt. The DNA template containing the RUNX2c.1550 site region was amplified by PCR technique and then detected using a probe.
In some examples, the kit may include a primer pair for amplifying the RUNX2 gene and/or a probe for detecting RUNX2c.1550 delt. Thus, the gene mutation of RUNX2c.1550delT can be detected.
In some examples, the primer pair may be a primer or primer pair for capturing/amplifying a human genomic DNA fragment including the region of RUNX2 c.1550. In some examples, there may be no particular requirement in the sequence of the primer or primer pair as long as a genomic DNA fragment including runx2c.1550 can be amplified.
In some examples, the probe may refer to a probe for detecting RUNX2c.1550 delt. In some examples, the probe may be designed based on the nucleotide sequence upstream and downstream of RUNX2c.1550 in the human genome, the sequence of the probe covering the nucleotides of RUNX2c.1550delt in the human genome.
In some examples, the kit further comprises dNTPs (deoxyribonucleoside triphosphates), a DNA polymerase, and a PCR reaction buffer. Thus, the RUNX2 gene fragment can be amplified.
In some examples, a primer pair may be used to PCR amplify a genomic DNA fragment including the region of RUNX2c.1550 to yield a PCR amplified product. Thus, the 1550 th base of the coding region of the RUNX2 gene can be captured and enriched. In some examples, the sequence of the resulting PCR amplification product may be detected using a probe for detecting RUNX2c.1550delT using the resulting PCR amplification product as a template. Thus, it was possible to determine whether or not there was a deletion mutation of RUNX2c.1550delT at 1550 th base of the coding region of the RUNX2 gene.
In some examples, the kit may be applied to at least one of the following techniques: DNA sequencing, restriction enzyme fragment length polymorphism, single-stranded conformational polymorphism, denaturing high performance liquid chromatography, SNP chip, microfluidic chip technology, taqMan probe technology and Sequenom MassArray technology. In other words, the RUNX2 gene mutation in the sample may be detected using at least one of the following techniques: DNA sequencing, restriction enzyme fragment length polymorphism, single-stranded conformational polymorphism, denaturing high performance liquid chromatography, SNP chip, microfluidic chip technology, taqMan probe technology and Sequenom MassArray technology. Thus, the RUNX2 gene mutation can be detected using these techniques.
In some examples, the kit may include any of the following components:
(a) Reagents and/or instrumentation required to determine the polymorphism or genotype of RUNX2c.1550delt using Sequenom MassArray technology, comprising at least one of the following components: PCR primer pairs, extension primers based on single base extension reactions, phosphatases, resins, chips, MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight), and/or other reagents and instrumentation required for Sequenom MassArray techniques.
(b) Reagents and/or instrumentation required for determining the polymorphism or genotype of RUNX2c.1550delt using SNP chip technology, comprising at least one of the following components: a chip based on a nucleic acid hybridization reaction, a chip based on a single base extension reaction, a chip based on an allele-specific primer extension reaction, a chip based on a "one-step" reaction, a chip based on a primer ligation reaction, a chip based on a restriction enzyme reaction, a chip based on a protein DNA binding reaction, and/or a chip based on a fluorescent molecule DNA binding reaction.
(c) Reagents and/or instrumentation required for determining the polymorphism or genotype of RUNX2c.1550delt using microfluidic chip technology, comprising at least one of the following components: a DNA extraction microfluidic module and reagent, a DNA amplification module and PCR primer pair, a nucleic acid marking module and related reagent, a SNP chip and related hybridization, elution and scanning microfluidic module and reagent.
(d) And (3) a real-time fluorescent quantitative detection reagent. In some examples, the real-time fluorescent quantitative detection reagent may include primers for real-time fluorescent quantitative detection of RUNX2c.1550delt, including an upstream primer and a downstream primer.
In some examples, the upstream primer for real-time fluorescent quantitative detection of RUNX2c.1550delt may be any one of the single stranded DNA as follows a1 to a 4:
a1)5’-CTCTTCCCAAAGCCAGAGTG-3’；
a2 Single-stranded DNA obtained by adding one or more nucleotides to the 5 '-end and/or the 3' -end of a 1;
a3 Single-stranded DNA having 85% or more identity with the single-stranded DNA defined in a1 or a 2;
a4 Single-stranded DNA hybridized with the single-stranded DNA defined in a1 or a2 under stringent conditions;
in some examples, the downstream primer for real-time fluorescent quantitative detection of RUNX2c.1550delt may be any one of the single stranded DNA of b1 to b4 as follows:
b1)：5’-GCAGACAGCTCACAAAACCAG-3’。
b2 Single-stranded DNA obtained by adding one or more nucleotides to the 5 '-end and/or the 3' -end of b 1;
b3 Single-stranded DNA having an identity of 85% or more to the single-stranded DNA defined in b1 or b 2;
b4 Single-stranded DNA hybridized with the single-stranded DNA defined in b1 or b2 under stringent conditions.
The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. In this embodiment, "identity" refers to a nucleotide sequence comprising 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence shown as the sequence of a1 or the sequence of b1 of the present disclosure. Identity may be assessed manually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to evaluate the identity between related sequences.
In this embodiment, stringent conditions mean hybridization and washing of the membrane 2 times in a solution of 2 XSSC (sodium citrate), 0.1% SDS (sodium dodecyl sulfate) at 68℃for 5min each; the membrane was hybridized and washed 2 times in 0.5 XSSC, 0.1% SDS solution at 68℃for 15min each. Alternatively, hybridization and washing were performed at 65℃in a solution of 0.1 XSSPE (saline sodium phosphate EDTA, physiological saline sodium phosphate EDTA) and 0.1% SDS. Alternatively, hybridization and washing of the membrane were performed in a solution of 0.1 XSSC, 0.1% SDS at 65 ℃.
In some examples, the identity above 85% may be, for example, 85%, 90%, or 95% identity, etc.
In some examples, in practice, the reagent component for detecting RUNX2c.1550delt mutation may also be used in combination with other components (e.g., the reagent component may be a component for detecting other mutations in genes associated with craniocaulous dysplasia) to prepare a kit for screening for craniocaulous dysplasia patients.
In some examples, in the above-described applications to which the present disclosure relates, the RUNX2 polypeptide mutant may be RUNX2p.w518 gfs. Wherein RUNX2p.W518Gfs refers to substitution of tryptophan, arginine, proline and tyrosine at positions 518, 519, 520 and 521 of the amino acid sequence encoded by the wild-type RUNX2 gene.
In some examples, the detection method used to detect RUNX2 polypeptide mutants in a sample may include at least one of: sequence analysis techniques for proteins and peptides, including chemical methods of N-terminal sequencing Edman, C-terminal enzymatic hydrolysis, C-terminal chemical degradation, and the like; protein detection techniques associated with mass spectrometry, such as matrix assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS) and electrospray ionization mass spectrometry (electro-spectral-ization mass spectrometry, ESI-MS); antibody-based detection methods, such as preparing antibodies that recognize different mutants, detect protein variations using immunoblotting (e.g., western blot) and/or enzyme-linked immunosorbent assay (ELISA). Thus, the RUNX2 polypeptide mutant can be detected.
The use of the reagents for detecting a mutation in the RUNX2 gene in a sample provided by the present disclosure in the preparation of a kit for screening for patients with craniocaudal dysplasia is described in detail below in connection with the examples, but they should not be construed as limiting the scope of the present disclosure.
Examples (example)
Clinical cases:
patient (first person): normal at birth, without obvious clinical symptoms. It was found to grow slower than the same age at age 2. Deciduous teeth fall off at 12 years old and 3 teeth are replaced by 15 years old. Patients were clinically diagnosed with craniocaudal dysplasia at age 15 (Cleidocranial Dysplasia, CCD). Clinical manifestations: has the advantages of good response and normal intelligence. In the physical examination of the patient, typical characteristics of craniocaudal dysplasia such as forehead midline depression, shoulder drop, short stature were observed.
Fig. 1 is an X-ray image of an oral cavity showing a prover according to an embodiment of the present disclosure, and fig. 2 is a chest X-ray image showing a prover according to an embodiment of the present disclosure. The first person underwent X-ray examination of the oral cavity, and as shown in FIG. 1, the mixed dentition and deciduous teeth were shown to be retained and blocked. Chest X-ray examination was performed on the first-person, and as shown in FIG. 2, right two-thirds of the lateral clavicle maldevelopment, left one-third of the lateral clavicle maldevelopment, conical chest, scoliosis, rib deformity was observed.
Patient father, was not found to have clinical manifestations of craniocaulous dysplasia.
The mother of the patient was not found to have a clinical manifestation of craniocaudal dysplasia.
Other members of the family were not found to have clinical manifestations of craniocaulous dysplasia.
Sample detection:
craniocaulous dysplasia is an autosomal dominant inherited disease involving bones and teeth. The venous blood samples of the precursor and its father and mother were collected and the white blood cell gDNA (genomic DNA) of the three venous blood samples were subjected to Whole Exon Sequencing (WES). Specifically, firstly extracting genome DNA fragments in venous blood samples of a prover, father and mother, then capturing a 50bp region in exons of all genes in the genome DNA and adjacent introns thereof by using a whole exon detection reagent, amplifying and purifying after capturing, sequencing by using an Illumina Hiseq sequencing platform, and finally analyzing sequencing data. The above detection and analysis are provided by Beijing Fujun Gene biotechnology Co.
The sequencing results are shown in FIG. 3, FIG. 3 is a graph showing the peak of the sequencing of the gene according to the embodiment of the present disclosure, wherein (a) of FIG. 3 is a partial sequence diagram of the prior RUNX2 gene, and the c.1550delT mutation (GenBank accession number: NM-001024630) is shown by using an arrow; FIG. 3 (b) is a partial sequence diagram of the parent RUNX2 gene, with arrows showing the normal unmutated state at c.1550; FIG. 3 (c) is a partial sequence diagram of the maternal RUNX2 gene, with arrows showing the normal unmutated state at c.1550. As shown in FIG. 3, the detection of the RUNX2 (c.1550 delT) mutant by the proband, none of the parents, suggests that the patient detected RUNX2 (c.1550 delT) mutant may be a clinically significant new mutation.
Functional analysis:
(1) Functional impact prediction:
RUNX2c.1550delt is a frameshift deletion mutation which results in a change in amino acid synthesis of RUNX2 from amino acid position 518 (RUNX 2p.trp518 glyfs) and thus, the mutation may have an effect on the function of RUNX2 protein.
In this example, the structural conformations of WT-RUNX2 (wild-type RUNX2 polypeptide) and W518Gfs-RUNX2 (RUNX 2 polypeptide carrying the p.W518Gfs mutation) were analyzed and predicted comprehensively using phyr 2 (http:// www.sbg.bio.ic.ac.uk/phyre2/html/page. Cgiid=index), and FIG. 4 is a view showing the RUNX2 domains according to the examples of the present disclosure, as shown in FIG. 4, with the RUNX2c.1550delT mutation aggregating at the VWRPY end of the highly conserved PST domain, and thus the mutation may affect the protein function of RUNX 2.
RUNX2 frameshift mutation analysis: and comprehensively analyzing and predicting the structure configurations of the WT-RUNX2 and the W518Gfs-RUNX2 by adopting a threading method and a heavy head prediction method respectively, and establishing a model of the WT-RUNX2 and the W518Gfs-RUNX 2.
In addition, SAVE5.0 3D structure viewers (https:// SAVEs. Mbi. Ucla. Edu /) were also used to visualize 3D structures, and FIG. 5 is a schematic diagram showing a structural model of the established wild-type RUNX2 protein and W518Gfs-RUNX2 protein according to embodiments of the present disclosure. It is predicted that mutation at this site may change the structure of the protein, as shown in fig. 5, thereby affecting the protein activity of RUNX 2.
Fig. 6 is a sequence alignment diagram illustrating RUNX2 in different species according to an embodiment of the present disclosure. As shown in FIG. 6, the 518 th amino acid of RUNX2 has cross species conservation.
(2) Functional impact verification:
cell culture and transfection: human embryonic kidney 293 (HEK 293) cells were cultured in DMEM medium (Gbico, C11995500 BT) supplemented with 10% fetal bovine serum (FBS, gbico, 10099141), penicillin (100 IU/mL) and streptomycin (100. Mu.g/mL). Plasmid carrying the desired gene was transfected into cells with Lipofectamine 2000 (Invitrogen corporation) to investigate protein expression and other related studies.
Western blot analysis: expression of WT-RUNX2 and W518Gfs-RUNX2 was studied using human embryonic kidney 293 (HEK 293) cells. In Western blotting analysis, cells were seeded at 10cm 2 Is arranged on the flat plate of the die. After 1 day, pcDNA3.1-GFP, pcDNA3.1-wt-RU was usedHEK293 cells were transfected with NX2-GFP and pcDNA3.1W 518Gfs-RUNX2-GFP, wherein GFP was a green fluorescent protein (Green fluorescent protein). After 48 hours of transfection, the cells were cultured and subjected to SDS-PAGE (polyacrylamide gel electrophoresis) detection. Western blotting uses rabbit anti-GFP polyclonal antibody (1:1000, proteontech, 50430-2-AP) and horseradish peroxidase labeled goat anti-rabbit IgG polyclonal antibody (1:1000, hong Shanjin bridge, ZB-2301) as secondary antibodies. Chemiluminescent kits (Millipore, CA, USA, WBKLS 0050) were used as detection signals. FIG. 7 is a graph showing results of detection of the expression level of RUNX2 gene of a prover according to an embodiment of the present disclosure, wherein (a) in FIG. 7 is the expression level of wild-type RUNX2 and mutant RUNX 2; fig. 7 (b) is a histogram analysis of RUNX2 expression levels. As shown in FIG. 7, the gene mutation of RUNX2c.1550delT did not result in a decrease in the expression level of RUNX 2.
To verify the nuclear localization of the RUNX2 mutation, pGFP-RUNX2-WT and pGFP-RUNX2-W518Gfs plasmids were transfected into HEK293T cells. In situ immunofluorescence microscopy observed subcellular localization of wild-type RUNX2 and RUNX2p.w518gfs mutations. Fig. 8 is a schematic diagram showing subcellular localization results of RUNX2 wild-type and RUNX2p.w518gfs mutations observed by an in situ immunofluorescence microscope according to the examples of the present disclosure, as shown in fig. 8, both RUNX2p.w518gfs mutations and RUNX2 wild-type accumulated in nuclei of HEK293T cells, indicating that subcellular separation of RUNX2p.w518gfs mutations was not affected.
Luciferase reporter assay: HEK-293 cells were inoculated into 96-well plates, 10000/well, 200ul of cell suspension was added, and the mixture was incubated at 37℃with 5% CO 2 Culturing. On day 2, pRL-TK and pgl 3-basic-osteocalcin promoter plasmids were added to each group and different concentrations (0 ug, 1ug, 2 ug) of pcDNA3.1, pcDNA3.1-wt-RUNX2, pcDNA3.1-W518Gfs-RUNX2 were transfected, respectively. After 48h, a double luciferase assay was performed. Specifically, a dual luciferase reporter gene detection system was employed and the results were analyzed. FIG. 9 is a schematic diagram showing results of detection of activity of osteocalcin promoter induced by variation of RUNX2p.W518Gfs according to examples of the present disclosure, and as shown in FIG. 9, luciferase assay shows transactivation activity of RUNX2 and transcriptional regulation of osteocalcin promoterAs shown in FIG. 9, the variation of RUNX2p.W518Gfs induced osteocalcin promoter activity was lower than that of RUNX2-wt.
Unless otherwise indicated, all reagents and equipment used in this example were commercially available.
The above results show that the RUNX2c.1550delt mutation or RUNX2p.w518gfs mutation impairs the function of RUNX2 protein. Thus, the RUNX2c.1550delt mutation is a pathogenic mutation of the craniocaudal dysplasia family in this example. This example shows that runx2c.1550delt is a novel causative gene for craniocaudal dysplasia and is inherited autosomally dominant.
In addition, genetic counseling is performed on the family of patients with the craniocaudal dysplasia found in the embodiment of the disclosure, which is beneficial to prenatal and postnatal care.
The skull and the collarbone dysplasia has different phenotypes, the light disease only has abnormal teeth, the clinical diagnosis is difficult, and the gene diagnosis can be carried out through gene detection. According to embodiments of the present disclosure, a novel craniocaudal dysplasia-related pathogenic gene mutation site can be provided. Therefore, the method is beneficial to enriching the pathogenic mutation library of the craniocaudal dysplasia, thereby being beneficial to the genetic diagnosis of the craniocaudal dysplasia. Is helpful for assisting in screening patients with craniocaudal dysplasia so as to conduct prenatal and postnatal care guidance, and early discovery, prevention and treatment of diseases.
While the foregoing description of the embodiments of the present disclosure has been presented in conjunction with the drawings, it should be understood that it is not intended to limit the scope of the disclosure, but rather to cover all modifications or variations within the scope of the present disclosure, as would be apparent to one skilled in the art without undue effort based on the technical solutions of the present disclosure.
The foregoing description of the preferred embodiments of the present disclosure is provided only and not intended to limit the disclosure so that various modifications and changes may be made to the present disclosure by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present disclosure should be included in the protection scope of the present disclosure.
While the disclosure has been described in detail in connection with the drawings and embodiments, it should be understood that the foregoing description is not intended to limit the disclosure in any way. Modifications and variations of the present disclosure may be made as desired by those skilled in the art without departing from the true spirit and scope of the disclosure, and such modifications and variations fall within the scope of the disclosure.

Claims (10)

1. The application of a reagent for detecting RUNX2 polypeptide mutant in a sample in preparing a kit for evaluating susceptibility of craniocaudal dysplasia is characterized in that the polypeptide mutant is RUNX2p.W518Gfs and RUNX2p.W518Gfs is positioned in a PST domain.
2. The use according to claim 1, wherein,
the sample is from a subject, the subject being a general population, an individual suspected of having a craniocaudal dysplasia, or a high risk population of having a craniocaudal dysplasia.
3. The use according to claim 2, wherein,
the polypeptide mutant is caused by RUNX2c.1550delT mutation, so that the 518 th, 519 th, 520 th and 521 th tryptophan, arginine, proline and tyrosine of the amino acid sequence coded by the RUNX2 gene are replaced.
4. The use according to claim 3, wherein,
the kit obtains the detection result of RUNX2c.1550delT mutation by extracting the genomic DNA of the detected person and detecting the genomic DNA.
5. The use according to claim 3 or 4, characterized in that,
at least one RUNX2c.1550delT mutation exists in the RUNX2 gene of the patient with the craniocaudal dysplasia, and the RUNX2c.1550delT mutation is deleted in a base T at 1550 th position of a nucleotide sequence of the wild-type RUNX2 gene.
6. The use according to claim 1, wherein,
RUNX2p.w518gfs is located at VWRPY at the end of the PST domain.
7. The use according to claim 1, wherein,
the reagent includes a reagent for detecting the polypeptide mutant by at least one of a sequence analysis technique of proteins and peptide fragments, a mass spectrometry-related protein detection technique, and an antibody-based detection method.
8. The use according to claim 7, wherein,
the sequence analysis technology of the protein and peptide fragment comprises a chemical method Edman method for N-terminal sequence determination, a C-terminal enzymolysis method and a C-terminal chemical degradation method;
the mass spectrum related protein detection technology comprises a matrix-assisted laser desorption ionization/time-of-flight mass spectrometry and an electron spray ionization mass spectrometry;
the detection method based on the antibody comprises an antibody method for preparing different mutants, an immunoblotting method and an enzyme-linked immunosorbent assay protein variation detection method.
9. The use according to claim 1, wherein,
the kit also comprises a system consisting of an instrument for detecting the polypeptide mutant.
10. Application of a reagent for detecting RUNX2 polypeptide mutant in a sample in preparation of a product for detecting single nucleotide polymorphism or genotype easily related to craniocaudal dysplasia, wherein the polypeptide mutant is RUNX2p.W518Gfs, and RUNX2p.W518Gfs is located in a PST domain.
CN202310549073.5A 2021-11-23 2022-06-20 Application of RUNX2 polypeptide mutant in preparation of kit for evaluating patient suffering from craniocaudal dysplasia Pending CN117248004A (en)

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