CN115094065A - Application of RUNX2 gene mutation in preparation of kit for screening cranioclavicular dysplasia patients - Google Patents

Application of RUNX2 gene mutation in preparation of kit for screening cranioclavicular dysplasia patients Download PDF

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CN115094065A
CN115094065A CN202210697983.3A CN202210697983A CN115094065A CN 115094065 A CN115094065 A CN 115094065A CN 202210697983 A CN202210697983 A CN 202210697983A CN 115094065 A CN115094065 A CN 115094065A
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CN115094065B (en
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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 RUNX2 gene mutation in a sample in preparation of a kit for screening a patient with cranial clavicle dysplasia, wherein the gene mutation is RUNX2c.1550delT. The disclosure also provides an application of a reagent for detecting RUNX2 gene mutation in a sample in preparing a kit for detecting susceptibility of cranioclavicular dysplasia, an 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 cranioclavicular dysplasia, and an application of a reagent for detecting RUNX2 polypeptide mutant in a sample in preparing a kit for screening patients with cranioclavicular dysplasia. According to the present disclosure, a new cranium clavicle dysplasia related pathogenic gene mutation site can be provided.

Description

Application of RUNX2 gene mutation in preparation of kit for screening cranioclavicular dysplasia patients
Technical Field
The disclosure relates to the technical field of molecular biology, in particular to application of a reagent for detecting RUNX2 gene mutation in a sample in preparation of a kit for screening patients with cranial clavicle dysplasia.
Background
The formation and remodeling of bone is mainly the differentiation, proliferation and extracellular matrix formation of osteoprogenitor cells and chondroprogenitor cells, and these processes are independent of the activation and inhibition of a number of related genes, which are important to be regulated by transcription factors. The dynamic balance between osteoblasts and osteoclasts is the basis for ensuring normal eruption of teeth, and imbalance of the balance may cause eruption failure of teeth. Skull clavicle Dysplasia (Cleidocranial dyslasia, CCD) is a typical tooth eruption disorder disease, belongs to an autosomal dominant hereditary disease involving teeth and bones, has high penetrance and different phenotypes, has obvious familial aggregative properties, and has no obvious difference in male and female morbidity. Typical clinical symptoms of cranioclavicular dysplasia include delayed fontanel closure, widened skull suture, calcified and lacked clavicle, short and small pelvis development caused by combined widening of cone breast and pubic bone, insufficient development of maxilla, more raw teeth, retained deciduous teeth, delayed eruption of permanent teeth and the like.
The RUNX2 gene (RUNX family transcription factor 2), also known as core tuberculosis factor α 1, polyoma virus enhancer binding protein or acute myeloid leukemia factor, is located in human autosome 6p21, approximately 220kb long. RUNX2 belongs to the RUNX-related factor (RUNt-related gene) family, which is a generic name for a class of transcription factors, all of which are heterodimers composed of alpha and beta subunits, the RUNX family mainly includes the RUNX1 gene, the RUNX2 gene and the RUNX3 gene, and RUNX2 has a similar RUNt domain to other family members. RUNX2 is the most critical transcription factor for regulating differentiation and maturation of bone marrow mesenchymal stem cells into osteoblasts during bone development, the expression of RUNX2 is the marker of osteoblasts to begin differentiation, so that it is the earliest and most specific marker gene during bone formation, and RUNX2 comprises a polyglutamine/alanine repeat domain (Q/A) at the N-terminal, a RUNT domain in the middle, and a PST domain rich in proline, serine and threonine at the C-terminal. Missense mutation, nonsense mutation, gene insertion/deletion mutation or frameshift mutation of RUNX2 gene are all important causes of cranial clavicle dysplasia.
Although a lot of researches on the pathogenesis of the cranioclavicular dysplasia and the RUNX2 gene exist, the screening, verification and analysis work of the mutation of the pathogenesis gene of the disease is far insufficient, and an unknown pathogenesis gene locus still exists. The identification of the new pathogenic gene locus of the cranial clavicle dysplasia has important significance for early diagnosis and management of diseases and guidance of prenatal and postnatal care. Therefore, there is an urgent need in the art to develop new pathogenic genes of skull clavicle dysplasia and their associated pathogenic mutation sites.
Disclosure of Invention
The present disclosure has been made in view of the above-mentioned state of the art, and an object thereof is to provide a novel site of mutation of a causative gene associated with cranial clavicle dysplasia.
Therefore, the first aspect of the present disclosure provides an application of a reagent for detecting a mutation of a RUNX2 gene in a sample in preparing a kit for screening a patient with cranioclavicular dysplasia, wherein the gene mutation is RUNX2c.1550delt, that is, the gene mutation is deletion of 1550 th base T of a nucleotide sequence of a wild-type RUNX2 gene. In the disclosure, a gene mutation RUNX2c.1550delT is used as a marker to screen a patient with cranioclavicular dysplasia, and further provides an application of a reagent for detecting the RUNX2 gene mutation in a sample in preparation of a kit for screening the patient with cranioclavicular dysplasia.
In the use according to the first aspect of the present disclosure, optionally, the kit may be applied to at least one of the following technologies: DNA sequencing, restriction enzyme fragment length polymorphism, single-strand conformation polymorphism, denaturing high performance liquid chromatography, SNP chip, microfluidic chip technology, TaqMan probe technology and Sequenom MassArray technology. Thus, the mutation site of RUNX2 gene can be detected by using these techniques.
In an application related to the first aspect of the present disclosure, optionally, the kit includes a primer pair for amplifying the RUNX2 gene and/or a probe for detecting RUNX2 c.1550delt. Thus, the primer set and/or the probe can be used to detect the RUNX2 gene.
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 the coding region of the RUNX2 gene in the human genome. This enables detection of a mutation, RUNX2 c.1550delT.
In the application related to the first aspect of the present disclosure, optionally, the kit further comprises dNTPs, a DNA polymerase and a PCR reaction buffer. This enables amplification of the RUNX2 gene.
In the application related to the first aspect of the present disclosure, optionally, the kit includes a real-time fluorescent quantitative detection reagent, the real-time fluorescent quantitative detection reagent includes a primer for real-time fluorescent quantitative detection of gene expression of a new mutation site of RUNX2, 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 derived from at least one of peripheral blood, saliva and tissue sample of the subject, and the RUNX2 gene mutation is a germline mutation of RUNX2 gene. Thus, by collecting a sample of peripheral blood, saliva or tissue from a subject, the germline mutation in 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 mutation of a RUNX2 gene in a sample in preparing a kit for detecting susceptibility to cranioclavicular dysplasia, wherein the gene mutation is deletion of 1550 th base T of a nucleotide sequence of a wild RUNX2 gene.
The third aspect of the present disclosure provides a use of a reagent for detecting a mutation of a RUNX2 gene in a sample in the preparation of a product for detecting a single nucleotide polymorphism or a genotype easily associated with cranioclavicular dysplasia, wherein the gene mutation is a deletion of the 1550 th base T of a nucleotide sequence of a wild-type RUNX2 gene.
The fourth aspect of the disclosure provides an application of a reagent for detecting a RUNX2 polypeptide mutant in a sample in preparation of a kit for screening patients with cranial clavicle dysplasia, wherein the polypeptide mutant is RUNX2 p.W518Gfs.
According to the present disclosure, a novel mutation of a pathogenic gene associated with cranial clavicle dysplasia can be provided.
Drawings
Fig. 1 is an oral X-ray image showing a proband according to an embodiment of the present disclosure.
Fig. 2 is a chest X-ray image showing a proband according to an embodiment of the present disclosure.
FIG. 3 is a diagram showing a gene sequencing peak according to an embodiment of the present disclosure.
Fig. 4 is a diagram illustrating the RUNX2 domain according to an embodiment of the present disclosure.
FIG. 5 is a schematic diagram showing the structural models of the established wild-type RUNX2 protein and W518Gfs-RUNX2 protein to which the disclosed embodiments relate.
Figure 6 is a diagram showing the alignment of RUNX2 in different species according to the embodiments of the present disclosure.
Fig. 7 is a diagram illustrating the results of detecting the expression level of RUNX2 gene of proband according to the example of the present disclosure.
Fig. 8 is a schematic diagram showing the result of subcellular localization of RUNX2 wild type and RUNX 2W 518Gfs mutation observed by in situ immunofluorescence microscopy according to an embodiment of the present disclosure.
FIG. 9 is a schematic diagram showing the detection result of osteocalcin promoter activity induced by RUNX2 p.W518Gfs variation according to the embodiment of the 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 components are denoted by the same reference numerals, and redundant description thereof is omitted. The drawings are schematic and the ratio of the dimensions of the components and the shapes of the components may be different from the actual ones.
It is noted that the terms "comprises," "comprising," and "having," and any variations thereof, in this disclosure, for example, 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, but may include or have other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
In addition, the headings and the like referred to in the following description of the present disclosure are not intended to limit the content or scope of the present disclosure, but merely serve as a reminder for reading. Such a subtitle should neither be understood as a content for segmenting an article, nor should the content under the subtitle be limited to only 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 the context clearly indicates otherwise. It will be further understood that the terms "comprises" and/or "comprising," when used in this specification, 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, components, and/or groups thereof.
For the purpose of promoting an understanding of the disclosure, the disclosure is further explained in conjunction with the drawings and the detailed description, and the drawings or the detailed description are not to be construed as limiting the embodiments of the disclosure. It should be understood by those skilled in the art that the drawings are merely schematic representations of embodiments and that the elements shown in the drawings are not necessarily required to practice the present disclosure.
The present disclosure relates to any one of the following applications:
the application of a reagent for detecting RUNX2 gene mutation in a sample in preparing a kit for screening patients with cranial clavicle dysplasia;
the application of a reagent for detecting RUNX2 gene mutation in a sample in preparing a kit for detecting susceptibility of skull clavicle dysplasia;
the 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 skull clavicle dysplasia;
the application of RUNX2 gene mutation in a detection sample in preparing a product for identifying or assisting in identifying single nucleotide polymorphism related to cranial clavicle dysplasia;
application of a reagent for detecting RUNX2 polypeptide mutant in a sample in preparation of a kit for screening patients with cranial clavicle dysplasia.
According to the application of the disclosure, a pathogenic mutation site of the cranial clavicle dysplasia can be provided, which is helpful for assisting in screening patients with the cranial clavicle dysplasia so as to facilitate prenatal and postnatal care guidance and early discovery, prevention and treatment of diseases.
In some examples, in the above applications related to the present disclosure, the RUNX2 gene may be detected by detecting at least one of peripheral blood, saliva and tissue sample of the subject. In some examples, the subject may be a general population, an individual suspected of having cranioclavicular dysplasia, or a high risk population of cranioclavicular dysplasia.
In some examples, germline mutations in the RUNX2 gene can be detected. Germ line mutation is also called germ cell mutation, and is derived from mutation carried by germ cells such as sperms or ova.
In some examples, the germline mutation result of RUNX2 gene can be obtained by extracting gDNA (genomic DNA) of the subject and detecting the genomic DNA.
In some examples, a mutation at base 1550 of the RUNX2 gene can be detected. Further, the RUNX2c.1550delT mutation can be detected. RUNX2c.1550delT means that the 1550 th base T of the nucleotide sequence of the wild-type RUNX2 gene is deleted.
In the embodiment, the gene mutation RUNX2c.1550delT is used as a marker to screen patients with cranioclavicular dysplasia, and further provides application of a reagent for detecting the gene mutation RUNX2 in a sample in preparation of a kit for screening patients with cranioclavicular dysplasia.
In some examples, the presence of only one RUNX2c.1550delt mutation in the RUNX2 gene of the detected person can assist in diagnosing that the detected person is a patient with cranial clavicle dysplasia. In other words, when the RUNX2c.1550delT of the detected person is detected as the heterozygous mutation, the diagnosis of the detected person as the skull clavicle dysplasia can be assisted. Of course, when the detected RUNX2c.1550delT is detected as the homozygous mutation, the method can also assist in diagnosing that the detected person is a patient with cranioclavicular dysplasia.
In some examples, mutation of the RUNX2c.1550delt gene resulted in mutation of the amino acid sequence of RUNX2, and the amino acid mutation of RUNX2 p.w518fs (also referred to as amino acid mutation or polypeptide mutation) was generated. Specifically, RUNX2 p.W518fs means that the 518 th site of an amino acid sequence encoded by a wild-type RUNX2 gene is subjected to frame shift mutation, and the 518 th site, the 519 th site, the 520 th site and the 521 th site of the amino acid sequence are all replaced by tryptophan, arginine, proline and tyrosine. In the embodiment, an amino acid mutation RUNX2 p.W518fs is used as a marker, so that a patient with cranioclavicular dysplasia can be screened, and further, an application of a reagent for detecting the amino acid mutation RUNX2 in a sample in preparation of a kit for screening the patient with cranioclavicular dysplasia is provided.
In some examples, other mutation sites besides the RUNX2c.1550delt mutation may also be detected. In some examples, the other mutations may include all currently known causative mutations and suspected causative mutations of cranial clavicular dysplasia. Therefore, all relevant sites of the cranial clavicle dysplasia can be detected, and the cranial clavicle dysplasia can be screened more comprehensively.
In some examples, in the above-mentioned applications to which the present disclosure relates, the kit may be in the form of a reagent or a kit of reagents. In some examples, the kit may further include 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 PCR reagents, DNA sequencing reagents and a DNA sequencer; the system consists of a TaqMan probe, a PCR primer pair, a quantitative PCR instrument, a genotyping module and other reagents required by the TaqMan probe technology; a system consisting of probes, PCR primer pairs and other reagents and instrumentation required for Ligase Detection Reaction (LDR); the kit comprises a PCR primer pair, a single-base extension primer, a chip, a PCR instrument, a genotyping module and/or other reagents and instruments required by the Sequenom MassArray technology.
In one embodiment of the present disclosure, a genotyping assay for RUNX2c.1550delt may be performed using taqman (thermo fisher) genotyping platform. 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 RUNX2 c.1550delt. Thus, a gene mutation such as 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 RUNX2 c.1550. In some examples, the primer or primer pair may not be specifically required in sequence, as long as it can amplify a genomic DNA fragment including RUNX2 c.1550.
In some examples, the probe may be a probe for detecting RUNX2 c.1550delt. In some examples, probes can be designed based on the nucleotide sequence upstream and downstream of RUNX2c.1550 in the human genome, with the sequence of the probe covering the nucleotides RUNX2c.1550delt in the human genome.
In some examples, the kit further comprises dNTPs (deoxyribonucleoside triphosphates), a DNA polymerase, and a PCR reaction buffer. This enables amplification of the fragment of RUNX2 gene.
In some examples, a primer pair can be used to perform PCR amplification on a genomic DNA fragment including the RUNX2c.1550 region to obtain a PCR amplification product. Thus, the 1550 th base in the coding region of the RUNX2 gene can be captured and enriched. In some examples, the obtained PCR amplification product may be used as a template, and the sequence of the obtained PCR amplification product may be detected by using a probe for detecting RUNX2 c.1550delt. Thus, it was possible to determine whether or not a deletion mutation of RUNX2c.1550delT was present at the 1550 th base in the coding region of the RUNX2 gene.
In some examples, the kit may be applied to at least one of the following technologies: DNA sequencing, restriction enzyme fragment length polymorphism, single-strand conformation polymorphism, denaturing high performance liquid chromatography, SNP chip, microfluidic chip technology, TaqMan probe technology and Sequenom MassArray technology. In other words, a mutation in the RUNX2 gene in a sample can be detected using at least one of the following techniques: DNA sequencing, restriction enzyme fragment length polymorphism, single-strand conformation polymorphism, denaturing high performance liquid chromatography, SNP chip, microfluidic chip technology, TaqMan probe technology and Sequenom MassArray technology. Thus, these techniques can be used to detect a mutation in the RUNX2 gene.
In some examples, the kit may include any one of the following:
(a) reagents and/or instrumentation required to determine the polymorphism or genotype of RUNX2c.1550delt using the Sequenom MassArray technique, comprising at least one of the following: PCR primer pairs, single base extension based extension primers, phosphatase, resin, chip, MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight mass spectrometry) and/or other reagents and instruments required by the Sequenom MassArray technique.
(b) Reagents and/or instruments 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 nucleic acid hybridization reaction, a chip based on single base extension reaction, a chip based on allele-specific primer extension reaction, a chip based on "one-step" reaction, a chip based on primer ligation reaction, a chip based on restriction endonuclease reaction, a chip based on protein DNA binding reaction, and/or a chip based on fluorescent molecule DNA binding reaction.
(c) Reagents and/or instruments required for determining the polymorphism or genotype of RUNX2c.1550delt using microfluidic chip technology, comprising at least one of the following components: the kit comprises a DNA extraction microfluidic module and a reagent, a DNA amplification module and a PCR primer pair, a nucleic acid marking module and a related reagent, an SNP chip and a related hybridization, elution and scanning microfluidic module and a related reagent.
(d) 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 single-stranded DNA of a1 to a4 as follows:
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) a single-stranded DNA having 85% or more identity to the single-stranded DNA defined by a1 or a 2;
a4) a single-stranded DNA which hybridizes with the single-stranded DNA defined by a1 or a2 under stringent conditions;
in some examples, the downstream primer for real-time fluorescent quantitative detection of RUNX2c.1550delt may be any 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) a single-stranded DNA having 85% or more identity to the single-stranded DNA defined in b1 or b 2;
b4) and (b) single-stranded DNA which hybridizes with the single-stranded DNA defined by 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 set forth in the sequence of a1 or the sequence of b1 of the present disclosure. Identity can be assessed by human or computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.
In the present embodiment, stringent conditions refer to hybridization and washing of the membrane 2 times, 5min each, in a solution of 2 XSSC (sodium citrate), 0.1% SDS (sodium dodecyl sulfate) at 68 ℃; the membrane was hybridized and washed 2 times for 15min each at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS. Alternatively, hybridization and membrane washing were carried out in a solution of 0.1 XSSPE (saline sodium phosphate EDTA), 0.1% SDS at 65 ℃. Alternatively, hybridization and membrane washing were carried out in a solution of 0.1 XSSC, 0.1% SDS at 65 ℃.
In some examples, 85% identity or greater, e.g., can be 85%, 90%, or 95% identity, etc.
In some examples, in practical applications, the reagent component for detecting RUNX2c.1550delt mutation can be combined with other components (other components may be, for example, reagent components for detecting other gene mutations related to cranioclavicular dysplasia) to prepare a kit for screening the cranioclavicular dysplasia patients.
In some examples, in the above applications to which the present disclosure relates, the RUNX2 polypeptide mutant may be RUNX2 p.w518gfs. Wherein, RUNX2 p.W518Gfs means that tryptophan, arginine, proline and tyrosine at the 518, 519, 520 and 521 position of an amino acid sequence coded by a wild-type RUNX2 gene are replaced.
In some examples, the detection methods used to detect a RUNX2 polypeptide mutant in a sample can include at least one of: protein and peptide sequence analysis techniques, including chemical method of N-terminal sequence determination, Edman method, C-terminal enzymolysis method, C-terminal chemical degradation method, etc.; protein detection techniques related to mass spectrometry, such as matrix-assisted laser desorption/ionization/time-of-flight mass spectrometry (MALDI-TOF MS) and electrospray ionization mass spectrometry (ESI-MS); antibody-based detection methods, such as preparing antibodies that recognize different mutants, and detecting protein variations using immunoblotting (e.g., western blots) and/or enzyme-linked immunosorbent assay (ELISA). Thus, the mutant RUNX2 polypeptide can be detected.
The following examples are provided to illustrate the application of the reagent for detecting mutation of RUNX2 gene in a sample provided by the present disclosure in the preparation of a kit for screening patients with cranial clavicle dysplasia, but should not be construed as limiting the scope of the present disclosure.
[ examples ]
Clinical cases:
patient (proband): normal at birth, with no obvious clinical symptoms. It was found to grow slower than the same age at age 2. Deciduous teeth were lost at age 12 and 3 teeth were replaced by age 15. Patients at 15 years of age were clinically diagnosed with cranial clavicle Dysplasia (Cleidocranial Dysplasia, CCD). The clinical manifestations are as follows: the reaction is mild and the intelligence is normal. In the physical examination of patients, typical features of cranial clavicle dysplasia, such as a depressed midline forehead, shoulder drop, short stature, were observed.
Fig. 1 is an oral X-ray image showing a proband to which an embodiment of the present disclosure relates, and fig. 2 is a chest X-ray image showing a proband to which an embodiment of the present disclosure relates. The proboscis was examined by oral X-ray, and as shown in fig. 1, the mixed dentition, the retention of deciduous teeth, and the impacted teeth were shown. The proband was subjected to chest X-ray examination, as shown in FIG. 2, showing that two-thirds of the right lateral clavicle was hypoplastic, one-third of the left lateral clavicle was hypoplastic, the tapered thorax, scoliosis, and rib deformity.
The father of the patient does not find the clinical manifestation of the cranial clavicle dysplasia.
The patient mother, found not to have cranial clavicle dysplasia clinical manifestations.
Other members of the family were not found to have clinical manifestations of cranial clavicle dysplasia.
Sample detection:
cranial clavicle dysplasia is an autosomal dominant hereditary disease involving bones and teeth. Venous blood samples of the proband father and mother thereof were collected, and leukocyte gDNA (genomic DNA) of the three venous blood samples was subjected to Whole Exon Sequencing (WES). Specifically, genomic DNA fragments in venous blood samples of a proband, a father and a mother of the proband are extracted, then a whole exon detection reagent is used for capturing the exons of all genes in the genomic DNA and the 50bp regions in the adjacent introns of the exons, amplification and purification are carried out after capture, then sequencing is carried out through an Illumina HiSeq sequencing platform, and finally sequencing data analysis is carried out. The detection and analysis are provided by Beijing Fujun Gene biotechnology, Inc.
The sequencing results are shown in FIG. 3, and FIG. 3 is a diagram showing the sequencing peaks of the gene according to the example of the present disclosure, wherein (a) of FIG. 3 is a partial sequence diagram of the proband RUNX2 gene, showing the c.1550delT mutation using an arrow (GenBank accession No.: NM-001024630); FIG. 3 (b) is a partial sequence diagram of the father RUNX2 gene, with the arrow indicating the normal unmutated state at c.1550; FIG. 3 (c) is a partial sequence diagram of the mother RUNX2 gene with arrows showing the normal unmutated state at c.1550. As shown in fig. 3, the proband detected the RUNX2(c.1550delt) mutant, which was not detected in the parents, suggesting that the RUNX2(c.1550delt) mutant detected by the patient may be a new clinically significant mutation.
Functional analysis:
(1) functional impact prediction:
RUNX2c.1550delT is a frame shift deletion mutation, and the frame shift mutation causes the change of amino acid synthesis of RUNX2 from the 518 th amino acid (RUNX2 p.Trp518Glyfs), so that the mutation possibly has an influence on the function of the RUNX2 protein.
In this example, the structural conformations of WT-RUNX2(wild-type-RUNX2, wild-type RUNX2 polypeptide) and W518Gfs-RUNX2 (RUNX2 polypeptide carrying p.w518gfs mutation) were comprehensively analyzed and predicted using phyer2(http:// www.sbg.bio.ic.ac.uk/phyre2/html/page. cgigid ═ index), and fig. 4 shows the RUNX2 domain involved in the disclosed example, and as shown in fig. 4, the RUNX2c.1550delt mutation aggregates VWRPY at the end of the highly conserved PST domain, and thus, the mutation may affect the protein function of RUNX 2.
RUNX2 frameshift mutation analysis: respectively adopting a threading method and a heavy head prediction method to comprehensively analyze and predict the structure configurations of WT-RUNX2 and W518Gfs-RUNX2, and establishing WT-RUNX2 and W518Gfs-RUNX2 structure models.
In addition, SAVE5.03D structure viewer (https:// saves. mbi. ula. edu /) was also used to visualize the 3D structure, and FIG. 5 is a schematic diagram showing the structural models of the wild-type RUNX2 protein and W518Gfs-RUNX2 protein established according to the embodiments of the present disclosure. It is predicted that mutation at this site may alter the structure of the protein, thereby affecting the protein activity of RUNX2, as shown in figure 5.
Fig. 6 is a diagram showing a sequence alignment of RUNX2 in different species to which embodiments of the present disclosure relate. As shown in fig. 6, the site at amino acid 518 of RUNX2 has cross-species conservation.
(2) Functional influence verification:
cell culture and transfection: human embryonic kidney 293(HEK293) cells were cultured in DMEM medium (Gbico, C11995500BT) supplemented with 10% fetal bovine serum (FBS, Gbico, 10099141), penicillin (100IU/mL) and streptomycin (100. mu.g/mL). The plasmid carrying the desired gene was transfected into cells using Lipofectamine TM 2000 (Invitrogen) to study protein expression and other related studies.
Western blot analysis: study of WT-RUN Using human embryonic Kidney 293(HEK293) cellsExpression of X2 and W518Gfs-RUNX 2. In Western blotting analysis, cells were seeded at 10cm 2 On a flat plate. After 1 day HEK293 cells were transfected with pcDNA3.1-GFP, pcDNA3.1-wt-RUNX2-GFP and pcDNA3.1W 518Gfs-RUNX2-GFP, where GFP is Green fluorescent protein. 48 hours after transfection, cells were cultured and examined by SDS-PAGE (polyacrylamide gel electrophoresis). Western blotting was performed using rabbit anti-GFP polyclonal antibody (1:1000, proteintech, 50430-2-AP) and horseradish peroxidase-labeled goat anti-rabbit IgG polyclonal antibody (1:1000, Hongshan bridge, ZB-2301) as secondary antibodies. As detection signal, a chemiluminescence kit (Millipore, CA, USA, WBKLS0050) was used. Fig. 7 is a schematic diagram illustrating the detection result of the expression level of RUNX2 gene of proband according to the 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 mutation in the gene RUNX2c.1550delt did not result in a decrease in 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 was used to observe the subcellular localization of wild-type RUNX2 and RUNX2 p.w518gfs mutations. Fig. 8 is a schematic diagram showing the subcellular localization results of the RUNX2 wild-type and RUNX2 p.w518gfs mutations observed by an in situ immunofluorescence microscope according to the embodiment of the present disclosure, and as shown in fig. 8, both the RUNX2 p.w518gfs mutation and the RUNX2 wild-type were accumulated in the nucleus of HEK293T cells, indicating that the subcellular separation of the RUNX2 p.w518gfs mutation was not affected.
Luciferase reporter assay: HEK-293 cells were seeded into 96-well plates at 10000/well, 200ul of cell suspension was added thereto at 37 ℃ with 5% CO 2 And (5) culturing. On day 2, pRL-TK and pgl 3-basic-osteocalcin promoter plasmids were added to each fraction, and pcDNA3.1, pcDNA3.1-wt-RUNX2, and pcDNA3.1-W518Gfs-RUNX2 were transfected at different concentrations (0ug, 1ug, 2ug), respectively. The dual luciferase detection operation was performed after 48 h. Specifically, a dual-luciferase reporter gene detection system is adopted and the results are analyzed. FIG. 9 shows RUNX2 p.W518Gfs variation induction according to the examples of the present disclosureThe results of the detection of osteocalcin promoter activity in (1) are shown in a schematic diagram, as shown in FIG. 9, luciferase detection shows that the trans-activation activity of RUNX2 and the transcriptional regulation of osteocalcin promoter are shown, as shown in FIG. 9, the activity of the osteocalcin promoter induced by RUNX2 p.W518Gfs variation is lower than that of RUNX 2-wt.
In the present example, the reagents and instruments used are commercially available unless otherwise specified.
The above results show that the function of RUNX2 protein is impaired by RUNX2c.1550delT mutation or RUNX2 p.W518Gfs mutation. Thus, the RUNX2c.1550delt mutation is a pathogenic mutation in the cranioclavicular dysplastic family in this example. This example found that RUNX2c.1550delT is a novel causative gene of cranial clavicle dysplasia and is inherited as autosomal dominant.
In addition, the family of the patient with the cranial clavicle dysplasia found in the embodiment of the disclosure is subjected to genetic consultation, so that the bearing and rearing are facilitated.
The cranial clavicle dysplasia has different phenotypes, mild symptoms only include tooth abnormality, and clinical diagnosis is difficult, and gene diagnosis can be performed through gene detection. According to the embodiment of the disclosure, a novel cranium clavicle dysplasia related pathogenic gene mutation site can be provided. Therefore, enrichment of a pathogenic mutation library of the cranial clavicle dysplasia can be facilitated, and genetic diagnosis of the cranial clavicle dysplasia is facilitated. Is helpful for assisting in screening patients with cranial clavicle dysplasia so as to facilitate the guidance of prenatal and postnatal care and the early detection, prevention and treatment of diseases
Although the present disclosure has been described with reference to specific embodiments, it should be understood that the scope of the present disclosure is not limited thereto, and those skilled in the art will appreciate that various modifications and variations can be made without departing from the spirit and scope of the present disclosure.
The above description is only a preferred embodiment of the present disclosure and is not intended to limit the present disclosure, and various modifications and changes may be made to the present disclosure by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present disclosure should be included in the protection scope of the present disclosure.
While the present disclosure has been described in detail above with reference to the drawings and the embodiments, it should be understood that the above description does not limit the present disclosure in any way. Variations and changes may be made as necessary by those skilled in the art without departing from the true spirit and scope of the disclosure, which fall within the scope of the disclosure.

Claims (10)

1. Application of a reagent for detecting RUNX2 gene mutation in a sample in preparation of a kit for screening patients with cranial clavicle dysplasia is characterized in that the gene mutation is RUNX2 c.1550delT.
2. Use according to claim 1, characterized in that the kit is used in at least one of the following technologies: DNA sequencing, restriction enzyme fragment length polymorphism, single-strand conformation polymorphism, denaturing high performance liquid chromatography, SNP chip, microfluidic chip technology, TaqMan probe technology and Sequenom MassArray technology.
3. The use according to claim 1, wherein the kit comprises a primer pair for amplifying the RUNX2 gene and/or a probe for detecting RUNX2 c.1550delT.
4. The use of claim 3, wherein the probe for detecting RUNX2c.1550delT is designed according to the nucleotide sequence upstream and downstream of the 1550 th base in the coding region of the RUNX2 gene in the human genome.
5. The use of claim 3, wherein the kit further comprises dNTPs, a DNA polymerase and PCR reaction buffer.
6. The use of claim 1, wherein the kit comprises a real-time fluorescent quantitative detection reagent comprising an upstream primer and a downstream primer for real-time fluorescent quantitative detection of the gene mutation, the sequence of the upstream primer comprises 5'-CTCTTCCCAAAGCCAGAGTG-3', and the sequence of the downstream primer comprises 5'-GCAGACAGCTCACAAAACCAG-3'.
7. The use of claim 1, wherein the sample is from at least one of peripheral blood, saliva, and tissue of the subject, and the RUNX2 gene mutation is a germline mutation in the RUNX2 gene.
8. Application of a reagent for detecting RUNX2 gene mutation in a sample in preparation of a kit for detecting susceptibility to skull clavicle dysplasia is characterized in that the gene mutation is RUNX2 c.1550delT.
9. Application of a reagent for detecting RUNX2 gene mutation in a sample in preparation of products for detecting single nucleotide polymorphism or genotype easily related to skull clavicle dysplasia is characterized in that the gene mutation is RUNX2 c.1550delT.
10. An application of a reagent for detecting a RUNX2 polypeptide mutant in a sample in preparation of a kit for screening a patient with cranial clavicle dysplasia is characterized in that the polypeptide mutant is RUNX2 p.W518Gfs.
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