CN108866197B - Application of PTCH2 gene mutation site in judging susceptibility of young breast cancer - Google Patents

Application of PTCH2 gene mutation site in judging susceptibility of young breast cancer Download PDF

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CN108866197B
CN108866197B CN201810963970.XA CN201810963970A CN108866197B CN 108866197 B CN108866197 B CN 108866197B CN 201810963970 A CN201810963970 A CN 201810963970A CN 108866197 B CN108866197 B CN 108866197B
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马飞
徐兵河
焦宇辰
钱海利
黎立喜
李春晓
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Cancer Hospital and Institute of CAMS and PUMC
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Abstract

The invention discloses application of a PTCH2 gene mutation site, wherein the mutation site is p.S391X (SNP rs 56126236). The invention discovers that the mutation site is related to the susceptibility of the young breast cancer for the first time, and therefore, on the basis, the invention provides a product for diagnosing the susceptibility risk of the young breast cancer by detecting the mutation.

Description

Application of PTCH2 gene mutation site in judging susceptibility of young breast cancer
Technical Field
The invention belongs to the field of biological medicine, relates to application of a PTCH2 gene mutation site, and particularly relates to application of a PTCH2 gene mutation site in judging susceptibility of young breast cancer.
Background
The breast cancer is the most common malignant tumor of women, the incidence rate of breast cancer of women in China is increased year by year, the incidence rate accounts for 7-10 percent of the malignant tumor, and about 120 ten thousand women all year round generate the breast cancer, which accounts for 18 percent of the female tumor.
Germline mutations in the BRCA1 and BRCA2 genes have been investigated to greatly increase the risk of women suffering from breast cancer. Under normal conditions, these two genes are mainly involved in homologous recombination repair of DNA. If they develop deleterious alterations, the cells proliferate uncontrollably leading to cancer formation. However, BRCA1 and BRCA2 can account for inheritance of only a portion of familial breast cancer, and the mutation frequencies of BRCA1 and BRCA2 in china are lower than in european and american countries.
Single Nucleotide Polymorphism (SNP) refers to DNA sequence polymorphism at the genomic level due to variation of a single nucleotide in a form including deletion, insertion, transition, transversion, and the like of a single base. Generally, the frequency of the least 1 allele in the above format in the population is not less than 1%, but may be less than 1% in a particular case (e.g., in cDNA). 1 SNP has a single nucleotide change at a certain locus in the genome, and mainly occurs in two forms: one is caused by the switching of single bases, i.e. the substitution of one pyrimidine for another pyrimidine or one purine for another purine; another form is transversion, i.e. purine is exchanged for pyrimidine. From a principle analysis, the base at the mutation may be C, G, A, T, whereas in reality SNPs mostly occur between T and C. Polymorphism of human genetic genes is essentially expressed in genetic information, and more than 90% of the polymorphism is caused by SNP. Because of the large amount of polypeptide information, the gene is currently a third generation genetic marker following a Restriction Fragment Length Polymorphism (RFLP) marker and a microsatellite, namely a Short Tandem Repeat (STR) marker.
The SNP marker is a molecular marker with the most development potential at present, and can realize large-scale high automation due to the large quantity and wide distribution in a genome and the fact that DNA does not need to be banded according to the size of a fragment in the gene analysis process, so that the SNP marker is more suitable for detection and analysis with large quantity and is widely applied to a plurality of fields of biology, agriculture, medicine, biological evolution and the like. The research of SNP provides more basis for gene diagnosis, especially for early diagnosis of diseases. SNPs are expected to play an important role in complex diseases such as cancer, diabetes, hypertension, depression, and asthma due to their wide distribution and high density, which are the result of the interaction of multiple genetic variation sites with environmental factors, and since the causes of the disease are complex, the number of involved genes is large, which has become the focus of the international research on disease genomics. At present, the SNP is applied to the judgment of the prognosis and susceptibility of the tumor in the existing experiments, for example, the susceptibility of the lung cancer carcinogen has individual difference, namely the genetic susceptibility of the lung cancer, and the research on the polymorphism of the metabolic enzyme genes is more frequent, such as human cytochrome P450-CYP450 and myeloperoxidase MPO, and the like. Through a traditional candidate gene research method, genes related to the breast cancer occurrence function are measured, and a plurality of susceptible genes such as an endoglin gene, a matrix metalloproteinase gene, an IR type collagen gene, an otl-antitrypsin gene, an elastin gene, an angiotensin converting enzyme gene, an apolipoprotein gene and the like are also found.
Disclosure of Invention
In order to make up for the deficiencies of the prior art, the present invention aims to provide a mutation site which can be used for diagnosing breast cancer susceptibility. The breast cancer is diagnosed with early warning by detecting the existence of mutation.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides application of a reagent for detecting SNP sites of PTCH2 gene in preparing a product for diagnosing breast cancer susceptibility.
Further, the SNP site of the PTCH2 gene is rs 56126236. Among them, when CT deletion exists in the SNP site rs56126236 of PTCH2, the susceptibility of breast cancer is increased.
The person skilled in the art can detect the SNP site of the PTCH2 gene by any method or technique for detecting SNP sites. For example, Taqman, Mass Spectrometry, DNA microarray, sequencing, micro-sequencing, hybridization, restriction fragment analysis, oligonucleotide ligation detection, allele-specific PCR-HRM or a combination thereof. Therefore, the reagent of the present invention includes a reagent for detecting the SNP site of the PTCH2 gene using the above method.
Further, the reagents comprise a primer pair for amplifying a nucleotide sequence including the rs56126236 site.
Preferably, the breast cancer as described above refers to a young breast cancer.
The invention also provides application of a reagent for detecting the mutation site of the PTCH2 protein in preparing a product for diagnosing the susceptibility of breast cancer, wherein the mutation site is 391 th site of the PTCH2 protein. When the 391 th position of the PTCH2 protein is changed from Ser to Ter, the susceptibility to breast cancer increases.
Further, the reagent for detecting the mutation site of the PTCH2 protein described above includes reagents used in methods for determining the amino acid type of the mutation site by N-terminal sequencing, C-terminal sequencing, intra-chain amino acid sequencing, mass spectrometry, and the like.
Preferably, the breast cancer as described above refers to a young breast cancer.
The invention also provides a product for diagnosing the susceptibility of the breast cancer, which comprises a reagent for detecting the genotype of the SNP locus rs56126236 of the PTCH2 gene.
The product of the invention may comprise reagents for detecting the genotype of the SNP site by any technique known in the art, as long as they are capable of detecting the genotype of the rs56126236 site in the sample.
Techniques or methods known to those skilled in the art that are capable of detecting the genotype of a SNP site include, but are not limited to, Taqman methods, mass spectrometry, DNA microarray methods, sequencing methods, micro-sequencing, hybridization, restriction fragment analysis, oligonucleotide ligation detection, allele-specific PCR-HRM, or combination applications.
Further, the reagent for detecting the SNP locus rs56126236 of the PTCH2 gene comprises a primer pair for amplifying a nucleotide sequence including the locus rs 56126236.
Preferably, the breast cancer as described above refers to a young breast cancer.
The invention provides a product for diagnosing breast cancer susceptibility, which comprises a reagent for detecting 391 th amino acid species of PTCH2 protein.
The product of the present invention may include a reagent for detecting the amino acid sequence of a protein by any technique known in the art, as long as it is a reagent capable of detecting the 391 th amino acid species of the PTCH2 protein.
Techniques or methods known to those skilled in the art that are capable of detecting protein amino acid sequences include N-terminal sequencing, C-terminal sequencing, intra-chain amino acid sequencing, mass spectrometry, and the like.
Preferably, the breast cancer as described above refers to a young breast cancer.
The invention also provides a product for diagnosing the susceptibility of the breast cancer, which comprises a reagent for detecting the SNP locus rs56126236 genotype of the PTCH2 gene and a reagent for detecting the 391 th amino acid type of the PTCH2 protein.
The above reagent species are not limited as long as they can detect the SNP site rs56126236 genotype of the PTCH2 gene or detect the 391 th amino acid species of the PTCH2 protein.
Preferably, the breast cancer as described above refers to a young breast cancer.
The definition of the specific reagents is as described above.
Further, the aforementioned product may be a chip, an array or a kit.
Still further, the chip or array comprises a solid support; and an oligonucleotide probe immobilized on the solid support, the oligonucleotide probe comprising a SNP site specifically corresponding to the PTCH2 gene described above.
Furthermore, the kit can also comprise a genomic DNA extraction reagent, a PCR reaction system reagent, a DHPLC related reagent or a protein extraction reagent.
Furthermore, the PCR reaction system reagent comprises dNTP and MgCl2Taq DNA polymerase, PCR reaction buffer solution and deionized water.
Furthermore, the kit also comprises an enzyme digestion conventional component, including reaction buffer solution and deionized water.
In the present invention, the oligonucleotide probe for the SNP site of the PTCH2 gene may be DNA, RNA, DNA-RNA chimera, PNA or other derivatives. The length of the probe is not limited, and any length may be used as long as specific hybridization and specific binding to the target nucleotide sequence are achieved. The length of the probe may be as short as 25, 20, 15, 13 or 10 bases in length. Also, the length of the probe can be as long as 60, 80, 100, 150, 300 base pairs or more, even for the entire gene. Since different probe lengths have different effects on hybridization efficiency and signal specificity, the length of the probe is usually at least 14 base pairs, and at most, usually not more than 30 base pairs, and the length complementary to the nucleotide sequence of interest is optimally 15 to 25 base pairs. The probe self-complementary sequence is preferably less than 4 base pairs so as not to affect hybridization efficiency.
In the present invention, the solid phase carrier includes plastic products, microparticles, membrane carriers, and the like. The plastic products can be combined with antibodies or protein antigens through a non-covalent or physical adsorption mechanism, and the most common plastic products are small test tubes, small beads and micro reaction plates made of polystyrene; the micro-particles are microspheres or particles polymerized by high molecular monomers, the diameter of the micro-particles is more than micron, and the micro-particles are easy to form chemical coupling with antibodies (antigens) due to the functional groups capable of being combined with proteins, and the combination capacity is large; the membrane carrier comprises microporous filter membranes such as a nitrocellulose membrane, a glass cellulose membrane, a nylon membrane and the like.
The term "young breast cancer" as used herein refers to a breast cancer in a person of 35 or less years of age.
In the present invention, the term "genotype" refers to the identity of the alleles present in an individual or sample. Typically, it refers to the genotype of the individual associated with a particular gene of interest; in a polyploid individual, it refers to an individual carrying various combinations of genetic alleles.
In the present invention, the term "primer" refers to a naturally occurring oligonucleotide (e.g., a restriction fragment) or a synthetically produced oligonucleotide that is capable of serving as a point of initiation of synthesis of a primer extension product that is complementary to a nucleic acid strand (template or target sequence) when subjected to appropriate conditions (e.g., buffer, salt, temperature, and pH) and in the presence of nucleotides and an agent for nucleic acid polymerization (e.g., a DNA-dependent or RNA-dependent polymerase).
In the present invention, an "array" or "microarray" is an ordered arrangement of hybridization array elements, such as polynucleotide probes (e.g., oligonucleotides) or binding agents (e.g., antibodies), on a substrate. The matrix may be a solid matrix, for example, a glass or silica slide, beads, a fiber optic binder, or a semi-solid matrix, for example, a nitrocellulose membrane. The nucleotide sequence may be DNA, RNA or any permutation thereof. Microarrays can be prepared from gene-specific oligonucleotide probes of known susceptibility to breast cancer. The array may contain different oligonucleotide probes for each gene SNP site, the array may also contain controls, and one or more appropriate controls for non-specific hybridization may also be included on the microchip.
The invention has the advantages and beneficial effects that:
the invention discovers that the SNP locus rs56126236 of the PTCH2 gene is related to breast cancer for the first time. Therefore, a product capable of detecting the susceptibility risk of the breast cancer is provided on the basis. The detection product of the invention has good sensitivity, stability and specificity. The detection product of the invention can effectively play roles in risk early warning and early diagnosis when screening the susceptibility of breast cancer to normal people.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are provided only for the purpose of illustration and are not meant to limit the scope of the present invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 SNP sites associated with susceptibility to Breast cancer
1. Study object
36 patients with ultra-young breast cancer (< 25 years old), 223 patients with sub-young breast cancer (>25 years old, < 35 years old), 571 patients with non-young breast cancer (>35 years old) were collected.
Inclusion criteria were: 1) young women with onset age less than or equal to 35 years old; 2) pathologically confirmed breast cancer; 3) the patient himself informed and voluntarily added the genetic testing of the project.
Exclusion criteria: (1) the patient himself refuses the gene test.
All patients signed informed consent with the consent of the ethical committee in this study.
2. DNA extraction step
The DNA kit was purchased from Tiangen Biochemical technology (Beijing) Ltd, cat #: DP 304-02. DNA extraction was performed according to the procedures described in the kit instructions:
1) treating the materials: 4ml of peripheral blood is extracted from the peripheral vein of the patient to be treated, and 200 mu l of peripheral blood added with anticoagulant is directly used;
2) adding 20 μ l proteinase K (protease K) solution, and mixing;
3) adding 200 μ l buffer solution GB, fully reversing and mixing, standing at 70 deg.C for 10min, making the solution clear and bright, and centrifuging briefly to remove water column on the inner wall of the tube cover;
4) adding 200 μ l of anhydrous ethanol, shaking thoroughly, mixing for 15sec, wherein flocculent precipitate may appear, and centrifuging briefly to remove water column on the inner wall of the tube cover;
5) adding the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3 (the adsorption column is placed into a collecting pipe), centrifuging at 12,000rpm (-13, 400 Xg) for 30 s, pouring the waste liquid, and placing the adsorption column CB3 back into the collecting pipe;
6) adding 500 μ l buffer GD (checking whether absolute ethanol is added before use) into adsorption column CB3, centrifuging at 12,000rpm (-13, 400 Xg) for 30 s, pouring off waste liquid, and placing adsorption column CB3 into a collection tube;
7) to adsorption column CB3, 600. mu.l of rinsing solution PW (to check whether or not absolute ethanol was added before use) was added, centrifuged at 12,000rpm (. about.13, 400 Xg) for 30 seconds, the waste liquid was discarded, and adsorption column CB3 was put into the collection tube.
8) Repeating the operation step 7);
9) the adsorption column CB3 was returned to the collection tube, centrifuged at 12,000rpm (. about.13, 400 Xg) for 20 minutes, the waste liquid was decanted, and CB3 was left at room temperature for several minutes to allow the rinse remaining on the adsorption material to air-dry completely.
Note that: the purpose of this step is to remove the residual rinsing liquid in the adsorption column, and the residual ethanol in the rinsing liquid can affect the subsequent enzyme reaction (enzyme digestion, PCR, etc.) experiments.
10) Transferring the adsorption column CB3 into a clean centrifuge tube, suspending and dripping 50-200 mu l of elution buffer TE into the middle part of the adsorption membrane, standing for 2-5min at room temperature, centrifuging for 2 min at 12,000rpm (13-400 Xg), and collecting the solution into the centrifuge tube.
3. Identification of mutation sites
Original image data obtained by high-throughput whole exon sequencing (Illumina HiSeqTM X Ten) is converted into an original sequencing sequence through base recognition analysis, and then data analysis is carried out, wherein the analysis comprises four parts:
1) preprocessing sequencing data: controlling and comparing the quality of data; removing low-quality data, and comparing the residual high-quality sequences to a human reference genome; counting data quantity, comparison rate, coverage depth, coverage degree and other information, evaluating whether the sequencing data meet the analysis standard, performing subsequent analysis according with the standard, and otherwise, rebuilding a library or adding a test;
2) basic variation detection: carrying out variation detection on the sample according to the sequence comparison result; performing germ line mutation detection on a single sample;
3) variant screening and annotation: filtering and annotating the results of the variant detection; screening high-quality mutation according to information such as coverage depth, mutation frequency and the like, and annotating the structure and function of a screening result by using ANNOVAR (Wang K et al.2010), oncotor and other software; and filtering the annotated mutation results (SNV and INDEL) according to the annotation information, and screening rare mutations with important meanings such as biological research and clinical research for subsequent comprehensive analysis. The flow of the filtration analysis was as follows: all the detection results retain missense mutation, nonsense mutation, frameshift mutation and mutation of a variable shearing region within 1-2bp near the upstream and downstream of an exon in a coding region, and the mutation result is marked as VAR 0: (1) results from VAR0 were subjected to mutation type statistics, SNP _ stat and INDEL _ stat; (2) the results of VAR0 were population frequency filtered, with 4 sites in the database (1000 genes, 1000 genes chinese, ESP6500, EXAC) where population frequencies were all less than 0.05, and the results were scored as VAR 1: the clinical significance of the gene mutation is then determined by annotating information (type of variation, influence of protein function, SIFT and other software predicted values) and relevant literature (please refer to American society for medical genetics and genomics ACMG judgment guidelines in detail).
4) And (3) comprehensively analyzing variation results: and judging that the PTCH2rs56126236 site is harmful mutation by the dbSNP database, judging that the mutation frequency in the 1000 gene and 1000 gene Chinese databases is lower than 0.05, and judging that the mutation is harmful according to the ACMG criterion.
4. Results
Through data analysis, the rs56126236 site of a part of breast cancer patients is subjected to CT deletion, or the 391 site of PTCH2 protein is subjected to amino acid change. The detailed data are shown in Table 1.
TABLE 1 mutation statistics
Figure BDA0001774468180000081
7. Data analysis
PTCH2 p.S391X (SNP rs56126236) site mutation has a frequency of 0.48% in Chinese healthy population (thousand human genomes) (data from https:// www.ncbi.nlm.nih.gov/variation/tools/1000 genes /). According to the results in table 1, the correlation of PTCH2 p.s391x (SNP rs56126236) site mutation with breast cancer susceptibility was analyzed. Fisher's exact test showed P0.014, indicating that PTCH2 p.s391x (SNP rs56126236) site mutation in young breast cancer patients (< 35 years) was significantly associated with susceptibility to young breast cancer.
TABLE 2 statistical analysis results
Figure BDA0001774468180000082
Figure BDA0001774468180000091
The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Claims (5)

1. Application of a reagent for detecting SNP sites of PTCH2 genes in preparation of products for diagnosing susceptibility of young breast cancer, wherein the SNP sites are rs 56126236.
2. The use of claim 1, wherein the reagents comprise reagents used in methods for genotyping the SNP sites using Taqman, Mass Spectrometry, DNA microarray, sequencing, micro sequencing, hybridization, restriction fragment analysis, oligonucleotide ligation detection, allele specific PCR-HRM.
3. The use according to claim 1 or 2, characterized in that the reagents comprise a primer pair for amplifying a nucleotide sequence comprising the rs56126236 site.
4. Application of a reagent for detecting a mutation site of PTCH2 protein in preparing a product for diagnosing susceptibility of young breast cancer, wherein the mutation site is 391 th site of PTCH2 protein, and the mutation is p.S391X.
5. The use of claim 4, wherein the reagents comprise reagents used in a method for determining the amino acid type of the mutation site by N-terminal sequencing, C-terminal sequencing, intra-chain amino acid sequencing, mass spectrometry sequencing.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013075059A1 (en) * 2011-11-18 2013-05-23 Vanderbilt University Markers of triple-negative breast cancer and uses thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013075059A1 (en) * 2011-11-18 2013-05-23 Vanderbilt University Markers of triple-negative breast cancer and uses thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Frameshift mutation in the PTCH2 gene can cause nevoid basal cell carcinoma syndrome.;Katsunori Fujii et al.;《Familial Cancer》;20130312;第12卷(第4期);611-614 *
Germline mutation PTCH2 1172-1173delCT in Chinese population with early-onset breast cancer.;Lixi Li et al.;《Journal of Clinical Oncology》;20190526;第37卷(第15期);e13049 *
Standards and Guidelines for the Interpretation of Sequence Variants: A Joint Consensus Recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.;Sue Richards et al.;《Genet Med》;20150305;第17卷(第5期);405-424 *

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