CN106868128B - Biomarker for auxiliary diagnosis of breast cancer and application thereof - Google Patents
Biomarker for auxiliary diagnosis of breast cancer and application thereof Download PDFInfo
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Abstract
The invention discloses a group of biomarkers for auxiliary diagnosis of breast cancer, which are a combination of SNP loci rs114337127, rs13306048, rs139477122, rs140787599, rs16859487, rs199972750, rs200315000, rs35115195, rs368392829, rs369896558, rs372909717 and rs 373617497. The invention can make the diagnosis of the breast cancer more convenient and easier by the research and the application of the SNP genotype diagnostic reagent and the diagnostic kit, lays a foundation for the clinical treatment effect evaluation for the clinician to quickly and accurately master the disease condition of the patient, and provides help for finding a novel micromolecule drug target with potential treatment value.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a group of biomarkers for auxiliary diagnosis of breast cancer and application thereof.
Background
Breast cancer is a systemic disease, and the occurrence and development of the breast cancer are complex processes involving multiple factors and multiple links, including the activation of oncogenes and the inactivation of cancer suppressor genes. Therefore, gene mutation plays an important role in the process of the occurrence and development of breast cancer.
Breast cancer is a multifactorial genetic variability disease, with less than 10% due to single gene defects. With the development of high-throughput gene technology, more and more genes related to breast cancer are discovered, and potential genetic variations (single nucleotide polymorphism and copy number variation) on the genes can cause the difference of the curative effect of breast cancer drugs. Due to the existence of genetic variation, metabolic pathways of the antitumor drugs and target genes of drug action can be influenced, and further, the curative effect and prognosis are influenced.
SNP (single nucleotide polymorphism) is a molecular genetic marker proposed by Lander in 1996, which is a chemical marker of human genome research center of the national academy of science and technology of Massachusetts, and mainly refers to DNA sequence polymorphism caused by variation of a single nucleotide at the genome level. SNPs exhibit polymorphisms involving only single base variations, including transitions, transversions, insertions, and deletions. Single nucleotide polymorphism is a third generation genetic marker, and many phenotypic differences in humans, susceptibility to drugs or diseases, and the like may be associated with SNP. Currently, the predictive research on prognosis and curative effect of different types of breast cancer mainly focuses on SNP level.
SNPs confer different responses to environmental exposure, drug treatment, etc. to individuals, thereby generating different phenotypes, and thus SNPs may be an important genetic basis for causing differences in development of individual diseases. The SNP spectrum which is susceptible to diseases is utilized to diagnose the diseases, and the method has the characteristics of rapidness, sensitivity, accuracy and the like, so the method has wide application prospect. In recent years, the development of disease diagnosis by using SNP has become a research hotspot of clinical and scientific researchers.
However, at present, there is no report of applying SNP to breast cancer diagnosis, and if SNP susceptible to breast cancer can be screened out as a biomarker and a corresponding diagnostic kit is developed, the current situation of early diagnosis of breast cancer in China is pushed strongly, and a new approach is developed for drug screening, drug efficacy evaluation and targeted therapy.
Disclosure of Invention
The invention aims to solve the technical problems and provides a group of biomarkers for assisting in diagnosing breast cancer.
The second purpose of the invention is to provide a breast cancer auxiliary diagnosis kit.
The inventor searches a group of SNP highly related to the breast cancer and having high specificity and sensitivity by separating and researching single nucleotide polymorphism in the peripheral blood DNA of breast cancer patients and healthy women matched with the breast cancer patients with age, develops a breast cancer auxiliary diagnosis kit convenient for clinical application, and provides data support for screening and diagnosing the breast cancer.
The purpose of the invention is realized by the following technical scheme:
a group of biomarkers for assisting in diagnosing breast cancer, wherein the biomarkers are a combination of SNP loci rs114337127, rs13306048, rs139477122, rs140787599, rs16859487, rs199972750, rs200315000, rs35115195, rs368392829, rs369896558, rs372909717 and rs 373617497.
The invention further provides specific amplification primers of the SNP sites of the biomarkers, and the sequences of the primers are as follows:
the sequence of the amplification primer of rs114337127 is SEQ ID No: 1 and SEQ ID No: 2;
the sequence of the amplification primer of rs13306048 is SEQ ID No: 3 and SEQ ID No: 4;
the amplification primer sequence of rs139477122 is SEQ ID No: 5 and SEQ ID No: 6;
the sequence of the amplification primer of rs140787599 is SEQ ID No: 7 and SEQ ID No: 8;
the sequence of the amplification primer of rs16859487 is SEQ ID No: 9 and SEQ ID No: 10;
the amplification primer sequence of rs199972750 is SEQ ID No: 11 and SEQ ID No: 12;
the sequence of the amplification primer of rs200315000 is SEQ ID No: 13 and SEQ ID No: 14;
the sequence of the amplification primer of rs35115195 is SEQ ID No: 15 and SEQ ID No: 16;
the sequence of the amplification primer of rs368392829 is SEQ ID No: 17 and SEQ ID No: 18;
the sequence of the amplification primer of rs369896558 is SEQ ID No: 19 and SEQ ID No: 20;
the amplification primer sequence of rs372909717 is SEQ ID No: 21 and SEQ ID No: 22;
the sequence of the amplification primer of rs373617497 is SEQ ID No: 23 and SEQ ID No: 24.
further, the invention also provides application of the biomarker in auxiliary detection, diagnosis, treatment and prognosis evaluation of breast cancer.
Further, the invention provides a kit for auxiliary diagnosis of breast cancer, which comprises a reagent for detecting the genotype of the SNP locus, wherein the combination of the SNP loci rs114337127, rs13306048, rs139477122, rs140787599, rs16859487, rs199972750, rs200315000, rs35115195, rs368392829, rs369896558, and rs372909717 and rs 373617497.
Preferably, the reagent comprises a specific primer for amplifying the SNP site, or comprises a specific primer and a restriction enzyme for amplifying the SNP site.
Preferably, the specific primer for amplifying the SNP site is a combination of the specific primers, and the combination of the specific primers has strong specificity and good amplification effect.
Preferably, the kit further comprises enzymes and reagents commonly used in PCR reactions, such as dNTPs, Taq enzyme, Mg2+PCR reaction buffer solution, etc.; standards and/or controls may also be included.
The invention has the beneficial effects that:
the invention researches the application prospect of the biomarker in breast cancer auxiliary diagnosis, explains the influence of SNP on the breast cancer progress, and reveals the diagnostic value of the biomarker. Therefore, the invention can make the diagnosis of the breast cancer more convenient and easier through the research and the application of the SNP genotype diagnostic reagent and the diagnostic kit, lays a foundation for the clinician to quickly and accurately master the illness state of the patient, and provides help for the clinical treatment effect evaluation and the discovery of a novel micromolecule drug target with potential treatment value.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
The technical scheme of the invention specifically comprises the following steps: collecting blood samples meeting the standard, and collecting complete demographic data and clinical data by a system; and (3) genotype detection: selecting a breast cancer case and a healthy female contrast matched with the breast cancer case in age, and finding out SNP (Single nucleotide polymorphism) related to the breast cancer by exon sequencing; further adopting genotyping to detect the screened positive associated SNP, and verifying the repeatability of the SNP applied to clinical diagnosis; development of an auxiliary diagnosis kit for breast cancer: the SNP auxiliary diagnostic kit is developed according to the SNP with obvious difference in the genotype distribution frequency in the breast cancer case and the healthy female contrast.
The values in the data analysis are expressed as follows:
1. ljb23_ sift: the SIFT score (version 2.3), which represents the effect of the variation on the protein sequence, contains three values, namely the SIFT initial score, the converted value (1-SIFT), and T or D. When the variation affects multiple protein sequences simultaneously, there is a SIFT value for each protein sequence, taking the minimum value. The smaller SIFT score, the more "harmful", indicating that the SNP has a high probability of causing structural or functional changes in the protein; d, Deleterious (sift < ═ 0.05); t: tolerated (sift > 0.05));
2. ljb23_ pp2 hvar: the effect of this variation on protein sequence was predicted based on the HumanVar database using PolyPhen2 for monogenic genetic diseases. This column contains two values, the first being the PolyPhen2 score, the larger the value the more "harmful", indicating a high probability that the SNP results in a change in protein structure or function; the second is D or P or B (D: basic damaging (> ═ 0.909), P: subscription damaging (0.447< ═ pp2_ hvar < ═ 0.909); B: benign (pp2_ hvar < ═ 0.446));
3. ljb23_ pp2 hdiv: the impact of this variation on protein sequence was predicted based on the HumanDiv database using PolyPhen2 for complex diseases. This column contains two values, the first being the PolyPhen2 score, the larger the value the more "harmful", indicating that the SNP has a high probability of causing a change in protein structure or function; the second is D or P or B (D: basic damaging (> ═ 0.957), P: marketing (0.453 ═ pp2_ hdiv ═ 0.956); B: benign (pp2_ hdiv ═ 0.452));
4. ljb23_ mt: the Mutation marker score (version 2.3), which represents the effect of the Mutation on the protein sequence, contains three values, one is the Mutation marker initial score, the other is the transformed value, and the third is A, D, N or P. The larger the second value, the more "detrimental", indicating that the SNP has a high probability of causing a structural or functional change in the protein, wherein "a"; "D" ("discrete using"); "N" ("polymorphism"); "P" ("polymorphism automatic").
The experimental method of research mainly includes the following parts:
1. selection of study samples
(1) 25 pathologically well-diagnosed breast cancer cases, of which 3 patients had a family history of cancer, and 10 age-matched healthy women served as controls;
(2) before blood collection, radiotherapy or chemotherapy is not received, and no history of previous tumor is available;
(3) healthy female controls matched to age of case
2. Extracting peripheral blood genome DNA by phenol-chloroform method, and performing conventional method. 20-50 ng/. mu.LDNA can be obtained in general, and the purity (UV 2600D: 2800D) is 1.6-2.0.
3. Whole exon chip detection
(1) Taking a whole genome DNA sample of a subject;
(2) scanning was performed on a full-exon chip (Beijing Nuo He-derived science and technology Co., Ltd., the same applies below);
(3) the individual differences of each genotype in breast cancer cases and healthy female controls were detected and compared.
4. Genotyping of a Single SNP
(1) Taking a DNA sample of a subject;
(2) designing specific amplification primers of single SNP;
(3) carrying out PCR reaction, and recycling a product for sequencing;
(4) the differences in the distribution of the different genotypes in breast cancer cases and healthy female controls were compared.
5. Method for preparing diagnostic kit
After the scanning and single SNP detection of the whole exon chip, the SNP with obvious difference in the genotype distribution frequency of the breast cancer case and the healthy female contrast is determined and used as the index of breast cancer diagnosis. The screened SNP auxiliary diagnostic kit related to the breast cancer onset comprises reagents for detecting SNP locus genotypes of rs114337127, rs13306048, rs139477122, rs140787599, rs16859487, rs199972750, rs200315000, rs35115195, rs368392829, rs369896558, rs372909717 and rs373617497, and the diagnostic kit can also comprise specific amplification primers of the SNPs, Taq enzyme, dNTPs and other reagents.
6. Example of clinical application
The breast cancer auxiliary diagnosis kit prepared by the inventor is used for detecting a breast cancer patient to be screened and comparing with actual clinical detection to determine the effectiveness of the breast cancer auxiliary diagnosis kit. The method specifically comprises the steps of measuring the specific amplification primers and other detection reagents of the SNP in the blood sample DNA of the subject, and provides support for clinicians to quickly and accurately master the disease state and the disease severity of the patient and to timely adopt a more personalized prevention and treatment scheme.
EXAMPLE 1 Collection of samples and working up of sample data
The inventor collects a large number of blood specimens of new breast cancer patients in Shenzhen second people hospital from 1 month to 2015 year 12, selects 25 samples meeting the following standards from the blood specimens through sorting sample data, and selects 10 healthy women aged 25-55 years as controls to carry out full-exon chip detection, wherein the sample selection standards are as follows:
1. cases of breast cancer clearly diagnosed by pathology, of which 3 patients had a family history of cancer and were labeled X1, X2, X3, respectively;
2. before blood collection, radiotherapy or chemotherapy is not received, and no history of previous tumor is available;
3. healthy female controls matched to age of case
And the system collects the conditions of demographic data, clinical data and the like of the samples.
Example 2 extraction and purification of peripheral blood DNA
In the above-mentioned eligible 25 breast cancer patients and 10 healthy female controls, the two groups were age-balanced and comparable.
The method comprises the following specific steps:
1. a hemolysis reagent (40 parts of lysate prepared by mixing 219.72g of sucrose, 2.02g of magnesium chloride and 20mL of TrisHcl solution in 20mL of TrisHcl solution was added to peripheral blood stored in a 2mL cryopreserved tube, and the volume was adjusted to 2000mL, as described below), and the mixture was inverted and mixed to complete the transfer.
2. Removing red blood cells: the 5mL centrifuge tube was made up to 4mL with the hemolysis reagent, mixed by inversion, centrifuged at 4000rpm for 10 minutes and the supernatant discarded. 4mL of the hemolysis reagent was added to the pellet, washed once again by inversion, centrifuged at 4000rpm for 10 minutes, and the supernatant was discarded.
3. Extracting DNA: to the precipitate were added 1mL of an extract (containing 122.5mL of 0.2M sodium chloride, 14.4mL of 0.5M ethylenediaminetetraacetic acid, 15mL of 10% sodium dodecylsulfate, 148.1mL of double distilled water, the same applies below) and 8. mu.L of proteinase K per 300mL, followed by shaking thoroughly on a shaker, mixing well, and water bath at 37 ℃ overnight.
4. Removing proteins: add 1mL of saturated phenol and mix well (shake gently for 15 minutes), centrifuge at 4000rpm for 10 minutes, and transfer the supernatant to a new 5mL centrifuge tube. To the supernatant was added a mixture of chloroform and isoamyl alcohol (chloroform: isoamyl alcohol: 24:1, v/v, same below), and after thoroughly mixing (shaking by hand for 15 minutes), the mixture was centrifuged at 4000rpm for 10 minutes, and the supernatant was collected (divided into two 1.5mL centrifuge tubes).
5. DNA precipitation: adding 3M sodium acetate 60 μ L into the supernatant, adding ice anhydrous ethanol with the same volume as the supernatant, shaking up and down to obtain white flocculent precipitate, and centrifuging at 12000rpm for 10 min.
6. DNA washing: adding 1mL of ice absolute ethyl alcohol into the precipitate, centrifuging at 12000rpm for 10min, removing the supernatant, and then vacuum-drying or placing in a clean and dry environment for evaporation.
7. And (3) measuring the concentration: 20-50 ng/. mu.LDNA can be obtained in general, and the purity (UV 2600D: 2800D) is 1.8-2.0.
Example 3 Whole exome detection of SNPs
The two groups of people in example 2 were tested by whole exon chip to obtain the relevant results.
1. Library construction
The Agilent liquid phase chip capture system is adopted by Beijing Nuo He-derived science and technology Co., Ltd to efficiently enrich human DNA in the whole exon region, and then high-throughput and high-depth sequencing is carried out on an IlluminaHiseq platform. The Agilent SureSelectHumanAll Exon V5 kit is adopted in the library building and capturing experiment, the reagents and consumables recommended by the instruction are strictly used, and the operation is carried out according to the latest optimized experiment flow.
The basic experimental process is as follows: randomly breaking the genome DNA into fragments with the length of 180-280bp by a Covaris breaker, and respectively connecting adapters at two ends of the fragments after end repair and A tail addition to prepare a DNA library. Carrying out liquid phase hybridization on the library pooling with the specific index and 543,872 biotin-labeled probes, capturing 334,378 exons of 20,965 genes by using magnetic beads with streptomycin, carrying out PCR linear amplification, carrying out library quality inspection, and carrying out on-machine sequencing if the library is qualified.
2. Warehouse inspection
After the library is constructed, firstly using Qubit2.0 to carry out preliminary quantification, diluting the library to 1 ng/. mu.L, then using Agilent 2100 to detect the insert size of the library, and after the insert size meets the expectation, using a Q-PCR method to accurately quantify the effective concentration of the library (the effective concentration of the library is more than 2nM) so as to ensure the quality of the library.
3. Sequencing on machine
And (4) if the library is qualified, carrying out Illumina Hiseq platform sequencing according to the effective concentration of the library and the data output requirement.
4. Data analysis and processing
Through data screening, deep processing, bioinformatics sequence comparison and genetic analysis, 53 SNP loci with significantly different genotype distribution frequencies in a breast cancer case group and a healthy female control group are finally determined to be preferred sensitive loci. The protein function prediction is carried out by using programs such as SIFT, Polyphen-2 and Mutation Taster, and the influence value result of the SNP Mutation site on the protein is shown in Table 1:
TABLE 112 values of the influence of SNP mutation sites of genes on proteins
| Serial number | Gene abbreviation | dbSNP_RS | ljb23_sift | ljb23_pp2hvar | ljb23_pp2hdiv | ljb23_mt |
| 1 | NGFR | rs114337127 | 0.41,0.59,T | 0.0,B | 0.0,B | 1.000,1.000,D |
| 2 | TBXA2R | rs13306048 | 0,1.00,D | . | . | 1,0.0,N |
| 3 | KITLG | rs139477122 | 0.06,0.94,T | 0.006,B | 0.003,B | 0.961,0.961,D |
| 4 | ADRM1 | rs140787599 | 0.03,0.97,D | 0.166,B | 0.763,P | 1,1.0,D |
| 5 | CYBRD1 | rs16859487 | 0.07,0.93,T | 0.998,D | 1.0,D | 1,1.0,D |
| 6 | ADAMTS10 | rs199972750 | 0.13,0.87,T | 0.3,B | 0.914,P | 1.000,1.000,D |
| 7 | KIAA1407 | rs200315000 | 0.25,0.75,T | 0.0,B | 0.0,B | 1,1.0,D |
| 8 | NRK | rs35115195 | 0,1.00,D | 0.998,D | 1.0,D | 1,1.0,D |
| 9 | ARHGAP18 | rs368392829 | . | 0.145,B | 0.482,P | 1,1.0,D |
| 10 | TBKBP1 | rs369896558 | 0.19,0.81,T | 0.049,B | 0.147,B | 0.670,0.670,D |
| 11 | CAPN15 | rs372909717 | 0,1.00,D | 0.998,D | 1.0,D | 1,1.0,D |
| 12 | ZNF598 | rs373617497 | 0.04,0.96,D | 0.99,D | 1.0,D | 1.000,1.000,D |
The sites can be identified as breast cancer candidate markers through bioinformatics analysis.
Example 4 further analysis of risk of SNP and Breast cancer onset Risk Using Risk score method
The inventor selects positively associated SNPs by comparing the genotype distribution frequency of 2 groups of samples (a breast cancer case group and a healthy female control group), further calculates the risk score by taking the regression coefficient of a single SNP in a whole exon scanning sample as a weight, and draws ROC to evaluate the sensitivity and specificity of diagnosis, thereby diagnosing the judgment capability of the SNPs on the breast cancer. The combined analysis of all SNP markers revealed SNP mutations in 13 genes in Table 1: rs114337127, rs13306048, rs139477122, rs140787599, rs16859487, rs199972750, rs200315000, rs35115195, rs368392829, rs369896558, rs372909717 and rs373617497, and the sensitivity and specificity of the kit are more than 60%.
Therefore, the inventors have demonstrated that the site marker can well distinguish healthy female controls from breast cancer patients.
Example 5 genotyping of Individual SNPs
1. The same DNA samples were taken from 5 breast cancer patients and 5 healthy women as in example 2;
2. PCR amplification
Using online primer design software provided by the NCBI website and primer premier 5.0, https:// www.ncbi.nlm.nih.gov/tools/primer-blast/index. cgi? Specific amplification primers for a single SNP designed for 12 genes by LINK _ LOC ═ BlastHo meAd are shown in table 2.
TABLE 2 primer sequences
The PCR reaction system is shown in Table 3. The PCR amplification procedure was: pre-denaturing at 95 ℃ for 10min, denaturing at 94 ℃ for 15s, annealing at 58-68 ℃ (according to different Tm values of different primers) for 15s, extending at 72 ℃ for 30s, performing 30 cycles, finally extending at 72 ℃ for 30min, storing at 4 ℃, and standing overnight for freezing at-20 ℃.
TABLE 3 reaction System
| Components | Amount of addition |
| 2×mix | 25μL |
| Upstream primer (10uM) | 3.0μL |
| Downstream primer (10uM) | 3.0μL |
| Form panel | 5μL |
| Adding sterilized distilled water | To 50 μ L |
3. Sequencing
After the PCR amplification is finished, taking 5 mu L of amplification product, carrying out 1% agarose gel electrophoresis, carrying out electrophoresis for 30min, dyeing for 20min, then placing the gel block in a gel imager for observation, and preliminarily judging whether the amplified fragment is correct or not according to the condition of comparing the size of the Marker fragment. And further purifying the amplification product which meets the requirements: the Mag-BindOligonucleotide labeling kit is adopted and operated according to the requirements of the kit. Loading and sequencing: adopting a BigDye3.1Sequeningkit kit of ABI company, and operating according to the requirements of the kit; sequencing was performed using an ABI model 3730 sequencer.
4. Analysis of results
And comparing the sequencing result with the standard sequence through Chromas sequence analysis software, searching for the SNP locus, and analyzing the type of the base at the SNP locus to obtain the genotype of the SNP locus. The results showed that the 12 SNP sites were true mutations.
Therefore, the 12 SNP loci can be further confirmed to be used for auxiliary diagnosis such as detection, treatment, diagnosis, prognosis evaluation and the like of the breast cancer.
EXAMPLE 6 preparation of SNP kit for breast cancer auxiliary diagnosis
Based on the primer set obtained in example 5, the kit for breast cancer of the present invention is assembled, the kit includes specific primers for amplifying the 12 SNP sites, and the kit may further include common reagents required by corresponding PCR technology, such as: dNTPs, MgCl2Double distilled water, Taq enzyme, etc., which are well known to those skilled in the art, and in addition, there may be a standard and a control (e.g., a genotype-determining standard and a blank, etc.). The kit has the value that only peripheral blood is needed, other tissue samples are not needed, SNP is detected through the simplest and most specific primer pair, and then breast cancer is judged through the assistance of the SNP spectrum, so that the kit is stable, convenient and accurate to detect, and greatly improves the sensitivity and specificity of disease diagnosis, and therefore the kit can help to guide diagnosis and more effective individualized treatment when put into practice.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> Shenzhen second people hospital
<120> a group of biomarkers for auxiliary diagnosis of breast cancer and application thereof
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Claims (5)
1. The biomarker is a combination of SNP loci rs114337127, rs13306048, rs139477122, rs140787599, rs16859487, rs199972750, rs200315000, rs35115195, rs368392829, rs369896558, rs372909717 and rs 371743697.
2. The use of claim 1, wherein the specific amplification primer sequences for amplifying the SNP sites of the biomarkers are:
the sequence of the amplification primer of rs114337127 is SEQ ID No: 1 and SEQ ID No: 2;
the sequence of the amplification primer of rs13306048 is SEQ ID No: 3 and SEQ ID No: 4;
the amplification primer sequence of rs139477122 is SEQ ID No: 5 and SEQ ID No: 6;
the sequence of the amplification primer of rs140787599 is SEQ ID No: 7 and SEQ ID No: 8;
the sequence of the amplification primer of rs16859487 is SEQ ID No: 9 and SEQ ID No: 10;
the amplification primer sequence of rs199972750 is SEQ ID No: 11 and SEQ ID No: 12;
the sequence of the amplification primer of rs200315000 is SEQ ID No: 13 and SEQ ID No: 14;
the sequence of the amplification primer of rs35115195 is SEQ ID No: 15 and SEQ ID No: 16;
the sequence of the amplification primer of rs368392829 is SEQ ID No: 17 and SEQ ID No: 18;
the sequence of the amplification primer of rs369896558 is SEQ ID No: 19 and SEQ ID No: 20;
the amplification primer sequence of rs372909717 is SEQ ID No: 21 and SEQ ID No: 22;
the sequence of the amplification primer of rs373617497 is SEQ ID No: 23 and SEQ ID No: 24.
3. the use of claim 1, wherein said product is a reagent for detecting the genotype of said SNP site, said reagent being capable of detecting a combination of rs114337127, rs13306048, rs139477122, rs140787599, rs16859487, rs199972750, rs200315000, rs35115195, rs368392829, rs369896558, rs372909717 and rs 371743697 in the peripheral blood DNA.
4. The use of claim 3, wherein the reagents comprise specific primers for amplifying the SNP site or comprise specific primers and restriction enzymes for amplifying the SNP site.
5. The use of claim 4, wherein the specific primer sequence has the sequence of SEQ ID NO: 1-24.
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| CN104178567A (en) * | 2014-07-22 | 2014-12-03 | 南京医科大学 | Mammary cancer assisted diagnosis related SNP (single-nucleotide polymorphism) marker and application thereof |
| CN106399304A (en) * | 2016-11-18 | 2017-02-15 | 深圳市第二人民医院 | Breast cancer related SNP marker |
| CN106636351A (en) * | 2016-11-11 | 2017-05-10 | 深圳大学 | SNP marker related to breast cancer and its application |
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| EP2933639A1 (en) * | 2014-04-16 | 2015-10-21 | Deutsches Krebsforschungszentrum Stiftung des Öffentlichen Rechts | S100p and Hyaluronic acid as biomarkers for metastatic breast cancer |
| CN106337081B (en) * | 2016-02-02 | 2020-01-21 | 中国医学科学院肿瘤医院 | Correlation of SNP locus rs1054135 of FABP4 gene and triple negative breast cancer prognosis |
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| CN104178567A (en) * | 2014-07-22 | 2014-12-03 | 南京医科大学 | Mammary cancer assisted diagnosis related SNP (single-nucleotide polymorphism) marker and application thereof |
| CN106636351A (en) * | 2016-11-11 | 2017-05-10 | 深圳大学 | SNP marker related to breast cancer and its application |
| CN106399304A (en) * | 2016-11-18 | 2017-02-15 | 深圳市第二人民医院 | Breast cancer related SNP marker |
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