CN106811528B

CN106811528B - A kind of breast cancer is cured the disease gene new mutation and its application

Info

Publication number: CN106811528B
Application number: CN201710101599.1A
Authority: CN
Inventors: 杨跃梅
Original assignee: Beijing Zhicheng Biomedical Technology Co Ltd
Current assignee: Beijing Zhicheng Biomedical Technology Co Ltd
Priority date: 2017-02-24
Filing date: 2017-02-24
Publication date: 2019-10-18
Anticipated expiration: 2037-02-24
Also published as: CN110144404B; CN110144403B; CN110144403A; CN110144404A; CN110205322B; CN110205322A; CN106811528A

Abstract

It cures the disease gene new mutation the invention discloses a kind of breast cancer, the new mutation includes four breast cancer susceptibility SNP sites, and specifying information is as shown in table 1.The present invention further discloses application of the new mutation SNP site in breast cancer detection, diagnosis, treatment or prognosis.The present invention has studied susceptible SNP mutation in the application prospect of Computer-aided Diagnosis of Breast Cancer, illustrates influence of the SNP for breast cancer progression, discloses its diagnostic value.Therefore, the present invention passes through the development and application of SNP gene type diagnostic preparation and diagnostic kit, it may make the diagnosis of breast cancer more convenient and easy, conditions of patients is quick and precisely grasped for clinician, it lays the foundation for clinical therapeutic efficacy evaluation, and provide help to be found to have the new small molecule drug targets of potential treatment value.

Description

A kind of breast cancer is cured the disease gene new mutation and its application

Technical field

The present invention relates to field of biomedicine technology, and in particular to a kind of breast cancer is cured the disease gene new mutation and its application.

Background technique

Breast cancer is that a kind of systemic disease, its occurrence and development are one and are related to multifactor, too many levels complex process, The inactivation etc. of activation and tumor suppressor gene including oncogene.Therefore, gene mutation rises in the generation, development process of breast cancer Very important effect.

Breast cancer is a multifactor hereditary variability disease, is only due to caused by single-gene defect less than 10%. It is more and more to be found with breast cancer related gene with the development of high-throughput gene technology, potential heredity on these genes Variation (mononucleotide polymorphic and copy number variation) may cause the difference of breast cancer medicines therapeutic effect.Due to hereditary variation May be affected in the presence of the metabolic pathway and pharmaceutically-active target gene for making anti-tumor drug, so affect the treatment and Prognosis.

SNP (singlenucleotidepolymorphism, SNP, i.e. single nucleotide polymorphism) is 1996 by the U.S. The molecule genetic marker that the human genome research center scholar Lander of the Massachusetts Institute of Technology is proposed, is primarily referred to as gene The horizontal DNA sequence polymorphism caused by a single nucleotide variation of group.The polymorphism that SNP is shown relates only to individually The variation of base, performance are that have conversion, transversion, insertion and missing etc..Single nucleotide polymorphism is third generation genetic marker, human body Many phenotypic differences, all may be related with SNP to neurological susceptibility of drug or disease etc..It is pre- for different parting breast cancer at present Afterwards, the predictive research of curative effect is concentrated mainly on SNP level.

SNP assigns differential responses of the individual to environmental exposure, drug therapy etc., to generate different phenotypes, therefore SNP It may be the important hereditary basis for leading to individual disease development difference.It is diagnosed the illness, is had using the SNP spectrum of disease-susceptible humans Quickly, the features such as sensitive, accurate, thus have a extensive future.In recent years, it is had become using the occurrence and development that SNP diagnoses the illness Clinical and researcher research hotspot.

However, there is presently no the reports that SNP is applied to breast cancer diagnosis, if the SNP of breast cancer susceptibility can be filtered out As biomarker, and corresponding diagnostic kit is developed, will effectively push the status of China's early diagnosing mammary cancer, And new approach is opened up for its drug screening, evaluating drug effect and targeted therapy.

Summary of the invention

The purpose of the present invention is in view of the above technical problems, propose that a kind of breast cancer is cured the disease gene new mutation.

A second object of the present invention is to provide the new mutation answering in breast cancer detection, diagnosis, treatment or prognosis With.

Third object of the present invention is to provide a kind of biological agents for detecting breast cancer susceptibility SNP site genotype.

Fourth object of the present invention is to provide Computer-aided Diagnosis of Breast Cancer kit.

Inventor is by separating and studying patient with breast cancer and compare in peripheral blood DNA with the healthy women of its age-matched Single nucleotide polymorphism, find the SNP of one group of high specific and sensibility highly relevant with breast cancer, and developing can be just In the Computer-aided Diagnosis of Breast Cancer kit of clinical application, data support is provided for the screening and diagnosis of breast cancer.

The purpose of the present invention is what is realized by following technical proposal:

It cures the disease gene new mutation present invention firstly provides a kind of breast cancer, the new mutation includes following four breast cancer Susceptible SNP site, specifying information are as shown in table 1.

1 four, table susceptible SNP site information

Further, the present invention provides new mutation SNP sites described in table 1 in breast cancer detection, diagnosis, treatment or pre- Application in afterwards.

Further, described the present invention provides a kind of biological agent for detecting breast cancer susceptibility SNP site genotype Biological agent includes the primer pair for expanding SNP site described in table 1, or includes for expanding drawing for SNP site described in table 1 Object to and restriction enzyme.

Preferably, expand the SNP site primer pair be according to 5.0 primer-design software of PrimerPremier or The primer pair for the online primer-design software design that ncbi database provides, the present invention selects such as in a preferred embodiment Four breast cancer susceptibility SNP site primer pairs shown in SEQIDNO:1-8.The primer pair high specificity that the present invention selects, amplification Effect is good, and the sequence of amplification can also be used as the biomarker of the Molecular tools such as breast cancer diagnosis, prediction, assessment.

Still further, the present invention provides a kind of kits of Computer-aided Diagnosis of Breast Cancer comprising SNP described in table 1 The reagent of point gene type.

Preferably, the reagent includes the primer pair for expanding SNP site described in table 1, or including for expanding The primer pair and restriction enzyme of the SNP site.

Preferably, expand the SNP site primer pair be according to 5.0 primer-design software of PrimerPremier or The primer pair for the online primer-design software design that ncbi database provides, the present invention selects such as in a preferred embodiment Four breast cancer susceptibility SNP site primer pairs shown in SEQIDNO:1-8, the primer pair high specificity that the present invention selects, amplification Effect is good.

Preferably, the kit further includes that PCR reacts common enzyme and reagent, such as dNTPs, Taq enzyme, Mg²⁺, PCR it is anti- Answer buffer etc.；Standard items and/or reference substance can also be contained.

The invention has the advantages that:

The present invention has studied susceptible SNP mutation in the application prospect of Computer-aided Diagnosis of Breast Cancer, illustrate SNP for breast cancer into The influence of exhibition discloses its diagnostic value.Therefore, the present invention by the development of SNP gene type diagnostic preparation and diagnostic kit and Using may make the diagnosis of breast cancer more convenient and easy, grasp conditions of patients quick and precisely for clinician, be clinical treatment Effect assessment lays the foundation, and provides help to be found to have the new small molecule drug targets of potential treatment value.

Specific embodiment

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment Used in the conventional means that are well known to those skilled in the art of technological means.

Technical solution of the present invention specifically includes: acquiring standard compliant blood sample, system collects complete demography Data and clinical data；Genotype detection: selection breast cancer case is compareed with the healthy women of breast cancer case age-matched, It is sequenced using exon, finds out SNP relevant to breast cancer；To the positive association SNP filtered out, Genotyping is further used It is detected, verifies its repeatability for being applied to clinical diagnosis；The development of Computer-aided Diagnosis of Breast Cancer kit: according to breast cancer The genotype distribution frequency SNP that there were significant differences develops SNP auxiliary diagnostic box in case and healthy women control.

Each numerical value is expressed as follows in data analysis:

1, ljb23_sift:SIFT score value (version 2.3) indicates influence of the variation to protein sequence, includes three A value, first is that SIFT initial value, second is that the value (1-SIFT) after conversion, third is that T or D.When the variation while influencing multiple When protein sequence, there is a SIFT value to every protein sequence, be minimized.SIFT score value is smaller more " nocuousness ", shows the SNP A possibility that causing protein structure or function to change, is big；D:Deleterious (sift≤0.05)；T:tolerated(sift> 0.05))；

2, ljb23_pp2hvar: predict the variation to protein sequence based on HumanVar database using PolyPhen2 It influences, is used for single gene inheritance disease.The column include two values, and first is PolyPhen2 score value, and the numerical value the big more " nocuousness ", Show that a possibility that SNP causes protein structure or function to change is big；Second is D or P or B (D:Probably damaging (>=0.909), P:possiblydamaging (0.447≤pp2_hvar≤0.909)；B:benign (pp2_hvar≤ 0.446))；

3, ljb23_pp2hdiv: predict the variation to protein sequence based on HumanDiv database using PolyPhen2 It influences, is used for complex disease.The column include two values, and first is 2 score value of PolyPhen, and the numerical value the big more " nocuousness ", are shown A possibility that SNP causes protein structure or function to change is big；Second be D or P or B (D:Probably damaging (>= 0.957), (0.453≤pp2_hdiv≤0.956) P:possiblydamaging；B:benign (pp2_hdiv≤ 0.452))；

4, ljb23_mt:Mutation Taster score value (version 2.3) indicates the variation to the shadow of protein sequence It rings, is worth comprising three, first is that Mutation Taster initial value, second is that the value after conversion, third is that A, D, N or P.Second A value more big more " nocuousness " shows that a possibility that SNP causes protein structure or function to change is big, wherein " A " (" disease causing automatic")；"D"("disease causing")；"N"("polymorphism")；"P"(" polymorphism automatic")。

The experimental method specifically studied mainly includes following components:

1. studying the selection of sample

(1) breast cancer case clarified a diagnosis through pathology 25 and healthy women 10 with breast cancer case age-matched Example wherein has 3 patients to have cancer family history as control in breast cancer case；

(2) radiotherapy or chemotherapy were not received, without the past tumour medical history before blood sampling；

(3) it is compareed with the healthy women of case age-matched

2. phenol-chloroform method extracts peripheral blood genomic DNA, operate according to a conventional method.Usually lead to 20-50ng/ μ LDNA, purity (ultraviolet 2600D:2800D) is in 1.6-2.0.

3. full Exon chip detection

(1) subject's complete genome DNA sample is taken；

(2) it is scanned in full Exon chip (Beijing source Nuo Hezhi Science and Technology Co., Ltd., similarly hereinafter)；

(3) detection and difference difference of more each genotype in breast cancer case is compareed with healthy women.

4. the Genotyping of single SNP

(1) subject's DNA sample is taken；

(2) specificity amplification primer of single SNP is designed；

(3) PCR reaction is carried out, recovery product is sequenced；

(4) compare breast cancer case compareed with healthy women in different genotype distributional difference.

5. diagnostic reagent box preparation method

Full Exon chip be scanned with determine breast cancer case after single SNP detection with healthy women and compare in gene The type distribution frequency SNP that there were significant differences, the index as breast cancer diagnosis.The SNP related with pathogenesis of breast carcinoma filtered out Auxiliary diagnostic box comprising detection is located at EEF1D gene NM_001130053:exon3:c.G791A and EEF2:NM_ The reagent of the SNP site genotype of 001961:exon14:c.A2341G, diagnostic kit can also be including the special of these SNP The property reagents such as amplimer and Taq enzyme, dNTPs.

6. clinical application example

Using the present inventor preparation Computer-aided Diagnosis of Breast Cancer kit detection to screening patient with breast cancer and with reality Clinical detection compares so that the validity of Computer-aided Diagnosis of Breast Cancer kit has been determined.Specifically include measurement subject's blood specimen The specificity amplification primer of above-mentioned SNP and other detection reagents in DNA, the disease shape of patient is quick and precisely grasped for clinician State and coincident with severity degree of condition take the control prece of more personalized to provide support in time.

The collection of 1 sample of embodiment and the arrangement of sample data

Inventor has collected a large amount of new hair-cream gland in Shenzhen City Second People's Hospital in January, 2010 in December, 2015 Cancer patient's blood specimen, by the arrangement to sample data, inventor has therefrom selected 25 samples for meeting following standard, simultaneously It selects 10 ages to compare in 25-55 years old healthy women and carries out full Exon chip detection, sample selection criteria is as follows:

1, the breast cancer case clarified a diagnosis through pathology, wherein having 3 patients that there is cancer family history and marking respectively For X1, X2, X3；

2, radiotherapy or chemotherapy were not received, without the past tumour medical history before blood sampling；

3, it is compareed with the healthy women of case age-matched

And situations such as demographic data and clinical data of system acquisition these samples.

The extraction and purifying of 2 peripheral blood DNA of embodiment

In above-mentioned qualified 25 patient with breast cancers and 10 healthy women controls, two groups of age equilibriums are comparable.

Specific steps are as follows:

1, to the peripheral blood addition hemolyzing reagent being stored in 2mL cryopreservation tube, (i.e. lysate, 40 deal configuration methods are such as Under: sucrose 219.72g, magnesium chloride 2.02g and triton x-100 (amresco0694) 20mL mixing after, with TrisHcl solution It is settled to 2000mL, similarly hereinafter), it is transferred to completely after being mixed by inversion.

2, it removing red blood cell: 5mL centrifuge tube being mended to 4mL with hemolyzing reagent, is mixed by inversion, 4000rpm is centrifuged 10 minutes, Abandon supernatant.4mL hemolyzing reagent is added into precipitating, is mixed by inversion cleaning again once, 4000rpm is centrifuged 10 minutes, abandons supernatant.

3, extract DNA: into precipitating plus 1mL extract (contains 122.5mL0.2M sodium chloride, 14.4mL in every 300mL 0.5M ethylenediamine tetra-acetic acid, 15mL10% lauryl sodium sulfate, 148.1mL distilled water, similarly hereinafter) and 8 μ L Proteinase Ks, oscillation Sufficiently oscillation mixes on device, and 37 DEG C of water-baths are stayed overnight.

4, it removes isolating protein: 1mL saturated phenol being added to mix well (hand jog 15 minutes), 4000rpm is centrifuged 10 minutes, is taken It is transferred to clearly in new 5mL centrifuge tube.Be added in supernatant isometric chloroform and isoamyl alcohol mixed liquor (chloroform: isoamyl alcohol=24: 1, v/v, similarly hereinafter), after mixing well (hand 15 minutes), 4000rpm is centrifuged 10 minutes, take supernatant (be divided into two 1.5mL from Heart pipe).

5, DNA is precipitated: the 60 μ L of sodium acetate of 3M being added in supernatant, adds the anhydrous second of the ice isometric with supernatant Alcohol, upper and lower jog, it is seen that white flock precipitate object, then 10min is centrifuged with 12000rpm.

6, DNA is washed: ice dehydrated alcohol 1mL being added in precipitating, 12000rpm is centrifuged 10min, and vacuum is taken out after abandoning supernatant It does or is placed in and be evaporated in cleaning dry environment.

7, it measures concentration: usually leading to 20-50ng/ μ LDNA, purity (ultraviolet 2600D:2800D) is in 1.8-2.0.

The full exon group of embodiment 3SNP detects

Two groups of crowds in embodiment 2 are detected through full Exon chip and obtain correlated results.

1, library construction

Beijing source Nuo Hezhi Science and Technology Co., Ltd. uses the liquid-phase chip capture systems of Agilent, to the complete outer of people Aobvious subregion DNA carries out efficiently concentrating, and high-throughput, high depth sequencing is then carried out on IlluminaHiseq platform.Build library and Capture experiment uses Agilent SureSelectHumanAll ExonV5 kit, the reagent that stringent operation instructions are recommended And consumptive material, and operated referring to the newest experiment flow by optimization.

Experiment basic procedure: genomic DNA is crushed instrument through Covaris and is broken into the piece that length is 180-280bp at random Section is separately connected top connection preparation DNA library at segment both ends after repairing through end and adding A tail.Library with special index With up to 543 after pooling, the probe of 872 biotin labelings carries out solution hybridization, and reusing the magnetic bead with streptomysin will The 334 of 20,965 genes, 378 exon trappings get off, and through the laggard style of writing library quality inspection of PCR linear amplification, qualification can be into Machine is sequenced on row.

2, library is examined

After the completion of library construction, tentatively quantitative, dilution library to 1ng/ μ L is first carried out using Qubit2.0, then use Agilent 2100 detects the insert size in library, after insert size meets expection, uses Q-PCR method pair The effective concentration in library carries out accurate quantitative analysis (library effective concentration > 2nM), to guarantee Library Quality.

3, upper machine sequencing

Library inspection is qualified, carries out the sequencing of Illumina Hiseq platform according to the effective concentration in library and data output demand.

4, data analysis and processing

It is final to determine " breast cancer case " group and " be good for by data screening, deep processing and bioinformatics sequence alignment The genotype distribution frequency found in health female control " group 53 SNP sites that there were significant differences are preferred sensitivity level site.Its In, the present invention has screened four susceptible SNP sites, it makes a variation as shown in table 2 to albumen influence value:

2 SNP mutation Mutation of table is to albumen influence value

Gene	ljb23_sift	ljb23_pp2hvar	ljb23_pp2hdiv	ljb23_mt
					MOGAT3	0.03,0.97,D	0.003,B	0.002,B	0.878,0.122,N
RBM12B	0,1.00,D	0.003,B	0.002,B	1.000,0.000,N
					SEC63	0.91,0.09,T	0.005,B	0.007,B	1.000,1.000,D
TFR2	0.31,0.69,T	0.001,B	0.001,B	1.000,1.000,D

Bioinformatic analysis is passed through in the site, can be confirmed as breast cancer candidate markers.

Embodiment 4 further analyzes the onset risk of SNP and breast cancer using risk score method

The present inventor passes through to 2 groups of samples (" breast cancer case group " and " healthy women control group ") genotype distribution frequency Comparison, select the SNP of positive association, using SNP regression coefficient single in full exon scanned samples as weight, further acquire Dangerous score value draws ROC to evaluate the sensitivity and specificity of diagnosis, and then diagnoses judgement of these SNP to pathogenesis of breast carcinoma Ability.The Conjoint Analysis of all SNP markers is found, four susceptible SNP mutations, sensitivity and spy listed by the present invention Different degree all reaches 60% or more.

Therefore, inventors demonstrated that the site marker can be well by healthy women control and patient with breast cancer area Point.

The Genotyping of the single SNP of embodiment 5

1, take 5 patient with breast cancers and 5 healthy women comparison DNA samples with embodiment 2；

2, PCR amplification

The online primer-design software https: //www.ncbi.nlm.nih.gov/tools/ provided using the website NCBI Primer-blast/index.cgi? LINK_LOC=BlastHomeAd designs the special of single SNP to 4 susceptible SNP sites Property amplimer is as shown in table 3.

3 primer sequence of table

PCR reaction system is as shown in table 4.PCR amplification program are as follows: 95 DEG C of initial denaturation 10min, 94 DEG C of denaturation 15s, 60 DEG C are moved back Fiery 15s, 72 DEG C of extension 30s carry out 30 circulations, and last 72 DEG C of extensions 30min is saved in 4 DEG C, need -20 DEG C of placement cold overnight Freeze.

4 reaction system of table

Component	Additional amount
		2×mix	25μL
Upstream primer (10uM)	3.0μL
		Downstream primer (10uM)	3.0μL
Template	5μL
		Sterile purified water is added	To 50 μ L

3, it is sequenced

After PCR amplification, 5 μ L amplified productions, 1% agarose gel electrophoresis are taken, electrophoresis 30min dyes 20min, so Gel piece is placed in gel imager afterwards and is observed, according to the clip size situation for comparing Marker, tentatively judges amplified fragments It is whether correct.And then satisfactory amplified production is purified: using Mag-BindOligonucleotidePurific AtionKit kit, and operated by kit requirement.Loading sequencing: ABI company is used BigDye3.1SequencingKit kit, and operated by kit requirement；It is carried out with 3730 type sequenator of ABI company Sequencing.

4, interpretation of result

By Chromas sequence analysis software, sequencing result is compared with standard sequence, finds SNP site, pass through Analyze the type of base at SNP site, so that it may obtain the genotype of SNP site.Above-mentioned 4 SNP sites are true as the result is shown Real existing variation.

It is auxiliary to further confirm that 4 SNP sites can be used for detection, treatment, diagnosis, prognosis evaluation of breast cancer etc. Help diagnosis.

Embodiment 6 is used for the production of Computer-aided Diagnosis of Breast Cancer SNP kit

Based on the primer sets that embodiment 5 obtains, the kit of the present invention for breast cancer, the kit are assembled Including the specific primer for expanding 4 SNP sites, the kit can also have normal needed for corresponding round pcr With reagent, such as: dNTPs, MgCl₂, distilled water, Taq enzyme etc., these common agents be all it is well known to those skilled in the art, separately Can also there are standard items and control (such as determining standard items and the blank control of genotype) outside.The value of this kit is only It needs peripheral blood without other tissue samples, detects SNP with special primer pair by most simplifying, then composed by SNP auxiliary Judgement breast cancer is helped, it is not only stable, easy to detect and accurate, the sensibility and specificity of medical diagnosis on disease is greatly improved, therefore will The investment practice of this kit, can help to instruct diagnosis and more effective individualized treatment.

Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.

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Claims

1. detecting the reagent of gene M OGAT3 new mutation SNP site genotype in preparation breast cancer detection, diagnosis, treatment or prognosis Application in product, the new mutation SNP site are SNP position of No. 7 the 100839317th bit base of chromosome of the mankind by G to A Point mutation, the site mutation are MOGAT3:NM_001287147:exon6:c.G733A:p.R245C.

2. application as described in claim 1, which is characterized in that the product is biological agent, and the biological agent includes using In the primer pair of amplification gene MOGAT3 new mutation SNP site, or including being used for amplification gene MOGAT3 new mutation SNP site Primer pair and restriction enzyme.

3. application as claimed in claim 2, which is characterized in that the primer pair of amplification gene MOGAT3 new mutation SNP site Nucleotide sequence is as shown in SEQ ID NO:1-2.

4. application as described in claim 1, which is characterized in that the product is kit, and the kit includes for expanding Increase the primer pair of the SNP site, or includes the primer pair and restriction enzyme for expanding the SNP site.

5. application as claimed in claim 4, which is characterized in that the nucleotide sequence of the primer pair such as SEQ ID NO:1-2 It is shown.

6. application as claimed in claim 4, which is characterized in that the kit further includes dNTPs, Taq enzyme, Mg²⁺It is anti-with PCR Answer buffer.