阅读说明：本技术 一种用于乳腺癌辅助诊断的突变基因及其应用 (Mutant gene for breast cancer auxiliary diagnosis and application thereof ) 是由 沈洪兵 胡志斌 马红霞 江玥 闻洋 于 2015-06-02 设计创作，主要内容包括：本发明属于基因工程及肿瘤医学领域,公开了一种用于乳腺癌辅助诊断的突变基因及其应用。该突变基因具有下列一个或多个突变位点：与正常BRCA1基因序列相比具有g.41215910delA、g.41244291delT、g.41223130A>T、g.41256139T>A突变位点；与正常BRCA2基因序列相比具有g.32912699A>G、g.32912799T>C、g.32929399dupT、g.32911757C>T、g.32911659-32911662delAAAA、g.32910963T>G等突变位点。本发明提供了乳腺癌致病的突变位点组合,可用于早期诊断乳腺癌。(The invention belongs to the field of genetic engineering and tumor medicine, and discloses a mutant gene for breast cancer auxiliary diagnosis and application thereof. The mutant gene has one or more of the following mutation sites: compared with the normal BRCA1 gene sequence, the gene has g.41215910delA, g.41244291delT, g.41223130A > T and g.41256139T > A mutation sites; compared with the normal BRCA2 gene sequence, the mutant sites of g.32912699A > G, g.32912799T > C, g.32929399dupT, g.32911757C > T, g.32911659-32911662delAAAA, g.32910963T > G and the like are provided. The invention provides a mutation site combination for breast cancer pathogenesis, which can be used for early diagnosis of breast cancer.)

1. A mutant gene for the auxiliary diagnosis of breast cancer in Han-nationality women, which is characterized in that the mutant gene has one or more of the following mutation sites:

has g.32911757C > T mutation site compared with the normal BRCA2 gene sequence.

2. The use of the mutant gene of claim 1 in the preparation of a Han female breast cancer auxiliary diagnostic kit.

Technical Field

The invention belongs to the field of genetic engineering and tumor medicine, and relates to a mutant gene for breast cancer auxiliary diagnosis and application thereof.

Background

Breast cancer is the second most common malignancy worldwide, with the top being among female malignancies. According to statistics of international cancer research center of world health organization, 167 thousands of new cases of breast cancer of women worldwide in 2012 account for 25% of malignant tumors of all women. Meanwhile, the breast cancer is the fifth common cause of death of malignant tumors worldwide and is the first cause of death of female malignant tumors, and 52 thousands of people worldwide die of breast cancer and account for 14.7 percent of all female malignant tumor deaths. In 2012, the number of new breast cancer cases of Chinese women is 18.7 ten thousand, and the number of the cases is second to that of lung cancer, wherein 4.8 ten thousand women die due to breast cancer and live in the sixth place of the death of malignant tumors of women.

The breast cancer is malignant tumor with strong genetic background, and 5-10% of breast cancers are caused by breast cancer susceptibility gene mutation with high penetrance rate. BRCA1, BRCA2, TP53, PALB2, PTEN, CHEK2, ATM, RAD50, etc. are identified as breast cancer susceptibility genes, and breast cancers bearing germline mutations in these genes are referred to as hereditary breast cancers, of which at least 30% are due to BRCA1 and BRCA2 mutations.

The presence of mutant sites is thought to confer different phenotypic traits to individuals, as well as different reactivities to environmental exposure, drug therapy, etc., and thus the mutant sites may be an important genetic basis for the differences in susceptibility of individuals to common diseases. The mutation site spectrum of the disease is used for the auxiliary diagnosis of the disease, and has wide application prospect. In recent years, the auxiliary diagnosis of diseases by using mutation sites becomes a research hotspot of clinical and scientific researchers, and the application value of the auxiliary diagnosis on common and serious diseases such as tumors, congenital diseases, cardiovascular and cerebrovascular diseases and the like is very obvious.

Hereditary breast cancer is more clinically characterized by familial clustering, advanced age, bilateral breast cancer and combination of other tumors, and therefore these people are considered to be high risk groups for breast cancer. Genetic risk assessment and genetic testing for high risk populations has become the standard medical choice in many countries in the europe and the united states. And a series of preventive measures can be taken aiming at mutation carriers, such as early entering breast cancer screening, and the screening frequency is improved, the use of preventive medicines and the implementation of various preventive operations are improved. About 30% of hereditary breast cancers are due to mutations in BRCA1 and BRCA2, and carriers of functional mutations in BRCA1 and BRCA2 are also highly likely to develop into breast cancer patients, especially carriers of family history of breast cancer. The chinese BRCA1 and BRCA2 gene tests were initiated late and despite several related studies, no sufficient number of mutation "hot spots" have been found, especially in chinese populations specific breast cancer-related mutations. Moreover, due to ethnic difference, the mutation spectrum of European and American countries is not completely suitable for Chinese population, if mutation sites related to the breast cancer onset can be screened out as biomarkers, and corresponding diagnostic kits are developed, the method can be used for effectively promoting the breast cancer screening and early diagnosis in China.

Disclosure of Invention

The invention aims to provide a mutant gene for breast cancer auxiliary diagnosis.

Another object of the present invention is to provide specific primers for detecting the above-mentioned mutation sites.

The third purpose of the invention is to provide the application of the mutant gene and the specific primer in the preparation of an auxiliary diagnostic kit for breast cancer.

The fourth purpose of the invention is to provide a breast cancer auxiliary diagnosis kit.

The inventor searches high-specificity mutation sites related to the breast cancer by separating and researching the mutation in the peripheral blood DNA of a breast cancer patient and a healthy control matched with the breast cancer patient in age, develops a breast cancer auxiliary diagnosis kit applicable to clinic or people, and provides support for screening and diagnosing the breast cancer.

The purpose of the invention is realized by the following technical scheme:

a mutant gene for breast cancer auxiliary diagnosis, which has one or more of the following mutation sites:

compared with the normal BRCA1 gene sequence, the gene has g.41215910delA, g.41244291delT, g.41223130A > T and g.41256139T > A mutation sites;

compared with the normal BRCA2 gene sequence, the mutant has mutation sites of g.32912699A > G, g.32912799T > C, g.32914767-32914768delTT, g.32906615-32906619del5, g.32929399dupT, g.32911757C > T, g.32911659-32911662delAAAA, g.32914173-32914174insA, g.32910963T > G and g.32929083C > A.

The normal BRCA1 gene is numbered NC-000017.10 in NCBI reference database GRCh37.p13 (41196312 and 41277500), and the normal BRCA2 gene is numbered NC-000013.10 in NCBI reference database GRCh37.p13 (32889617 and 32973809).

The specific primers for detecting the mutation sites in the mutant genes are as follows:

g.41215910delA primers are SEQ ID NO.1 and SEQ ID NO. 2;

g.41244291delT primers are SEQ ID NO.3 and SEQ ID NO. 4;

g.41223130A > T primers are SEQ ID NO.5 and SEQ ID NO. 6;

g.41256139T > A primers are SEQ ID NO.7 and SEQ ID NO. 8;

g.32912699A > G primers are SEQ ID NO.9 and SEQ ID NO. 10;

g.32912799T > C primers are SEQ ID NO.11 and SEQ ID NO. 12;

g.32914767-32914768delTT primers are SEQ ID NO.13 and SEQ ID NO. 14;

g.32906615-32906619del5 primers SEQ ID NO.15 and SEQ ID NO. 16;

g.32929399dupT primers are SEQ ID NO.17 and SEQ ID NO. 18;

g.32911757C > T primers are SEQ ID NO.19 and SEQ ID NO. 20;

g.32911659-32911662delAAAA primers SEQ ID NO.21 and SEQ ID NO. 22;

g.32914173-32914174insA primers are SEQ ID NO.23 and SEQ ID NO. 24;

g.32910963T > G primers are SEQ ID NO.25 and SEQ ID NO. 26;

the primers of 32929083C > A are SEQ ID NO.27 and SEQ ID NO. 28.

The mutant gene is applied to the preparation of an auxiliary diagnostic kit for breast cancer.

The primer is applied to the preparation of an auxiliary breast cancer diagnosis kit.

An auxiliary diagnostic kit for breast cancer, which is used for detecting the mutant gene in peripheral blood DNA. I.e. detecting whether there is one or more of the following mutation sites in the peripheral blood DNA: whether the BRCA1 gene has g.41215910delA, g.41244291delT, g.41223130A > T and g.41256139T > A mutation sites or not; whether the BRCA2 gene has mutation sites of g.32912699A > G, g.32912799T > C, g.32914767-32914768delTT, g.32906615-32906619del5, g.32929399dupT, g.32911757C > T, g.32911659-32911662delAAAA, g.32914173-32914174insA, g.32910963T > G and g.32929083C > A.

The diagnostic kit contains one or more of the primers.

The diagnostic kit can also comprise reagents commonly used in PCR reaction.

Specifically, the method comprises the following steps:

according to the invention, the Sanger sequencing technology is adopted to find that 1 patient has g.41215910delA mutation on BRCA1 gene in 70 Chinese Han female breast cancer patients with family history of breast cancer; 1 patient presented with g.41244291delT mutation in BRCA1 gene; 1 patient had the g.41223130a > T mutation in BRCA1 gene; 1 patient presented with the g.41256139t > a mutation in the BRCA1 gene; 1 patient had the g.32912699A > G mutation in the BRCA2 gene; 1 patient had the g.32912799T > C mutation in the BRCA2 gene; 1 patient presented with a g.32914767-32914768delTT mutation in the BRCA2 gene; 1 patient had the g.32906615-32906619del5 mutation in the BRCA2 gene; 1 patient presented with the g.32929399dupT mutation in the BRCA2 gene; 1 patient presented with the g.32911757c > T mutation in the BRCA2 gene; 1 patient presented with the g.32911659-32911662delAAAA mutation in the BRCA2 gene; 1 patient presented with the g.32914173-32914174insA mutation in BRCA2 gene; 1 patient presented with the g.32910963t > G mutation in the BRCA2 gene; in 1 patient there was a mutation in the BRCA2 gene of g.32929083C > A.

Then, family history-free Chinese Han nationality breast cancer sample verification is carried out, and in 3000 family history-free Chinese Han nationality breast cancer patients, 1 patient is found to have g.41215910delA mutation on BRCA1 gene; 8 patients had the g.41244291delT mutation in the BRCA1 gene; 3 patients had the g.41223130a > T mutation in the BRCA1 gene; 5 patients had the g.41256139t > a mutation in the BRCA1 gene; 3 patients had the g.32912699A > G mutation in the BRCA2 gene; 8 patients had the g.32912799T > C mutation in the BRCA2 gene; 3 patients had g.32914767-32914768delTT mutations in the BRCA2 gene; 1 patient had the g.32906615-32906619del5 mutation in the BRCA2 gene; 5 patients had the g.32929399dupT mutation in the BRCA2 gene; 4 patients had the g.32911757c > T mutation in the BRCA2 gene; 6 patients had g.32911659-32911662delAAAA mutations in the BRCA2 gene; 3 patients had the g.32914173-32914174insA mutation in BRCA2 gene; 3 patients had the g.32910963t > G mutation in the BRCA2 gene; 2 patients had a mutation in the BRCA2 gene of g.32929083c > a; no mutant individuals were found in 3000 age-matched control populations of normal women from Han nationality of China with no family history of tumors. Therefore, the positive predictive value of the group of mutant compositions for breast cancer is 100%, the incidence rate in the human familial breast cancer patients is 20%, and the incidence rate in the sporadic breast cancer patients is 1.83%.

The technical scheme for solving the problems comprises the following steps: (1) screening new mutations related to breast cancer of BRCA1 and BRCA2 genes in the population, providing a base sequence (2) after mutation of sites, and establishing a unified standard specimen library and a database: standard procedures (SOP) were used to collect blood samples meeting the standards and the system collected complete demographic and clinical data. (3) Mutation screening and validation of its effect: in 70 Chinese Han female breast cancer patients with family history of breast cancer, the Sanger sequencing technology is adopted, and 1 patient is found to have g.41215910delA mutation on BRCA1 gene; 1 patient presented with g.41244291delT mutation in BRCA1 gene; 1 patient had the g.41223130a > T mutation in BRCA1 gene; 1 patient presented with the g.41256139t > a mutation in the BRCA1 gene; 1 patient had the g.32912699A > G mutation in the BRCA2 gene; 1 patient had the g.32912799T > C mutation in the BRCA2 gene; 1 patient presented with a g.32914767-32914768delTT mutation in the BRCA2 gene; 1 patient had the g.32906615-32906619del5 mutation in the BRCA2 gene; 1 patient presented with the g.32929399dupT mutation in the BRCA2 gene; 1 patient presented with the g.32911757c > T mutation in the BRCA2 gene; 1 patient presented with the g.32911659-32911662delAAAA mutation in the BRCA2 gene; 1 patient presented with the g.32914173-32914174insA mutation in BRCA2 gene; 1 patient presented with the g.32910963t > G mutation in the BRCA2 gene; in 1 patient there was a mutation in the BRCA2 gene of g.32929083C > A. Subsequently, family history-free breast cancer sample verification is carried out, and in 3000 Chinese Han family female breast cancer patients without family history, 1 patient is found to have g.41215910delA mutation on BRCA1 gene; 8 patients had the g.41244291delT mutation in the BRCA1 gene; 3 patients had the g.41223130a > T mutation in the BRCA1 gene; 5 patients had the g.41256139t > a mutation in the BRCA1 gene; 3 patients had the g.32912699A > G mutation in the BRCA2 gene; 8 patients had the g.32912799T > C mutation in the BRCA2 gene; 3 patients had g.32914767-32914768delTT mutations in the BRCA2 gene; 1 patient had the g.32906615-32906619del5 mutation in the BRCA2 gene; 5 patients had the g.32929399dupT mutation in the BRCA2 gene; 4 patients had the g.32911757c > T mutation in the BRCA2 gene; 6 patients had g.32911659-32911662delAAAA mutations in the BRCA2 gene; 3 patients had the g.32914173-32914174insA mutation in BRCA2 gene; 3 patients had the g.32910963t > G mutation in the BRCA2 gene; 2 patients had a mutation in the BRCA2 gene of g.32929083c > a; no mutant individuals were found in 3000 age-matched control populations of normal women from Han nationality of China with no family history of tumors. (3) The screened mutation sites need to meet the condition of no frequency in normal population and are not reported by various databases (such as dbSNP, HGMD, COSMIC and the like). (4) Development of an auxiliary diagnosis kit for breast cancer: and developing a mutation site auxiliary diagnosis kit according to the unique mutation site of the breast cancer case.

The present inventors collected blood samples meeting the standard using Standard Operating Procedures (SOPs), collected complete demographic data, clinical data, etc., systematically, and scanned the exon-coding regions of BRCA1 gene and BRCA2 gene using Sanger sequencing.

The experimental method of research mainly includes the following parts:

1. selection of research sample (all Chinese Han nationality)

(1) Cases of breast cancer with a family history of breast cancer clearly diagnosed by pathology;

(2) sporadic breast cancer cases clearly diagnosed by pathology;

(3) healthy female controls matched to the age of the case;

6070 samples meeting the standard are adopted for the research.

2. Extracting peripheral blood genome DNA by phenol-chloroform method, and performing conventional method. 20-50 ng/. mu.l DNA can be obtained usually, and the purity (the ratio of ultraviolet 260OD to 280 OD) is 1.6-2.0.

Sanger sequencing technology for screening mutation sites

(1) Taking a whole genome DNA sample of a subject;

(2) designing a Primer on line by adopting Primer 3 software;

(3) the exon coding regions of the BRCA1 and BRCA2 genes were scanned using Sanger sequencing;

(4) the individual differences of each genotype in breast cancer cases and healthy female controls were detected and compared.

4. Combination of mutation sites genotyping Using Sanger sequencing platform

(1) Taking a DNA sample of a subject;

(2) designing a Primer on line by adopting Primer 3 software;

(3) the mutated exon coding regions of BRCA1 gene and BRCA2 gene were scanned using Sanger sequencing;

(4) differences in the distribution of different genotypes between breast cancer cases and healthy female controls were detected and compared.

5. Method for preparing diagnostic kit

The mutation sites with genotype distribution difference between breast cancer cases and healthy controls are determined after the exon coding regions of BRCA1 and BRCA2 genes are scanned and single mutation site detection is carried out by adopting Sanger sequencing, and the mutation sites are used as the index for breast cancer diagnosis. The finally screened mutation site combinations related to the breast cancer onset form an auxiliary diagnostic kit (g.41215910delA, g.41244291delT, g.41223130A > T, g.41256139T > A on BRCA1 gene, g.32912699A > G, g.32912799T > C, g.32914767-32914768delTT, g.32906615-32906619del5, g.329399dupT, g.32911757C > T, g.32911659-32911662delAAAA, g.32419173-32914174 insA, g.32910963T > G, g.32928383838383A > A). The diagnostic reagent may include a primer specific to the mutation site, and reagents such as Taq enzyme and dNTP.

6. Statistical analysis method

Differences in the distribution of demographic characteristics, etc. among groups of study subjects were compared using the chi-square test (for categorical variables) or student t-test (for continuity variables).

The following is a further description of the invention:

in the 70 family history breast cancer cases described above we scanned the exon coding regions of the BRCA1 and BRCA2 genes using Sanger sequencing for relevant results.

According to the Sanger sequencing detection result, the inventor detects that 1 of 70 Chinese Han female breast cancer patients with family history of breast cancer has g.41215910delA mutation on BRCA1 gene; 1 patient presented with g.41244291delT mutation in BRCA1 gene; 1 patient had the g.41223130a > T mutation in BRCA1 gene; 1 patient presented with the g.41256139t > a mutation in the BRCA1 gene; 1 patient had the g.32912699A > G mutation in the BRCA2 gene; 1 patient had the g.32912799T > C mutation in the BRCA2 gene; 1 patient presented with a g.32914767-32914768delTT mutation in the BRCA2 gene; 1 patient had the g.32906615-32906619del5 mutation in the BRCA2 gene; 1 patient presented with the g.32929399dupT mutation in the BRCA2 gene; 1 patient presented with the g.32911757c > T mutation in the BRCA2 gene; 1 patient presented with the g.32911659-32911662delAAAA mutation in the BRCA2 gene; 1 patient presented with the g.32914173-32914174insA mutation in BRCA2 gene; 1 patient presented with the g.32910963t > G mutation in the BRCA2 gene; in 1 patient there was a mutation in the BRCA2 gene of g.32929083C > A.

Subsequently, family history-free breast cancer sample verification is carried out, and in 3000 family history-free Chinese Han family breast cancer patients, 1 patient is found to have g.41215910delA mutation on BRCA1 gene; 8 patients had the g.41244291delT mutation in the BRCA1 gene; 3 patients had the g.41223130a > T mutation in the BRCA1 gene; 5 patients had the g.41256139t > a mutation in the BRCA1 gene; 3 patients had the g.32912699A > G mutation in the BRCA2 gene; 8 patients had the g.32912799T > C mutation in the BRCA2 gene; 3 patients had g.32914767-32914768delTT mutations in the BRCA2 gene; 1 patient had the g.32906615-32906619del5 mutation in the BRCA2 gene; 5 patients had the g.32929399dupT mutation in the BRCA2 gene; 4 patients had the g.32911757c > T mutation in the BRCA2 gene; 6 patients had g.32911659-32911662delAAAA mutations in the BRCA2 gene; 3 patients had the g.32914173-32914174insA mutation in BRCA2 gene; 3 patients had the g.32910963t > G mutation in the BRCA2 gene; 2 patients had a mutation in the BRCA2 gene of g.32929083c > a; no mutant individuals were found in 3000 age-matched normal female control populations without family history of tumors.

Based on the above experimental results, the present inventors have found a group of mutation site combinations that can be used for breast cancer auxiliary diagnosis.

Particularly, the combination of the mutations is helpful for auxiliary diagnosis of breast cancer, and provides support for clinicians to quickly and accurately master the disease state and the disease severity of patients and to timely adopt more personalized prevention and treatment schemes.

The invention has the beneficial effects that:

the mutation site sequence change provided by the invention is used as a marker for breast cancer auxiliary judgment, and has the advantages that:

(1) the mutation site is a novel gene biomarker which is different from the traditional biomarker, is stable, minimally invasive and easy to detect, can greatly improve the sensitivity and specificity of disease diagnosis, and the successful development of the biomarker can create a brand new situation for the diagnosis and treatment of breast cancer and provide reference for the development of other disease biomarkers.

(2) By adopting a rigorous verification and evaluation system, the inventor scans exon coding regions of BRCA1 and BRCA2 genes by adopting Sanger sequencing in the initial stage to obtain a mutation site spectrum related to diseases, and verifies the mutation site spectrum in a large sample of patients with sporadic breast cancer by applying the Sanger sequencing method; the application of the method and the strategy accelerates and ensures the clinical application of the mutation site biomarker and the diagnostic kit, and provides a method and a reference for the strategy for the development of other disease biomarkers.

According to the invention, by controlling the influence factors of age on disease development, the application prospect of the mutation site in breast cancer auxiliary diagnosis is researched, the influence of the mutation site on breast cancer progress is explained, and the diagnosis value of the mutation site is disclosed. Therefore, the invention obtains the mutation site spectrum and the specific marker related to the breast cancer onset; the development and application of the related diagnosis kit are carried out through the change of the mutation site sequence, so that the diagnosis of the breast cancer is more convenient and feasible, the clinical doctor can quickly and accurately master the disease condition of the patient, the foundation is laid for the evaluation of the clinical treatment effect, and the help is provided for finding a novel micromolecule drug target with potential treatment value.

Detailed Description