WO2016019633A1 - 基因多态性变异位点早期评估乳腺癌风险的诊断试剂盒 - Google Patents

基因多态性变异位点早期评估乳腺癌风险的诊断试剂盒 Download PDF

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WO2016019633A1
WO2016019633A1 PCT/CN2014/089024 CN2014089024W WO2016019633A1 WO 2016019633 A1 WO2016019633 A1 WO 2016019633A1 CN 2014089024 W CN2014089024 W CN 2014089024W WO 2016019633 A1 WO2016019633 A1 WO 2016019633A1
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primer
sequencing
comt
shbg
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潘志文
付志璇
徐笑红
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浙江省肿瘤医院
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

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  • the invention belongs to the field of molecular biology, relates to medicine and biotechnology, and particularly relates to a genetic testing kit for risk of breast cancer in women, and estimates the risk of breast cancer in women from the genetic level according to the test result, and serves as a breast cancer prevention target for women. Suggested guidance.
  • Breast cancer is one of the most common malignant tumors in women.
  • the incidence rate is 7-10% of all kinds of malignant tumors in the body, ranking first in the incidence of female malignant tumors.
  • the incidence of breast cancer has increased significantly. Early detection and timely treatment are the key to breast cancer prevention and treatment.
  • SNP Single nucleotide polymorphism
  • SNP research is an important step in the application of the Human Genome Project. Because SNPs can be used as a powerful tool for the discovery of high-risk groups, identification of disease-related genes, drug design and testing, and basic research in biology. Case-control studies play an important role not only in the discovery of cancer-related genes and in elucidating their mechanisms of action, but also in the early diagnosis of risk and individualized treatment of cancer susceptibility.
  • the seven single nucleotide polymorphism loci genotypes based on ESR1 (rs2046210, rs9383951), COMT (rs4680) SHBG (rs6259) GSTP1 (rs1695) CYP19A1 (rs10046, rs700519) can be used to assess breast cancer risk.
  • the invention provides a genetic detection kit for genetic susceptibility of female breast cancer.
  • a diagnostic kit for early assessment of breast cancer risk by a genetic polymorphism variation site comprising seven single nucleotide polymorphism genotype specific primers and seven DNA sequencing primers,
  • the detection genes are five estrogen metabolic pathway-related genes: ESR1, GSTP1, COMT, SHBG and CYP19A1 genes.
  • the specific primers are seven single nucleotide polymorphisms of ESR1 (rs2046210, rs9383951), COMT (rs4680), SHBG (rs6259), GSTP1 (rs1695), CYP19A1 (rs10046, rs700519). Primer pairs of DNA fragments containing these seven SNPs sites were amplified.
  • the DNA sequencing primers are designed for seven single nucleotide polymorphism sites of ESR1 (rs2046210, rs9383951), COMT (rs4680), SHBG (rs6259), GSTP1 (rs1695), CYP19A1 (rs10046, rs700519). DNA sequencing primers capable of specifically detecting the genotypes of the above seven SNPs sites by DNA sequencing technology.
  • the seven pairs of specific primer sequences are as follows:
  • ESR1rs2046210 forward primer 5'CCATTTCTCCCTTCTTGTTGTGA 3' (SEQ ID NO: 1); reverse primer 5'AAGGCATGCTGGAAGAGTGT 3' (SEQ ID NO: 2);
  • ESR1rs9383951 forward primer 5'AGTGGCGCCAACTCTTATTGA 3' (SEQ ID NO: 3); reverse primer 5' CTAAGGTTGGGAGGGCAAGT 3' (SEQ ID NO: 4);
  • COMT forward primer: 5'CGAGGCTCATCACCATCGAG3' (SEQ ID NO: 5); reverse primer 5' ACTGAGGGGCCTGGTGATAG 3' (SEQ ID NO: 6);
  • SHBG rs6259 forward primer: 5' TTGAGGGGAAGGAAACCTCTG3' (SEQ ID NO: 7); reverse primer 5'GTGGAGCTTTAATGGGAAGCG3' (SEQ ID NO: 8);
  • GSTP1 (rs1695) forward primer: 5'TCATCCTTCCACGCACATCC3' (SEQ ID NO: 9); reverse primer 5'TTCTTTGTTCAGCCCCCAGT3' (SEQ ID NO: 10);
  • CYP19A1 rs10046 forward primer 5'ACAGTGTTCTGACTGACCCAT3' (SEQ ID NO: 11); reverse primer 5'CATGGGCCACTGAGTGTTCA3' (SEQ ID NO: 12);
  • CYP19A1 rs700519 forward primer 5'ACAGGCTTGATTTCGCTACCA3' (SEQ ID NO: 13); reverse primer 5'TCAACTCAGTGGCAAAGTCCA3' (SEQ ID NO: 14).
  • the seven pairs of DNA sequencing primer sequences are as follows:
  • ESR1rs9383951 sequencing primer 5'TTGGGAGGGCAAGTCCTAGT3' (SEQ ID NO: 16);
  • GSTP1 (rs1695) sequencing primer: 5'CCCCAGGGCTCTATGGGAAG3' (SEQ ID NO: 19);
  • Each person of the kit includes:
  • PCR reaction system 10 ⁇ PCR reaction buffer 2.5 ul; 25 mM dNTP mixture 0.2 ul; 5 U/ul Taq DNA polymerase 0.125 ul; 20 uM specific primer pair, each primer 0.25 ul; dd H 2 O 19.175ul;
  • PCR product purification system 1 U/ul SAP enzyme 0.75 ul; 10 U/ul ExoI enzyme 0.375 ul; dd H 2 O 3.875 ul;
  • the common risk assessment for breast cancer is mostly analyzed by single or several SNP loci. It has poor practicability, sensitivity and specificity, and can rarely be used for actual detection and application.
  • the detection sites used in the present invention are all related to the susceptibility of breast cancer, and the selected sites have certain representativeness, and the detection can effectively predict the risk of breast cancer.
  • the ESR1 gene encodes an estrogen receptor.
  • COMT encodes catechol-O-methyltransferase, which is involved in the inactivation of estrogen.
  • a G>A polymorphism mutation in the exon of COMT resulted in the amino acid encoded by codon 158, which has Val changed to Met.
  • the Val and Met alleles of the COMT gene are associated with the activity of catechol-O-methyltransferase, the Val/Val genotype enzyme has high activity, the Val/Met genotype has moderate enzyme activity, and the Met/Met genotype has enzymatic activity. lowest. Studies have shown that this polymorphism is associated with the onset of breast cancer.
  • the CYP19A1 gene encodes a cytochrome P450 aromatase which is an estrogen synthase, rs10046 is located in the 3'UTR region of the CYP19A1 gene, and the rs700519 polymorphism site causes R264C mutation.
  • SHBG encodes estrogen-binding globulin
  • GSTP1 encodes a phase II metabolizing enzyme, glutathione-transferase.
  • haplotype TGGGGTC significantly increased the risk of breast cancer, compared with common haplotypes, the risk of breast cancer increased by 20 times, P ⁇ 0.0001, with significant differences.
  • the venous blood was taken or the oral mucosal epithelial cells of the recipient were scraped, and the genomic DNA was extracted by phenol chloroform method.
  • ESR1rs2046210 forward primer 5'CCATTTCTCCCTTCTTGTTGTGA 3' (SEQ ID NO: 1); Primer 5'AAGGCATGCTGGAAGAGTGT 3' (SEQ ID NO: 2);
  • ESR1rs9383951 forward primer 5'AGTGGCGCCAACTCTTATTGA 3' (SEQ ID NO: 3); reverse primer 5' CTAAGGTTGGGAGGGCAAGT 3' (SEQ ID NO: 4);
  • COMT forward primer: 5'CGAGGCTCATCACCATCGAG3' (SEQ ID NO: 5); reverse primer 5' ACTGAGGGGCCTGGTGATAG 3' (SEQ ID NO: 6);
  • SHBG rs6259 forward primer: 5' TTGAGGGGAAGGAAACCTCTG3' (SEQ ID NO: 7); reverse primer 5'GTGGAGCTTTAATGGGAAGCG3' (SEQ ID NO: 8);
  • GSTP1 (rs1695) forward primer: 5'TCATCCTTCCACGCACATCC3' (SEQ ID NO: 9); reverse primer 5'TTCTTTGTTCAGCCCCCAGT3' (SEQ ID NO: 10);
  • CYP19A1 rs10046 forward primer 5'ACAGTGTTCTGACTGACCCAT3' (SEQ ID NO: 11); reverse primer 5'CATGGGCCACTGAGTGTTCA3' (SEQ ID NO: 12);
  • CYP19A1 rs700519 forward primer 5'ACAGGCTTGATTTCGCTACCA3' (SEQ ID NO: 13); reverse primer 5'TCAACTCAGTGGCAAAGTCCA3' (SEQ ID NO: 14).
  • the PCR amplification reaction system was: 10 ⁇ reaction buffer 2.5 ul; 25 mM dNTP mixture 0.2 ul; 5 U/ul Taq DNA polymerase 0.125 ul; DNA template 1 ul; 20 uM specific primer pair (0.25 ul per primer) ); ddH 2 O19.175ul;
  • the reaction conditions were: denaturing enzyme activation at 94 ° C for 4 min, denaturation at 94 ° C for 30 sec, annealing at 55 ° C for 30 sec, extension at 72 ° C for 1 min, 25 cycles, and finally extension at 72 ° C for 5 minutes.
  • the kit was purified using the PCR product in the kit.
  • the reaction system was a total volume of 25 ul, containing 20 ul of PCR product, 0.75 ul of 1 U/ul SAP enzyme, 0.375 ul of 10 U/ul ExoI enzyme, and 3.875 ul of dd H 2 O.
  • reaction was carried out on an ABI 2720 PCR instrument at 37 ° C, 15 min, 72 ° C, 20 min.
  • the sequencing reaction kit in the detection kit is used, which contains the following DNA sequencing primers:
  • ESR1rs9383951 sequencing primer 5'TTGGGAGGGCAAGTCCTAGT3' (SEQ ID NO: 16);
  • GSTP1 (rs1695) sequencing primer: 5'CCCCAGGGCTCTATGGGAAG3' (SEQ ID NO: 19);
  • the total system of reaction was 5 ul, including 1 ul of PCR purified product, 2 ul of 25% Big Dye mix; 1 ul of 3.2 uM DNA sequencing primer; dd H 2 O 2 ul.
  • reaction was carried out on an ABI 2720 PCR instrument at a reaction temperature of 98 ° C for 2 min, followed by 25 cycles of 96 ° C for 30 s, 55 ° C for 30 s, and 60 ° C for 4 min.
  • the sequencing map was analyzed to identify the genotype of the detected SNP locus.
  • Example 3 Providing genetic testing services for breast cancer risk in women
  • Blood samples or oral epithelial cells were sampled by the hospital's laboratory physician, and the samples were subjected to DNA extraction using the phenol chloroform method.
  • ESR1 Seven single nucleotide polymorphisms of ESR1 (rs2046210, rs9383951), COMT (rs4680) SHBG (rs6259) GSTP1 (rs1695) CYP19A1 (rs10046, rs700519) of the subject's genome using the kit provided by the present invention
  • the DNA sequencing was performed separately to determine the genotypes of the seven SNP loci.
  • the report details the genotype information and pathogenesis of seven single nucleotide polymorphisms in ESR1 (rs2046210, rs9383951) and COMT (rs4680) SHBG (rs6259) GSTP1 (rs1695) CYP19A1 (rs10046, rs700519). Risk assessment results.

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Abstract

本发明公开了一种基因多态性变异位点早期评估乳腺癌风险的诊断试剂盒，所述的试剂盒包括七对单核苷酸多态性位点基因型的特异性引物与七个DNA测序引物，针对的检测基因为五个雌激素代谢通路相关基因：ESR1，GSTP1，COMT，SHBG和CYP19A1基因。

Description

基因多态性变异位点早期评估乳腺癌风险的诊断试剂盒 技术领域
本发明属于分子生物学领域，涉及医学和生物技术，具体涉及一种女性乳腺癌发病风险的基因检测试剂盒，根据检测结果从基因层面评估女性乳腺癌发病的风险，并作为预防女性乳腺癌的建议指导。
背景技术
乳腺癌是女性最常见的恶性肿瘤之一，发病率占全身各种恶性肿瘤的7-10％，位居女性恶性肿瘤发病率的第一位。近年随着环境和人口老龄化的影响，乳腺癌的发病率有明显增高的趋势。早期发现并及时治疗是乳腺癌防治的关键。
人类基因组计划的完成使我们对癌症发生的分子机制认识进入了新的阶段。基因多态性(single nucleotide polymorphism，SNP)普遍存在于机体DNA序列中，是第三代遗传标记。SNP研究是人类基因组计划走向应用的重要步骤。因为SNP可作为一个强有力的工具，用于高危群体的发现、疾病相关基因的鉴定、药物的设计和测试以及生物学的基础研究等。通过病例对照研究不仅对于发现癌症相关基因及阐明其作用机制起着重要作用，而且对于癌症易感性早期风险诊断和个体化医疗具有重要意义。
与乳腺癌的发病直接或间接相关的基因众多，其多态性位点变异可影响乳腺癌的发病风险，因此建立简单、可靠、快速的乳腺癌遗传易感性的无创检测方法，能够检测出乳腺癌发生相关的基因单核苷酸位点基因型，及时筛查出乳腺癌易感高危人群，从而根据检测结果尽早采取预防措施，这对于提高乳腺癌的早诊早治有重要意义。
发明内容
本发明基于ESR1(rs2046210，rs9383951)、COMT(rs4680)SHBG(rs6259)GSTP1(rs1695)CYP19A1(rs10046，rs700519)的7个单核苷酸多态性位点基因型可用于评估乳腺癌风险基础上，本发明提供一种女性乳腺癌遗传易感性基因检测试剂盒。
一种基因多态性变异位点早期评估乳腺癌风险的诊断试剂盒，所述的试剂盒包括七个单核苷酸多态性位点基因型的特异性引物与七个DNA测序引物，针对的检测基因为五个雌激素代谢通路相关基因：ESR1，GSTP1，COMT，SHBG和CYP19A1基因。
所述的特异性引物是指针ESR1(rs2046210，rs9383951)、COMT(rs4680)、SHBG(rs6259)、GSTP1(rs1695)、CYP19A1(rs10046，rs700519)七个单核苷酸多态性位点，能特异性扩增出包含这七个SNPs位点的DNA片段的引物对。
所述的DNA测序引物是针对ESR1(rs2046210，rs9383951)、COMT(rs4680)、SHBG(rs6259)、GSTP1(rs1695)、CYP19A1(rs10046，rs700519)七个单核苷酸多态性位点而设计，能通过DNA测序技术特异性检测出上述七个SNPs位点基因型的DNA测序引物。
所述的七对特异性引物序列如下：
(1)ESR1rs2046210正向引物：5’CCATTTCTCCCTTCTTGTTGTGA 3’(SEQ ID NO：1)；反向引物5’AAGGCATGCTGGAAGAGTGT 3’(SEQ ID NO：2)；
(2)ESR1rs9383951正向引物：5’AGTGGCGCCAACTCTTATTGA 3’(SEQ ID NO：3)；反向引物5’CTAAGGTTGGGAGGGCAAGT 3’(SEQ ID NO：4)；
(3)COMT(rs4680)正向引物：5’CGAGGCTCATCACCATCGAG3’(SEQ ID NO：5)；反向引物5’ACTGAGGGGCCTGGTGATAG 3’(SEQ ID NO：6)；
(4)SHBG(rs6259)正向引物：5’TTGAGGGGAAGGAAACCTCTG3’(SEQ ID NO：7)；反向引物5’GTGGAGCTTTAATGGGAAGCG3’(SEQ ID NO：8)；
(5)GSTP1(rs1695)正向引物：5’TCATCCTTCCACGCACATCC3’(SEQ ID NO：9)；反向引物5’TTCTTTGTTCAGCCCCCAGT3’(SEQ ID NO：10)；
(6)CYP19A1 rs10046正向引物：5’ACAGTGTTCTGACTGACCCAT3’(SEQ ID NO：11)；反向引物5’CATGGGCCACTGAGTGTTCA3’(SEQ ID NO：12)；
(7)CYP19A1 rs700519正向引物：5’ACAGGCTTGATTTCGCTACCA3’(SEQ IDNO：13)；反向引物5’TCAACTCAGTGGCAAAGTCCA3’(SEQ ID NO：14)。
所述的七对DNA测序引物序列如下：
(a)ESR1rs2046210测序引物：5’GATGGAGGTACAGAAAGGCAT3’(SEQ ID NO：15)；
(b)ESR1rs9383951测序引物：5’TTGGGAGGGCAAGTCCTAGT3’(SEQ IDNO：16)；
(c)COMT(rs4680)测序引物：5’GTGGGTTTTCAGTGAACGTGG3’(SEQ ID NO：17)；
(d)SHBG(rs6259)测序引物：5’GGAGGAGTGGAAAAGTGGGG3’(SEQ ID NO：18)；
(e)GSTP1(rs1695)测序引物：5’CCCCAGGGCTCTATGGGAAG3’(SEQ IDNO：19)；
(f)CYP19A1 rs10046测序引物：5’TCTGGCTAACTGTCTGATCATTTTC3’(SEQ ID NO：20)；
(g)CYP19A1 rs700519测序引物：5’AGGCTTGATTTCGCTACCAGT 3’(SEQ ID NO：21)。
所述的试剂盒的每一人份包括：
(i)PCR反应体系：10×PCR反应缓冲液2.5ul；25mM dNTP混合液0.2ul；5U/ul Taq DNA聚合酶0.125ul；20uM特异性引物对，每条引物各0.25ul；dd H2O 19.175ul；
(ii)PCR产物纯化体系：1U/ul SAP酶0.75ul；10U/ul ExoI酶0.375ul；dd H2O 3.875ul；
(iii)测序反应体系：25％Big Dye mix 1ul；3.2uM DNA测序引物1ul；125Mm EDTA溶液1ul；无水乙醇15ul；70％乙醇溶液30ul；HIDI溶液8ul；dd H2O 2ul。
本发明的有益效果：
常见的对乳腺癌进行发病风险估计多用单个或几个SNP位点进行分析，存在实用性差，敏感性和特异性都存在问题，很少能用于实际检测和应用。本发明所用的检测位点都与乳腺癌易感性相关，选择的位点有一定的代表性，通过检测，能够有效预测乳腺癌的发病风险。
具体实施方式
ESR1基因编码雌激素受体。COMT编码儿茶酚-O-甲基转移酶，与雌激素的灭活相关。COMT第4号外显子上一个G＞A多态性位点突变，导致其第158位密码子编码的氨基酸有Val变为Met。COMT基因的Val和Met等位基因与儿茶酚-O-甲基转移酶活性高低相关，Val/Val基因型酶具有高活性，Val/Met基因型酶活性中等，Met/Met基因型酶活性最低。研究表明该多态性位点与乳腺癌发病相关。CYP19A1基因编码细胞色素P450芳香化酶是雌激素合成酶，rs10046位于CYP19A1基因的3’UTR区，rs700519多态性位点导致R264C变异。SHBG编码雌激素结合球蛋白，GSTP1编码II相代谢酶谷胱甘肽转硫酶。
下面结合具体实施例，进一步阐释本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，或按照制造厂商所建议的条件。
实施例1.
通过对330例乳腺癌患者和400例正常对照进行基因分型，分析由七个位点组成的单体型在病例组和正常组的频率。结果发现单体型TGGGGTC显著提高乳腺癌发病风险，与常见单体型相比，乳腺癌发病风险提高20倍，P＜0.0001，具有显著性差异。
实施例2.检测试剂盒的应用
1、DNA抽提
抽取静脉血或刮取受捡者口腔黏膜上皮细胞，用酚氯仿法抽提基因组DNA。
2、PCR反应
使用检测试剂盒中PCR反应组件，其中含下列引物对：
(1)ESR1rs2046210正向引物：5’CCATTTCTCCCTTCTTGTTGTGA 3’(SEQ ID NO：1)；反 向引物5’AAGGCATGCTGGAAGAGTGT 3’(SEQ ID NO：2)；
(2)ESR1rs9383951正向引物：5’AGTGGCGCCAACTCTTATTGA 3’(SEQ ID NO：3)；反向引物5’CTAAGGTTGGGAGGGCAAGT 3’(SEQ ID NO：4)；
(3)COMT(rs4680)正向引物：5’CGAGGCTCATCACCATCGAG3’(SEQ ID NO：5)；反向引物5’ACTGAGGGGCCTGGTGATAG 3’(SEQ ID NO：6)；
(4)SHBG(rs6259)正向引物：5’TTGAGGGGAAGGAAACCTCTG3’(SEQ ID NO：7)；反向引物5’GTGGAGCTTTAATGGGAAGCG3’(SEQ ID NO：8)；
(5)GSTP1(rs1695)正向引物：5’TCATCCTTCCACGCACATCC3’(SEQ ID NO：9)；反向引物5’TTCTTTGTTCAGCCCCCAGT3’(SEQ ID NO：10)；
(6)CYP19A1 rs10046正向引物：5’ACAGTGTTCTGACTGACCCAT3’(SEQ ID NO：11)；反向引物5’CATGGGCCACTGAGTGTTCA3’(SEQ ID NO：12)；
(7)CYP19A1 rs700519正向引物：5’ACAGGCTTGATTTCGCTACCA3’(SEQ ID NO：13)；反向引物5’TCAACTCAGTGGCAAAGTCCA3’(SEQ ID NO：14)。
PCR扩增的反应体系为：10×反应缓冲液2.5ul；25mM的dNTP混合液0.2ul；5U/ul Taq DNA聚合酶0.125ul；DNA模板1ul；20uM特异性引物对(每条引物各0.25ul)；ddH2O19.175ul；
反应条件为：94℃4min变性酶激活，94℃30sec变性，55℃30sec退火，72℃1min延伸，25个循环，最后72℃延长5分钟。
3、PCR产物纯化
使用试剂盒中的PCR产物纯化组件，反应体系为总体积25ul，包含PCR产物20ul，1U/ul SAP酶0.75ul；10U/ul ExoI酶0.375ul；dd H2O 3.875ul。
在ABI2720型PCR扩增仪上进行反应，反应条件为37℃、15min，72℃、20min。
4、DNA测序反应
使用检测试剂盒中的测序反应组件，其中，含有下列DNA测序引物：
(a)ESR1rs2046210测序引物：5’GATGGAGGTACAGAAAGGCAT3’(SEQ ID NO：15)；
(b)ESR1rs9383951测序引物：5’TTGGGAGGGCAAGTCCTAGT3’(SEQ ID NO：16)；
(c)COMT(rs4680)测序引物：5’GTGGGTTTTCAGTGAACGTGG3’(SEQ ID NO：17)；
(d)SHBG(rs6259)测序引物：5’GGAGGAGTGGAAAAGTGGGG3’(SEQ ID NO：18)；
(e)GSTP1(rs1695)测序引物：5’CCCCAGGGCTCTATGGGAAG3’(SEQ ID NO：19)；
(f)CYP19A1 rs10046测序引物：5’TCTGGCTAACTGTCTGATCATTTTC3’(SEQ ID NO：20)；
(g)CYP19A1 rs700519测序引物：5’AGGCTTGATTTCGCTACCAGT 3’(SEQ ID NO：21)。
反应的总体系为5ul，包括PCR纯化产物1ul，25％Big Dye mix 1ul；3.2uM DNA测序引物1ul；dd H2O 2ul。
在ABI2720型PCR扩增仪上进行反应，反应条件为98℃2min，后25个循环的96℃30s，55℃30s，60℃4min。
反应结束后加入125mM EDTA溶液1ul和无水乙醇15ul，室温下沉淀15min，4℃，3600rpm/min离心30min，去除上清液，加入70％乙醇溶液30ul，3600rpm/min离心15min，去除上清液，室温放置20min后加入HIDI溶液8ul，放入测序仪。
5、基因型分析
对测序图谱进行分析辨认所检测的SNP位点的基因型。
实施例3.提供女性乳腺癌发病风险基因检测服务
1、采样机抽提DNA
由医院检验科医师对受检者进行血样或口腔上皮细胞采样，采用酚氯仿法对标本进行DNA抽提。
2、基因型检测
使用本发明提供的试剂盒，对受检者基因组的ESR1(rs2046210，rs9383951)、COMT(rs4680)SHBG(rs6259)GSTP1(rs1695)CYP19A1(rs10046，rs700519)的7个单核苷酸多态性位点分别进行DNA测序，确定这7个SNP位点的基因型。
3、乳腺癌发病风险的评估
通过对受检者SNPs基因型的分析，出具乳腺癌发病风险的评估分析报告。报告详细说明了受检者ESR1(rs2046210，rs9383951)、COMT(rs4680)SHBG(rs6259)GSTP1(rs1695)CYP19A1(rs10046，rs700519)的7个单核苷酸多态性位点的基因型信息及发病风险评估结果。

Claims (6)

  1. 一种基因多态性变异位点早期评估乳腺癌风险的诊断试剂盒，其特征在于，所述的试剂盒包括七个单核苷酸多态性位点基因型的特异性引物与七个DNA测序引物，针对的检测基因为五个雌激素代谢通路相关基因：ESR1，GSTP1，COMT，SHBG和CYP19A1基因。
  2. 根据权利要求1所述的试剂盒，其特征在于：所述的特异性引物是指针ESR1(rs2046210，rs9383951)、COMT(rs4680)、SHBG(rs6259)、GSTP1(rs1695)、CYP19A1(rs10046，rs700519)七个单核苷酸多态性位点，能特异性扩增出包含这七个SNPs位点的DNA片段的引物对。
  3. 根据权利要求1所述的试剂盒，其特征在于：所述的DNA测序引物是针对ESR1(rs2046210，rs9383951)、COMT(rs4680)、SHBG(rs6259)、GSTP1(rs1695)、CYP19A1(rs10046，rs700519)七个单核苷酸多态性位点而设计，能通过DNA测序技术特异性检测出上述七个SNPs位点基因型的DNA测序引物。
  4. 根据权利要求2所述的试剂盒，其特征在于：所述的七对特异性引物序列如下：
    (1)ESR1rs2046210正向引物：5’CCATTTCTCCCTTCTTGTTGTGA3’(SEQ ID NO：1)；反向引物5’AAGGCATGCTGGAAGAGTGT3’(SEQ ID NO：2)；
    (2)ESR1rs9383951正向引物：5’AGTGGCGCCAACTCTTATTGA3’(SEQ ID NO：3)；反向引物5’CTAAGGTTGGGAGGGCAAGT3’(SEQ ID NO：4)；
    (3)COMT(rs4680)正向引物：5’CGAGGCTCATCACCATCGAG3’(SEQ ID NO：5)；反向引物5’ACTGAGGGGCCTGGTGATAG3’(SEQ ID NO：6)；
    (4)SHBG(rs6259)正向引物：5’TTGAGGGGAAGGAAACCTCTG3’(SEQ ID NO：7)；反向引物5’GTGGAGCTTTAATGGGAAGCG3’(SEQ ID NO：8)；
    (5)GSTP1(rs1695)正向引物：5’TCATCCTTCCACGCACATCC3’(SEQ ID NO：9)；反向引物5’TTCTTTGTTCAGCCCCCAGT3’(SEQ ID NO：10)；
    (6)CYP19A1rs10046正向引物：5’ACAGTGTTCTGACTGACCCAT3’(SEQ ID NO：11)；反向引物5’CATGGGCCACTGAGTGTTCA3’(SEQ ID NO：12)；
    (7)CYP19A1rs700519正向引物：5’ACAGGCTTGATTTCGCTACCA3’(SEQ IDNO：13)；反向引物5’TCAACTCAGTGGCAAAGTCCA3’(SEQ ID NO：14)。
  5. 根据权利要求3所述的试剂盒，其特征在于：所述的七对DNA测序引物序列如下：
    (a)ESR1rs2046210测序引物：5’GATGGAGGTACAGAAAGGCAT3’(SEQ ID NO：15)；
    (b)ESR1rs9383951测序引物：5’TTGGGAGGGCAAGTCCTAGT3’(SEQ ID NO：16)；
    (c)COMT(rs4680)测序引物：5’GTGGGTTTTCAGTGAACGTGG3’(SEQ ID NO：17)；
    (d)SHBG(rs6259)测序引物：5’GGAGGAGTGGAAAAGTGGGG3’(SEQ ID NO：18)；
    (e)GSTP1(rs1695)测序引物：5’CCCCAGGGCTCTATGGGAAG3’(SEQ ID NO：19)；
    (f)CYP19A1rs10046测序引物：5’TCTGGCTAACTGTCTGATCATTTTC3’(SEQ ID NO：20)；
    (g)CYP19A1rs700519测序引物：5’AGGCTTGATTTCGCTACCAGT3’(SEQ ID NO：21)。
  6. 根据权利要求1所述的试剂盒，其特征在于，所述的试剂盒的每一人份包括：
    (i)PCR反应体系：10×PCR反应缓冲液2.5ul；25mM dNTP混合液0.2ul；5U/ul Taq DNA聚合酶0.125ul；20uM特异性引物对，每条引物各0.25ul；dd H2O19.175ul；
    (ii)PCR产物纯化体系：1U/ul SAP酶0.75ul；10U/ul ExoI酶0.375ul；ddH2O3.875ul；
    (iii)测序反应体系：25％Big Dye mix 1ul；3.2uM DNA测序引物1ul；125Mm EDTA溶液1ul；无水乙醇15ul；70％乙醇溶液30ul；HIDI溶液8ul；ddH2O2ul。
PCT/CN2014/089024 2014-08-08 2014-10-21 基因多态性变异位点早期评估乳腺癌风险的诊断试剂盒 WO2016019633A1 (zh)

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