CN101608225A - Kit for genetic detection of breast cancer - Google Patents

Abstract

The invention discloses a kind of test kit that detects the mammary cancer genetic risk.This test kit comprise the genotypic Auele Specific Primer of mononucleotide polymorphism site that detects on estrogen receptor alpha gene (ER-α), catechol-O-methyl transferase (COMT) gene (COMT), glutathione S-transferase P1 gene (GSTP1), human breast carcinoma tumor susceptibility gene 1 (BRCA1), the human breast carcinoma tumor susceptibility gene 2 (BRCA2) to and the specificity fluorescent probe to, quantification PCR normal assembly etc.Test kit of the present invention is assessed individual mammary cancer genetic risk by detection simultaneously and the pleomorphism site genotype on the closely-related gene of mammary cancer genetic risk.

Description

Kit for genetic detection of breast cancer

Technical field

The present invention relates to molecular biology and medical field, more specifically, the present invention relates to a kind of test kit that detects individual mammary cancer genetic predisposition, assess individual mammary cancer genetic predisposition by detection simultaneously and the mononucleotide polymorphism site genotype on the closely-related estrogen receptor alpha gene of mammary cancer (ER-α), catechol-O-methyl transferase (COMT) gene (COMT), glutathione S-transferase P1 gene (GSTP1), human breast carcinoma tumor susceptibility gene 1 (BRCA1), the human breast carcinoma tumor susceptibility gene 2 (BRCA2).

Background technology

Mammary cancer is the breast duct epithelial cell under the effect of various inside and outside carcinogenic factors, and cell loses normal characteristic and paraplasm, so that surpasses the limit of self-regeneration and the disease of canceration takes place, and clinical be mainly to show with the mammary gland tumor.Mammary cancer is one of modal malignant tumour of women, sickness rate height, tool aggressive is arranged but characteristics such as the course of disease is made slow progress, nature lifetime is long.

Mammary cancer is the disease of main harm body of women health, and the male sex also can suffer from breast cancer, but the chance that the male sex suffers from breast cancer is lacked 100 times than the women.The predilection site of mammary cancer occupies the majority on outside the breast.Breast carcinoma of early stage can not have any subjective symptoms, and pathology mammary gland tumor can occur late period, and quality is tough firmly, and the border is very not clear, no coating sense, the movability that pushes away is little, most no obvious pain, retraction, off normal appear in nipple, nipple overflow yellow water or watery blood, and the cancerous eczema sample changes.

Estrogen receptor (ER) is a kind of transcription factor of ligand-dependent, belong to nuclear receptor superfamily, part (as, oestrogenic hormon) its conformation changes under the keying action, form homology or heterodimer, be attached to and contain the oestrogenic hormon response element (estrogen response element on promotor ERE), thereby regulates target gene expression.This is the classical pathway that ER relies on part, has found other three approach of ER afterwards again, does not promptly rely on the approach of part, does not rely on the approach of transcribing and the non-transcribed signal pathway of ERE.ER-α gene PvuII site is No. 1 C/T polymorphic site (C397T) on the intron, can cut by being limited property restriction endonuclease PvuII enzyme behind this site mutation.The genotype of this polymorphic site has CC (PP), CT (Pp), TT (pp), and p represents and can be cut by the PvuII enzyme.Wherein TT and CT are confirmed as the risk genes type, and be relevant with the morbidity of mammary cancer.ER-α gene Xba I site is No. 1 G/A polymorphic site (G351A) on the intron, can cut by being limited property restriction endonuclease Xba I enzyme behind this site mutation.The genotype of this polymorphic site has GG (XX), GA (Xx), AA (xx), and x represents and can be cut by Xba I enzyme.Wherein AA and GA are confirmed as the risk genes type, and be relevant with the morbidity of mammary cancer.The PvuII of ER-α and Xba I gene polymorphism sites all are positioned at the gene transcription control region, so this two places site mutation has certain influence to genetic expression, but up-regulated gene is expressed.Discover that in a large number two polymorphic sites (C397T and G351A) of ER-α gene are relevant with breast cancer susceptibility.(397TT/CT, in the time of 351AA/GA), the ER-alpha levels raises, and strengthens the TGF-alpha expression, thereby promotes the cell growth, the rising of mammary cancer occurrence risk when carrying the risk genes type.

There is quite a few to be and the relevant gene of estrogen metabolism regulation and control in the inheritance susceptible factor of mammary cancer.Natural oestrogenic hormon is 17 β estradiol (E2).E2 and other hormone acting in conjunction have the mammary epithelial cell of promotion hyperplasia function.A G/A polymorphic site sudden change on No. 4 exon of COMT gene causes its 158th bit codon amino acids coding to become Met by Val.The genotype in this site has GG, GA, AA, and wherein A allelotrope is relevant with the generation of mammary cancer, and AA genotype (being the Met/Met genotype) is confirmed as the mammary cancer risk genotype.The Val of COMT gene, Met allelotrope are relevant with the high and low activity of enzyme respectively, and are codominant inheritance, and promptly the genotypic enzyme of Val/Val has high reactivity, and the genotypic enzyme of Val/Met has moderate activity, and the genotypic enzymic activity of Met/Met is minimum.Between Val/Val and the Met/Met genotype, enzyme work differs 3-4 doubly.When carrying risk genes type (Met/Met, i.e. AA), the COMT enzymic activity descends, and has influenced the deactivation of catechol estrogen, and promptly catechol estrogen increases, and the mammary cancer occurrence risk rises.

GSTP1 is a member in (GSTs) family of glutathione S-transferase system, belong to pi class wherein, mainly be present in lung and the placenta, has similar function to other member of GSTs, promptly mediate all kinds of electrophilic compounds,, combine with gsh as medicine, environmental toxin, oxidative chain product etc., enter next step metabolism step, these materials excrete the most at last.There is an A/G polymorphic site on No. 5 exon of GSTP1 gene, causes that the 105th bit codon coded amino acid becomes Val by Ile.There is multiple method for expressing in the Ile105Val site, is expressed as A342G in ncbi database, is expressed as A313G in some document again.The genotype of this polymorphic site has AA, AG, GG, and wherein GG (Val/Val) genotype is the risk genes type of mammary cancer.Studies show that at present, the amino acid change that the Ile105Val site mutation causes, amino acid whose space length of electrophilic binding site and structure in the GSTP1 polypeptide crystal structure have been had influence on, thereby influence combining of albumen electrophilic binding site and substrate, therefore gene function reduces, cause carcinogens accumulating in vivo, finally easily cause the generation of mammary cancer.Detect the genotype in GSTP1 Ile105Val site, for the early warning of pathogenesis of cancer, the drug screening of cancer therapy and prognosis all have important effect.When carrying GG (Val/Val) risk genes type, the ill risk of mammary cancer increases.

A kind of phosphocarnic acid albumen of BRCA1 genes encoding, this albumen is in that to keep genome stable and suppress to play an important role aspect the tumour generation.The BRCA1 gene has multiple montage mode, can produce the mRNA fragment and the albumen of different lengths and function.BRCA1 with normal physiological function can participate in the reparation of dna damage; Act on " check point of cell cycle ", thereby cell cycle is regulated and control; And can interact with rna plymerase ii and transcriptional activators p53 and c-myc, thereby regulate transcribing of cell.The 1100delAT site mutation of BRCA1 gene is that 1100 of nucleotide sequences are gone up disappearance AT, and phase shift mutation has caused at 328 translation terminations of codon, produces truncated protein, and the BRCA1 protein function is had a strong impact on.Therefore, when carrying this mutator gene type (--/--or AT/--), mammary cancer risk rises.The 589delCT site mutation of BRCA1 gene is that 589 of nucleotide sequences are gone up disappearance CT, and phase shift mutation has caused at 157 translation terminations of codon, produces truncated protein, and the BRCA1 protein function is had a strong impact on.Therefore, when carrying this mutator gene type (--/--or CT/--), the mammary cancer occurrence risk rises.The T5319A site mutation of BRCA1 gene is that the T on 5319 of this gene nucleotide series is replaced by A, causes 1734 coded amino acids of codon to become Ser by Phe, is the amino acid missense mutation.This sudden change may influence the function of BRCA1, and when therefore carrying this mutator gene type (AA or TA), the mammary cancer occurrence risk rises.

BRCA2 is familial breast cancer/ovarian cancer tumor susceptibility gene of finding after BRCA1.BRCA2 is high expression level in mammary gland and parathyroid tissue, and its expression level is low in lung, ovary and the spleen tissue.This mrna length is about 2 times of BRCA1, is made up of 27 exons, and wherein exons 11 accounts for 50% of whole encoding sequences, and mRNA length is about 10kb, and its coded product albumen contains 3,448 amino acid.Studies show that BRCA2 and BRCA1 albumen in a restricted areas (the amino acid/11 783-1863 district of the amino acid/11 394-1474 district of BRCA1 and BRCA2) have faint similarity.The BRCA2 transgenation occurs in the male breast carcinoma patient more, has improved the occurrence risk of male breast carcinoma greatly, is about 6%.On the contrary, carry the risk that the male sex of BRCA1 transgenation does not but suffer from breast cancer.No matter the male sex or women carry the individuality generation carcinoma of the pancreas of BRCA2 transgenation and the risk of other tumour and increase.The 5950delCT site mutation of BRCA2 gene is that 5950 of nucleotide sequences are gone up disappearance CT, is positioned at the o.11 exon.This sports phase shift mutation, has caused at 1909 translation terminations of codon, produces truncated protein.When carrying this mutator gene type (--/--or CT/--), the BRCA2 function is affected, and can't keep the stable of genome genetic information by the DNA repair mode, and the mammary cancer occurrence risk rises.

Summary of the invention

Can be used to assess on the basis of individual mammary cancer genetic predisposition based on 8 SNP loci polymorphisms on estrogen receptor alpha gene (ER-α), catechol-O-methyl transferase (COMT) gene (COMT), glutathione S-transferase P1 gene (GSTP1), human breast carcinoma tumor susceptibility gene 1 (BRCA1), the human breast carcinoma tumor susceptibility gene 2 (BRCA2), the invention provides a kind of test kit that detects individual mammary cancer genetic predisposition.

This test kit comprises:

The genotypic Auele Specific Primer of Pvu II SNP polymorphism is right to reaching the specificity fluorescent probe on the detection ER-α gene;

The genotypic Auele Specific Primer of Xba I SNP polymorphism is right to reaching the specificity fluorescent probe on the detection ER-α gene;

The genotypic Auele Specific Primer of Val158Met SNP polymorphism is right to reaching the specificity fluorescent probe on the detection COMT gene;

The genotypic Auele Specific Primer of Ile105Val SNP polymorphism is right to reaching the specificity fluorescent probe on the detection GSTP1 gene;

The genotypic Auele Specific Primer of 1100delAT SNP polymorphism is right to reaching the specificity fluorescent probe on the detection BRCA1 gene;

The genotypic Auele Specific Primer of 589delCT SNP polymorphism is right to reaching the specificity fluorescent probe on the detection BRCA1 gene;

The genotypic Auele Specific Primer of T5319A SNP polymorphism is right to reaching the specificity fluorescent probe on the detection BRCA1 gene;

The genotypic Auele Specific Primer of 5950delCTSNP polymorphism is right to reaching the specificity fluorescent probe on the detection BRCA2 gene;

The quantitative fluorescent PCR reaction component (comprises Taq enzyme, dNTP mixed solution, MgCl ₂Solution, reaction buffer, deionized water etc.).

Primer described in this test kit is to being that those skilled in the art can finish design easily, and the synthetic technology of available routine is synthesized.

Specificity fluorescent probe described in this test kit is to being that those skilled in the art can finish design easily, and the specificity fluorescent probe synthesizes the probe synthetic technology of available routine.

The component and the content of test kit of the present invention comprise:

10X quantitative fluorescent PCR reaction buffer 8 μ l,

25mM dNTP mixed solution 0.8 μ l,

25mM MgCl2 solution 4.8 μ l,

5units/ μ l Taq archaeal dna polymerase 0.02 μ l,

20 μ M Auele Specific Primers are to each 0.225 μ l of every primer,

10 μ M specificity fluorescent probes are to each 0.25 μ l of every probe,

Deionized water 42.6 μ l.

This test kit detects for a person-portion and uses, and the storage temperature of test kit is-20 ℃.

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.

The use of embodiment 1. detection kit

The extraction of step 1:DNA template

Genomic dna with silica gel adsorption extracting mouth epithelial cells.

Step 2: quantitative fluorescent PCR reaction

Use the quantitative fluorescent PCR suit in the detection kit, carry out 8 independently quantitative fluorescent PCR reactions respectively, the system of each reaction is cumulative volume 10 μ l, and comprising concentration is dna profiling 2 μ l, l μ l 10 X quantitative fluorescent PCR reaction buffers, 0.1 μ l 25mM dNTP mixed solution, the 0.6 μ l 25mM MgCl of 20ng/ μ l ₂The band VIC fluorescent probe that adopted primer and antisense primer each 0.225 μ l, 10 μ M are arranged of solution, 0.025 μ l (5units/ μ l) Taq archaeal dna polymerase, 20 μ M and each 0.25 μ l of band FAM fluorescent probe, deionized water 5.325 μ l.

React on the pcr amplification instrument, reaction conditions is 50 ℃, 2 minutes, 95 ℃, 10 minutes, carries out 95 ℃ of 60 round-robin, 30 seconds, 60 ℃, 1 minute.Reaction finishes the back and read the fluorescent amount on quantitative real time PCR Instrument.

Step 4: gene type assay

According to the gene type diagram that the test kit working instructions indicate gene type assay is carried out in the SNP site.

The those skilled in the art that are familiar with fluorescent quantitative PCR technique can be by the final sample fluorescence volume that shows on the identification quantitative real time PCR Instrument, can determine the genotype in the SNP site detected according to the power of different sequence probe VIC and FAM fluorescent signal.

Embodiment 2. applying detection test kits carry out the service that individual mammary cancer genetic predisposition detects

Step 1:DNA extracts

Instructing the examinee to use the oral cavity sampling to wipe away by the hospital laboratory doctor carries out the mouth epithelial cells sampling, adopts silica gel adsorption to carry out the DNA extracting of mouth epithelial cells.

Step 2: genotype tests

Use test kit provided by the invention, fluorescence quantitative PCR detection is carried out in 5950delCT SNP site on T5319A SNP site, the BRCA2 gene on 589delCT SNP site, the BRCA1 gene on 1100delATSNP site, the BRCA1 gene on Ile105Val SNP site, the BRCA1 gene on Val158Met SNP site, the GSTP1 gene on XbaI SNP site, the COMT gene on Pvu II SNP site, the ER-α gene on the ER-α gene of detected person's genomic dna, determine the genotype in these 8 SNP sites.

Step 3: the analysis of individual mammary cancer genetic predisposition

By to the genotypic analysis of detected person SNP, provide the analysis report list of individual mammary cancer genetic predisposition.Describe the height of detected person's mammary cancer genetic risk in the report in detail, and describe and understand individual mammary cancer genetic predisposition analysis report list in detail to the examinee by the doctor.

Claims (2)

1. a test kit that detects individual mammary cancer genetic predisposition is characterized in that: comprise that detecting estrogen receptor alpha gene (ER-α) simultaneously goes up PvuII SNP site, estrogen receptor alpha gene (ER-α) is gone up Xba I SNP site, catechol-O-methyl transferase (COMT) gene (COMT) is gone up Val158Met SNP site, glutathione S-transferase P1 gene (GSTP1) is gone up Ile105Val SNP site, human breast carcinoma tumor susceptibility gene 1 (BRCA1) is gone up 1100delAT SNP site, human breast carcinoma tumor susceptibility gene 1 (BRCA1) is gone up 589delCT SNP site, human breast carcinoma tumor susceptibility gene 1 (BRCA1) is gone up T5319A SNP site, it is right to reaching the specificity fluorescent probe that human breast carcinoma tumor susceptibility gene 2 (BRCA2) is gone up the Auele Specific Primer in 5950delCT SNP site, the Taq enzyme, the dNTP mixed solution, MgCl ₂Solution, quantitative fluorescent PCR reaction buffer, deionized water.

2. test kit according to claim 1, it is characterized in that: the component and the content of test kit comprise: 10X quantitative fluorescent PCR reaction buffer 8 μ l, 25mM dNTP mixed solution 0.8 μ l, 25mM MgCl2 solution 4.8 μ l, 5units/ μ l Taq archaeal dna polymerase 0.02 μ l, 20 μ M Auele Specific Primers are to each 0.225 μ l of every primer, and 10 μ M specificity fluorescent probes are to each 0.25 μ l of every probe, deionized water 42.6 μ l.

This test kit detects for a person-portion and uses, and the storage temperature of test kit is-20 ℃.