CN102864234A - Molecular marker for identifying susceptibility of ovarian cancer - Google Patents

Abstract

The invention discloses a molecular marker for identifying susceptibility of ovarian cancer. The nucleotide sequence of the molecular marker is presented as SEQ ID NO: 1 and single nucleotide polymorphism (SNP) C) T exists at the 1556 position. A kit for detecting the susceptibility of the ovarian cancer comprises a pair of specific primers provided with sequences of SEQ ID NO: 2 and SEQ ID NO: 3. A method for in vitro detecting whether the SNP of a GADD45A gene exists in a sample includes the steps of amplifying the GADD45A gene of the sample by using the specific primers to obtain amplification products; and performing sequencing on the amplification products to detect whether the SNP 1556C) T exists in the amplification products. According to the molecular marker, the method is simple, accurate and rapid in detection, the specificity is high, and the significance in effective early diagnosis, treatment and individual prevention for high risk groups, susceptible individuals and ovarian cancer patients is provided.

Description

A kind of molecule marker for identification of ovarian cancer susceptibility

Technical field

The present invention relates to a kind of molecule marker for identification of ovarian cancer susceptibility, test kit and detection method thereof, single nucleotide polymorphism (the single nucleotide polymorphism that relates to particularly the GADD45A gene, SNP) and with the dependency of ovarian cancer, and method and the test kit of these SNP of detection, belong to molecular biology and medical field.

Background technology

Ovarian cancer is the highest female reproductive system tumour of lethality rate, global new cases 225500 people in 2008, dead 140200 people.Because onset concealment, symptom is not obvious, late period when most of patients is found, 5 years survival rates of Patients with Advanced Ovarian Carcinoma only are about 30～45%, poor prognosis, and 5 years survival rates of early ovarian cancer patient are up to 90%, this key of pointing out us to improve ovarian cancer patients prognosis and existence is early to examine, early control, therefore inquire into the biological mechanism of ovarian cancer genesis, seek the molecule marker of identifying ovarian cancer Susceptible population, the molecule marker of ovarian cancer patients molecule parting and prognosis prediction is implemented effectively early to examine for high risk population or susceptible individual and ovarian cancer patients, early control with the individuation control significant.

In the prior art, to surpass 200 candidate genes relevant with the ovarian cancer susceptibility with 1100 heritable variations in 20 heredity zones although had, with the very strong heritable variation of ovarian cancer dependency but seldom.And the report that does not have GADD45A gene and ovarian cancer dependency does not more have the report of GADD45A gene SNP and ovarian cancer dependency.Therefore, ovarian cancer susceptible gene is badly in need of further seeking in this area, and exploitation detects method, the test kit of ovarian cancer high risk population and susceptible individual, and relevant medicine.

Summary of the invention

For above-mentioned prior art, the present inventor is through deeply and widely research, SNP to a large amount of candidate genes measures and analyzes, find that first GADD45A is closely related with the ovarian cancer susceptibility, can be used as the molecule marker of the detection of ovarian cancer susceptibility or early diagnosis, therefore, the invention provides a kind of test kit for detection of the ovarian cancer susceptibility and method thereof, and whether the vitro detection sample exists the method for the single nucleotide polymorphism of GADD45A gene.

The present invention is achieved by the following technical solutions:

A kind of molecule marker for identification of ovarian cancer susceptibility, its nucleotide sequence is shown in SEQ ID NO:1, it is compared with normal GADD45A gene, difference is: the 1556th exists single nucleotide polymorphism a: C〉T, namely the 1556th base comprises base C and two kinds of situations of base T, when being base C, is normal GADD45A gene, when being base T, be the GADD45A gene of sudden change:

cagtggctgg taggcagtgg ctgggaggca gcggcccaat tagtgtcgtg cggcccgtgg 60

cgaggcgagg tccggggagc gagcgagcaa gcaaggcggg aggggtggcc ggagctgcgg 120

cggctggcac aggaggagga gcccgggcgg gcgaggggcg gccggagagc gccagggcct 180

gagctgccgg agcggcgcct gtgagtgagt gcagaaagca ggcgcccgcg cgctagccgt 240

ggcaggagca gcccgcacgc cgcgctctct ccctgggcga cctgcagttt gcaatatgac 300

tttggaggaa ttctcggctg gagagcagaa gaccgaaagg tgagtcggcc tgcggactct 360

tccggcccga acttctctta cctaccccgc gctccccggt gcagccgggc tgtggaaggc 420

ttgcagggga ggaagctaaa aagtttgcac agggcaactc ccgcccttgc tccctcggga 480

ctctccgtgg agctcccacg gactgaaaga gcgtgccccc caacccgaac gagccccgcc 540

ggggcctttg caaagggcag cagtggccgt cgctgcccgt gcggctcccg tggctggcag 600

cctgtggcag gggcactctc gggacttctc acgggacgcc cggtccttgg gcgtgcaggg 660

gtcatggggg gtgacggggc cgcgggagcg ccgggttttc gtagagccca ggtgcgcggt 720

ggtgcttgca ttcgagaggg aggggcgtgg taccggacga ggggggcggc gatggccccg 780

agggcaccgg ggctgacggg acccctcgcc cttgcccgcg tgtaggatgg ataaggtggg 840

ggatgccctg gaggaagtgc tcagcaaagc cctgagtcag cgcacgatca ctgtcggggt 900

gtacgaagcg gccaagctgc tcaacgtgta agtggggccc ttgcgcgtcc cccatggcac 960

cccttcccgc cccagcccgg gaggtcgcct tggctgggcg cccctcgccc ggccgcgcca 1020

cttcctgtcg cttttctgcc tgtctcggaa gggagggggc gagcgggccg ggcggcgacc 1080

cccagggacc cgggcagggg ttgagggcgc ccgcgcttct gcgctcactg gccccgcccg 1140

ctgcccccag cgaccccgat aacgtggtgt tgtgcctgct ggcggcggac gaggacgacg 1200

acagagatgt ggctctgcag atccacttca ccctgatcca ggcgttttgc tgcgagaacg 1260

acatcaacat cctgcgcgtc agcaacccgg gccggctggc ggagctcctg ctcttggaga 1320

ccgacgctgg ccccgcggcg agcgagggcg ccgagcagcc cccggacctg cactgcgtgc 1380

tggtgacggt aagggactgg gggactgcag cctgcagggt agagccccgg aaggacggga 1440

gtcagggctg ggttgcctga ttgtggatct gtggtaggtg ggggtcagga gggtggctgc 1500

ctttgtccga ctagagtgtg gctggacttt cagccgagat gtgctagttt catcarcagg 1560

1556

attttctgtg gtacagaaca tgtctaagca tgctggggac tgccagcagc ggaagagatc 1620

cctgtgagtc agcagtcagc ccagctactc cctacctaca tctgcactgc ctcccgtgac 1680

taattccttt agcagggcag attagataaa gccaaatgaa ttcctggctc acccctcatt 1740

aaggagtcag cttcattctc tgccagtcag agctaaaaat agaaattgtg taggagacaa 1800

accttgttaa ttccctagaa atacattaag aggatagagt ggaatttttt ttctctgcaa 1860

tcttgcattt ttttaatggc tctttttttt tttcctgata aaaacctttg gtaggtaggg 1920

aagttatgtt ttcaggggta aatgtgctac ttttgtcttc taaattttgc tcttttttga 1980

ctggtctagt caagtgacag cccgattatt ttgctactcc ttaaaagtac tattctgtct 2040

cttggagtat ggttgatggc aattccagtt aactgctgtg cagctctcat ctcattgtgc 2100

acacagcatg gaaatctttc tcaaaactgt ttcactcagg tcagggtaac aagtttggta 2160

gagcaaaccg gtgaatgata ctctcatgca aaactgaaca gatatgcaaa catatgtatg 2220

tggttcagct tgggttgcat gggttcagac tttgcaatgt gtagtttaat aggtaattac 2280

ccttaacgct tttgcaggga acccaactac cttgaagaaa ctttaatttt tttgtgcttc 2340

taatttgtct ccatgtcaca tagccaaaat atagaatgtt caagtgtttt ctcctcaaaa 2400

gtataattac tagaatatac tggtttttaa aataagttta tttttataaa tttgtttcca 2460

g 2461

A kind of test kit for detection of the ovarian cancer susceptibility, comprise Auele Specific Primer, this primer is a pair of, has the sequence of SEQ ID NO:2 and SEQ ID NO:3, can be specific the primer of amplification GADD45A gene, can amplify specifically length 200～2700bp.

Further, test kit also comprises the PCR reaction solution, and the PCR reaction solution is by dNTP, Mg ²⁺, Taq enzyme and Buffer form.Each component is conventional with magnitude relation in the PCR reaction solution, is common practise for one of ordinary skill in the art.

Whether a kind of vitro detection sample exists the method for the single nucleotide polymorphism of GADD45A gene, and step is as follows:

(1) with the GADD45A gene of GADD45A gene-specific primer amplification sample, obtains amplified production; Described primer is a pair of, has the sequence of SEQ ID NO:2 and SEQ ID NO:3;

(2) amplified production is carried out sequencing, detect in the amplified production whether have following single nucleotide polymorphism: 1556C T; 1556C〉T sudden change Position Number is based on the sequence of SEQ ID NO:1.

A kind of method that ovarian cancer susceptibility or the ill risk of individuality are detected or diagnose, step is as follows:

Detect this individual GADD45A gene, transcript and/or albumen, and compare with normal GADD45A gene, transcript and/or albumen, just there are differences and show that the possibility that this individuality has ovarian cancer is lower than normal population.Described difference refers to whether there is single nucleotide polymorphism: 1556C〉T; 1556C〉T sudden change Position Number is based on the sequence of SEQ ID NO:1.

Molecule marker for detection of the ovarian cancer susceptibility of the present invention, test kit and method thereof, and whether the vitro detection sample exists the method for the single nucleotide polymorphism of GADD45A gene, detect simple, accurate, quick, high specificity is implemented effectively early to examine, early controls with the individuation control significant for high risk population or susceptible individual and ovarian cancer patients.

Embodiment

The present invention is further illustrated below in conjunction with embodiment.

The detection of embodiment 1GADD45A transgenation reaches the association analysis with ovarian cancer

1.1 research object

Choose ovarian cancer patients 139 examples, treatment is made a definite diagnosis in Shandong Qilu Hospital year April in September, 2008～2011, and clinical data obtains from case history.Contrast patient 189 examples of age-matched are the Physical Examination Center Physical Examination women of Shandong hospital.Most subjects is Donors in the Shandong Province.Get experimenter person's peripheral blood 1.5ml, in-80 ℃ of stored refrigerated.All tested personnel all require informed consent according to Ethics Committee of Shandong Qilu Hospital.

1.2DNA extract

Extract experimenter's peripheral blood DNA with conventional phenol chloroform method, specific as follows:

(1) getting 400ul blood is added in the 1.5ml centrifuge tube.

(2) add 800ul PBS, mixing, 3500g, 15min abandons supernatant; Come again.

(3) add 400ul lysate, 37 ℃, 1h.

(4) add Proteinase K 4ul, concentration 200ug/ml.

(5) 55 ℃ of water-baths digest spend the night (4-12h).

(6) the saturated phenol of Tris of adding 400ul mixes and shakes 10min.

(7) 5000g, 15min, water intaking phase.

(8) add 100ul NaAc, 800ul dehydrated alcohol ,-20 ℃, 20min.

(9) 12000g, 5min abandons supernatant.

(10) 600ul 70% ice ethanol, 12000g, 5min abandons supernatant, repeats once.

(11) 12000rpm is centrifugal, 10 minutes.Abandon supernatant, dry.Be dissolved in 100 μ l TE.

(12) measure concentration and purity, packing, frozen in-80 ℃.

1.3 primer, pcr amplification and order-checking

Download GADD45A genome sequence (GenBank sequence from GenBank; AY135686.1; GI:22122007), with primer premier5.0 design primer.Concrete primer details see Table 1.

Table 1 primer sequence table

The primer title	Sequence	SEQ ID NO：
			Upstream primer	5′AGTTTGCACAGGGCAACTCC3′	2
Downstream primer	5′CCTGCTAAAGGAATTAGTCACG3′	3

The pcr amplification condition: 94 ℃ 3 minutes, (94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 40 seconds) * 35,72 ℃ 7 minutes, 10 ℃ of insulations.Pcr amplification product is 1255bp.

Pcr amplification product is delivered the order-checking of Shanghai Bo Shang biotech company, and sequencing result application software Meglign 7.0 and Chromas 2.33 test and analyze, and backward sequencing has been adopted in this enforcement, by analysis, finds to exist following SNP:1556C〉T.

1.4SNP somatotype and association analysis

Application card side (X ²) check statistical study is carried out in the distribution in tested personnel site; Using the Fisher rigorous examination when the expectation number of a cell is less than 5 analyzes.All P values are the bilateral probability, think that remarkable statistical significance is arranged when P＜0.05.Using the dependency of unconditional logistic regression analysis between ill to genotype frequency, gene frequency and ovarian cancer assesses, calculate its odds ratio (Odds ratio, OR) and 95% credibility interval (confidence interval, CI), and application SPSS17.0 software (SPSS Inc.Chicago, Illinois, USA) carry out statistical study.

Found that rs532446 among the SEQ ID NO:1 (1556C〉T) site and ovarian tumors significant correlation, wherein the P value of additive inheritance model (C/C vs.C/T vs.T/T) is 0.03.The P value of dominant inheritance model (C/C+C/T vs T/T) is 0.011(OR=1.228,95%CI[1.043 ~ 1.445).The P value of recessive inheritance model (T/T vs T/C+C/C) is 0.144(OR=1.817,95%CI[1.112 ~ 2.968]), T allelotrope is tumor susceptibility gene (P=0.0101, OR=1.503,95%CI[1.101 ~ 2.053]).Detailed results sees Table 2.

Table 21556C〉T loci gene type and the distribution of allelotrope in ovarian cancer group and normal healthy controls group

Embodiment 2 ovarian cancer susceptibility detection kit

Because 1556C among the SEQ ID NO:1〉sudden change and the ovarian cancer of T deliver height correlation, and therefore can design the GADD45A gene-specific primer based on this sudden change and expand detection take patient's DNA as template again.

Prepare a test kit (100 person-times), it contains material as shown in table 3:

Table 3

Extract experimenter's peripheral blood 2ml, ordinary method is extracted DNA.Utilize ovarian cancer susceptibility detection kit to carry out PCR reaction, reaction product is checked order, utilize Meglign 7.0 and Chromas 2.33 softwares to test and analyze sequencing result.Experimenter's ovarian cancer susceptibility that detected result contains rs532446 (1556C〉T) is lower than normal population.

Should be understood that after having read foregoing of the present invention those skilled in the art can make various modifications or change to the present invention, but the equivalent form of value of these changes or modification drops on equally in the limited range of the present invention.

Claims (4)

1. molecule marker that is used for identification of ovarian cancer susceptibility, it is characterized in that: its nucleotide sequence is shown in SEQ ID NO:1, and the 1556th exists single nucleotide polymorphism a: C〉T, namely the 1556th base comprises base C and two kinds of situations of base T:

cagtggctgg taggcagtgg ctgggaggca gcggcccaat tagtgtcgtg cggcccgtgg 60

cgaggcgagg tccggggagc gagcgagcaa gcaaggcggg aggggtggcc ggagctgcgg 120

cggctggcac aggaggagga gcccgggcgg gcgaggggcg gccggagagc gccagggcct 180

gagctgccgg agcggcgcct gtgagtgagt gcagaaagca ggcgcccgcg cgctagccgt 240

ggcaggagca gcccgcacgc cgcgctctct ccctgggcga cctgcagttt gcaatatgac 300

tttggaggaa ttctcggctg gagagcagaa gaccgaaagg tgagtcggcc tgcggactct 360

tccggcccga acttctctta cctaccccgc gctccccggt gcagccgggc tgtggaaggc 420

ttgcagggga ggaagctaaa aagtttgcac agggcaactc ccgcccttgc tccctcggga 480

ctctccgtgg agctcccacg gactgaaaga gcgtgccccc caacccgaac gagccccgcc 540

ggggcctttg caaagggcag cagtggccgt cgctgcccgt gcggctcccg tggctggcag 600

cctgtggcag gggcactctc gggacttctc acgggacgcc cggtccttgg gcgtgcaggg 660

gtcatggggg gtgacggggc cgcgggagcg ccgggttttc gtagagccca ggtgcgcggt 720

ggtgcttgca ttcgagaggg aggggcgtgg taccggacga ggggggcggc gatggccccg 780

agggcaccgg ggctgacggg acccctcgcc cttgcccgcg tgtaggatgg ataaggtggg 840

ggatgccctg gaggaagtgc tcagcaaagc cctgagtcag cgcacgatca ctgtcggggt 900

gtacgaagcg gccaagctgc tcaacgtgta agtggggccc ttgcgcgtcc cccatggcac 960

cccttcccgc cccagcccgg gaggtcgcct tggctgggcg cccctcgccc ggccgcgcca 1020

cttcctgtcg cttttctgcc tgtctcggaa gggagggggc gagcgggccg ggcggcgacc 1080

cccagggacc cgggcagggg ttgagggcgc ccgcgcttct gcgctcactg gccccgcccg 1140

ctgcccccag cgaccccgat aacgtggtgt tgtgcctgct ggcggcggac gaggacgacg 1200

acagagatgt ggctctgcag atccacttca ccctgatcca ggcgttttgc tgcgagaacg 1260

acatcaacat cctgcgcgtc agcaacccgg gccggctggc ggagctcctg ctcttggaga 1320

ccgacgctgg ccccgcggcg agcgagggcg ccgagcagcc cccggacctg cactgcgtgc 1380

tggtgacggt aagggactgg gggactgcag cctgcagggt agagccccgg aaggacggga 1440

gtcagggctg ggttgcctga ttgtggatct gtggtaggtg ggggtcagga gggtggctgc 1500

ctttgtccga ctagagtgtg gctggacttt cagccgagat gtgctagttt catcarcagg 1560

1556

attttctgtg gtacagaaca tgtctaagca tgctggggac tgccagcagc ggaagagatc 1620

cctgtgagtc agcagtcagc ccagctactc cctacctaca tctgcactgc ctcccgtgac 1680

taattccttt agcagggcag attagataaa gccaaatgaa ttcctggctc acccctcatt 1740

aaggagtcag cttcattctc tgccagtcag agctaaaaat agaaattgtg taggagacaa 1800

accttgttaa ttccctagaa atacattaag aggatagagt ggaatttttt ttctctgcaa 1860

tcttgcattt ttttaatggc tctttttttt tttcctgata aaaacctttg gtaggtaggg 1920

aagttatgtt ttcaggggta aatgtgctac ttttgtcttc taaattttgc tcttttttga 1980

ctggtctagt caagtgacag cccgattatt ttgctactcc ttaaaagtac tattctgtct 2040

cttggagtat ggttgatggc aattccagtt aactgctgtg cagctctcat ctcattgtgc 2100

acacagcatg gaaatctttc tcaaaactgt ttcactcagg tcagggtaac aagtttggta 2160

gagcaaaccg gtgaatgata ctctcatgca aaactgaaca gatatgcaaa catatgtatg 2220

tggttcagct tgggttgcat gggttcagac tttgcaatgt gtagtttaat aggtaattac 2280

ccttaacgct tttgcaggga acccaactac cttgaagaaa ctttaatttt tttgtgcttc 2340

taatttgtct ccatgtcaca tagccaaaat atagaatgtt caagtgtttt ctcctcaaaa 2400

gtataattac tagaatatac tggtttttaa aataagttta tttttataaa tttgtttcca 2460

g 2461。

2. test kit for detection of the ovarian cancer susceptibility, it is characterized in that: comprise Auele Specific Primer, this primer is a pair of, has the sequence of SEQ ID NO:2 and SEQ ID NO:3.

3. the test kit for detection of the ovarian cancer susceptibility according to claim 1, it is characterized in that: also comprise the PCR reaction solution, the PCR reaction solution is by dNTP, Mg ²⁺, Taq enzyme and Buffer form.

4. whether a vitro detection sample exists the method for the single nucleotide polymorphism of GADD45A gene, and it is characterized in that: step is as follows:

(1) with the GADD45A gene of GADD45A gene-specific primer amplification sample, obtains amplified production; Described primer is a pair of, has the sequence of SEQ ID NO:2 and SEQ ID NO:3;

(2) amplified production is carried out sequencing, detect in the amplified production whether have following single nucleotide polymorphism: 1556C T; 1556C〉T sudden change Position Number is based on the sequence of SEQ ID NO:1.