CN103911440B - SNP marker related to liver toxicity of platinum type chemotherapeutic medicines and applications thereof - Google Patents
SNP marker related to liver toxicity of platinum type chemotherapeutic medicines and applications thereof Download PDFInfo
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Abstract
The invention belongs to the fields of genetic engineering and oncology, and discloses an SNP marker related to liver toxicity of platinum type chemotherapeutic medicines and applications thereof. The marker is a composition of rs6681909, rs4140932, rs13131227, rs4446279, rs17053350, rs9402873, rs16878272, rs13267737, rs7008590, rs947853, rs2838566, rs1383888, rs9642723, rs3822296, rs2160046, rs10002931, rs10032941, rs6449138, rs1048037 and rs10086636. The marker and primer probes thereof can be used for preparing a diagnosis kit of the liver toxicity of platinum type chemotherapeutic medicines, and are good in sensitivity and specificity.
Description
Invention field
The invention belongs to genetically engineered and oncology, relate to the relevant SNP mark of a kind of platinum-based chemotherapy medicine hepatotoxicity and application thereof.
Background technology
Since the sixties in 20th century, take cis-platinum as the treatment that the platinum kind anti-cancer drugs of representative is widely used in the malignant tumours such as nonsmall-cell lung cancer (non smallcell lung cancer, NSCLC), become indispensable medicine in NSCLC chemotherapy.Platinum medicine is distributed to whole body rapidly after entering human body, both can grow and diffusion by Tumor suppression, and also can kill the tumour of distant metastasis, all have therapeutic action to primary tumor, metastasis and subclinical metastasis.But because platinum-based chemotherapy medicine is poor to cell selective, while killing off tumor cells, also there is very large destruction to normal cell, often cause some toxic side effecties.
Although under standard dose, platinum-based chemotherapy is less occur that the liver histological such as steatosis or cholestasis changes, the functional of the livers such as transaminase rising more often occurs, and when platinum medicine dosage is larger, liver injury occurs frequently.Platinum medicine hepatotoxicity main manifestations is the rising of serum bilirubin, glutamic-oxal(o)acetic transaminase, gpt and alkaline phosphatase levels, and according to these indexs, hepatotoxicity severity can be divided into 0 ~ 4 five grade.Hepatotoxicity adds physiological load and the mental anguish of patient, reduces the quality of life of patient, is also often therefore forced to stop chemotherapy, thus have impact on the prognosis of NSCLC patient.Therefore, to the diagnosis that the NSCLC patient accepting platinum-based chemotherapy carries out hepatotoxicity onset risk, sfgd. is targetedly taked in advance to high-risk individuals, really realizes individualized treatment, seem particularly important.
Many factors can affect the onset risk of platinum medicine toxic side effects, as age, sex, smoking, race etc.But, in the NSCLC patient accepting platinum-based chemotherapy, even if the situations such as some patient's age, sex, smoking state, histological type, clinical stages are similar, whether hepatotoxicity occurs for it and hepatotoxic severity still has very big-difference, point out under equal ambient exposes, the susceptibility with the individual platinum medicine hepatotoxicity of NSCLC of different genetic background is different.
Research shows, single nucleotide polymorphism (single nucleotide polymorphism, SNP) is the important inherited genetic factors causing platinum medicine toxic side effects.SNP refers to the DNA sequence polymorphism in genomic level caused by the variation of single core thuja acid, and it is modal one in the heritable variation of the mankind.The existence of SNP is considered to impart individual different phenotypic character, and for the differential responses of the factor such as environmental exposure, pharmacological agent, therefore SNP causes individuality to common disease generation, prognosis, and the important hereditary basis to drug reaction susceptibility difference.The SNP of disease-susceptible humans is utilized to compose the generation diagnosed the illness, not only sensitive, accurate and quick, have broad application prospects.In recent years, the generation development utilizing SNP to diagnose the illness has become clinical and study hotspot that is researcher, the using value first meeting clue in the common major disease diagnosis such as tumour and cardiovascular and cerebrovascular diseases.
But, also SNP is not applied to the report of platinum-based chemotherapy medicine hepatotoxicity auxiliary diagnosis at present, if the SNP of platinum-based chemotherapy medicine hepatotoxicity susceptible can be filtered out as biomarker, and develop corresponding auxiliary diagnostic box, will be once strong promotion to the present situation of China's platinum-based chemotherapy medicine hepatotoxicity auxiliary diagnosis, also for its chemoprophylaxis with control to open new approach.
Summary of the invention
The object of the invention is for above-mentioned technical problem, propose a kind of SNP mark relevant to platinum-based chemotherapy medicine hepatotoxicity.
Second object of the present invention is to provide the Auele Specific Primer of above-mentioned SNP mark.
3rd object of the present invention is to provide the specificity fluorescent probe pair of above-mentioned SNP mark.
4th object of the present invention is to provide above-mentioned SNP mark and Auele Specific Primer and/or specificity fluorescent probe thereof and is preparing the application in platinum-based chemotherapy medicine hepatotoxicity auxiliary diagnostic box.
The present invention's the 5th object is to provide platinum-based chemotherapy medicine hepatotoxicity auxiliary diagnostic box.
Contriver accepts platinum-based chemotherapy and the single nucleotide polymorphism occurred in the NSCLC peripheral blood in patients DNA of hepatotoxicity in various degree by being separated and studying, find one group with the high specific of platinum-based chemotherapy medicine hepatotoxicity height correlation and the SNP of susceptibility, and develop the platinum-based chemotherapy medicine hepatotoxicity auxiliary diagnostic box can being convenient to clinical application, for the prevention and corntrol of platinum-based chemotherapy medicine hepatotoxicity provides Data support, for finding that the new small molecule medicine with potential therapeutic value provides Data support.
The object of the invention is to be realized by following technical proposal:
A SNP mark relevant to platinum-based chemotherapy medicine hepatotoxicity, this mark is the combination of rs6681909, rs4140932, rs13131227, rs4446279, rs17053350, rs9402873, rs16878272, rs13267737, rs7008590, rs947853, rs2838566, rs1383888, rs9642723, rs3822296, rs2160046, rs10002931, rs10032941, rs6449138, rs1048037 and rs10086636.
The Auele Specific Primer of described SNP mark, these primers are:
The primer sequence of rs6681909 is SEQ ID No:1 and SEQ ID No:2; The primer sequence of rs4140932 is SEQ ID No:5 and SEQ ID No:6; The primer sequence of rs13131227 is SEQ ID No:9 and SEQ IDNo:10; The primer sequence of rs4446279 is SEQ ID No:13 and SEQ ID No:14; The primer sequence of rs17053350 is SEQ ID No:17 and SEQ ID No:18; The primer sequence of rs9402873 is SEQ ID No:21 and SEQID No:22; The primer sequence of rs16878272 is SEQ ID No:25 and SEQ ID No:26; The primer sequence of rs13267737 is SEQ ID No:29 and SEQ ID No:30; The primer sequence of rs7008590 is SEQ ID No:33 and SEQ ID No:34; The primer sequence of rs947853 is SEQ ID No:37 and SEQ ID No:38; The primer sequence of rs2838566 is SEQ ID No:41 and SEQ ID No:42; The primer sequence of rs1383888 is SEQ ID No:45 and SEQ ID No:46; The primer sequence of rs9642723 is SEQ ID No:49 and SEQ ID No:50; The primer sequence of rs3822296 is SEQ ID No:53 and SEQ ID No:54; The primer sequence of rs2160046 is SEQ ID No:57 and SEQ ID No:58; The primer sequence of rs10002931 is SEQ ID No:61 and SEQ ID No:62; The primer sequence of rs10032941 is SEQ ID No:65 and SEQ ID No:66; The primer sequence of rs6449138 is SEQ ID No:69 and SEQ ID No:70; The primer sequence of rs1048037 is SEQ ID No:73 and SEQ IDNo:74; The primer sequence of rs10086636 is SEQ ID No:77 and SEQ ID No:78.
The specificity fluorescent probe pair of described SNP mark, these fluorescent probes are:
The fluorescent probe of rs6681909 is SEQ ID No:3 and SEQ ID No:4 to sequence; The fluorescent probe of rs4140932 is SEQ ID No:7 and SEQ ID No:8 to sequence; The fluorescent probe of rs13131227 is SEQ IDNo:11 and SEQ ID No:12 to sequence; The fluorescent probe of rs4446279 is SEQ ID No:15 and SEQ IDNo:16 to sequence; The fluorescent probe of rs17053350 is SEQ ID No:19 and SEQ ID No:20 to sequence; The fluorescent probe of rs9402873 is SEQ ID No:23 and SEQ ID No:24 to sequence; The fluorescent probe of rs16878272 is SEQ ID No:27 and SEQ ID No:28 to sequence; The fluorescent probe of rs13267737 is SEQ ID No:31 and SEQ ID No:32 to sequence; The fluorescent probe of rs7008590 is SEQ ID No:35 and SEQ ID No:36 to sequence; The fluorescent probe of rs947853 is SEQ ID No:39 and SEQ ID No:40 to sequence; The fluorescent probe of rs2838566 is SEQ ID No:43 and SEQ ID No:44 to sequence; The fluorescent probe of rs1383888 is SEQ IDNo:47 and SEQ ID No:48 to sequence; The fluorescent probe of rs9642723 is SEQ ID No:51 and SEQ IDNo:52 to sequence; The fluorescent probe of rs3822296 is SEQ ID No:55 and SEQ ID No:56 to sequence; The fluorescent probe of rs2160046 is SEQ ID No:59 and SEQ ID No:60 to sequence; The fluorescent probe of rs10002931 is SEQ ID No:63 and SEQ ID No:64 to sequence; The fluorescent probe of rs10032941 is SEQ ID No:67 and SEQ ID No:68 to sequence; The fluorescent probe of rs6449138 is SEQ ID No:71 and SEQ ID No:72 to sequence; The fluorescent probe of rs1048037 is SEQ ID No:75 and SEQ ID No:76 to sequence; The fluorescent probe of rs10086636 is SEQ ID No:79 and SEQ ID No:80 to sequence.
Described SNP mark is preparing the application in platinum-based chemotherapy medicine hepatotoxicity auxiliary diagnostic box.
The Auele Specific Primer of described SNP mark is preparing the application in platinum-based chemotherapy medicine hepatotoxicity auxiliary diagnostic box.
The specificity fluorescent probe of described SNP mark is to preparing the application in platinum-based chemotherapy medicine hepatotoxicity auxiliary diagnostic box.
A kind of platinum-based chemotherapy medicine hepatotoxicity auxiliary diagnostic box, this test kit is for detecting rs6681909, rs4140932, rs13131227, rs4446279, rs17053350, rs9402873, rs16878272, rs13267737, rs7008590, rs947853, rs2838566, rs1383888, rs9642723, rs3822296, rs2160046, rs10002931, rs10032941, rs6449138, rs1048037 and rs10086636 in peripheral blood DNA.
Described auxiliary diagnostic box, this test kit contains Auele Specific Primer and/or the specificity fluorescent probe of above-mentioned SNP mark.
Described auxiliary diagnostic box, the Auele Specific Primer of the SNP mark that this test kit contains is:
The primer sequence of rs6681909 is SEQ ID No:1 and SEQ ID No:2; The primer sequence of rs4140932 is SEQ ID No:5 and SEQ ID No:6; The primer sequence of rs13131227 is SEQ ID No:9 and SEQ IDNo:10; The primer sequence of rs4446279 is SEQ ID No:13 and SEQ ID No:14; The primer sequence of rs17053350 is SEQ ID No:17 and SEQ ID No:18; The primer sequence of rs9402873 is SEQ ID No:21 and SEQID No:22; The primer sequence of rs16878272 is SEQ ID No:25 and SEQ ID No:26; The primer sequence of rs13267737 is SEQ ID No:29 and SEQ ID No:30; The primer sequence of rs7008590 is SEQ ID No:33 and SEQ ID No:34; The primer sequence of rs947853 is SEQ ID No:37 and SEQ ID No:38; The primer sequence of rs2838566 is SEQ ID No:41 and SEQ ID No:42; The primer sequence of rs1383888 is SEQ ID No:45 and SEQ ID No:46; The primer sequence of rs9642723 is SEQ ID No:49 and SEQ ID No:50; The primer sequence of rs3822296 is SEQ ID No:53 and SEQ ID No:54; The primer sequence of rs2160046 is SEQ ID No:57 and SEQ ID No:58; The primer sequence of rs10002931 is SEQ ID No:61 and SEQ ID No:62; The primer sequence of rs10032941 is SEQ ID No:65 and SEQ ID No:66; The primer sequence of rs6449138 is SEQ ID No:69 and SEQ ID No:70; The primer sequence of rs1048037 is SEQ ID No:73 and SEQ IDNo:74; The primer sequence of rs10086636 is SEQ ID No:77 and SEQ ID No:78.
Described auxiliary diagnostic box, the specificity fluorescent probe of the SNP mark that this test kit contains to for:
The fluorescent probe of rs6681909 is SEQ ID No:3 and SEQ ID No:4 to sequence; The fluorescent probe of rs4140932 is SEQ ID No:7 and SEQ ID No:8 to sequence; The fluorescent probe of rs13131227 is SEQ IDNo:11 and SEQ ID No:12 to sequence; The fluorescent probe of rs4446279 is SEQ ID No:15 and SEQ IDNo:16 to sequence; The fluorescent probe of rs17053350 is SEQ ID No:19 and SEQ ID No:20 to sequence; The fluorescent probe of rs9402873 is SEQ ID No:23 and SEQ ID No:24 to sequence; The fluorescent probe of rs16878272 is SEQ ID No:27 and SEQ ID No:28 to sequence; The fluorescent probe of rs13267737 is SEQ ID No:31 and SEQ ID No:32 to sequence; The fluorescent probe of rs7008590 is SEQ ID No:35 and SEQ ID No:36 to sequence; The fluorescent probe of rs947853 is SEQ ID No:39 and SEQ ID No:40 to sequence; The fluorescent probe of rs2838566 is SEQ ID No:43 and SEQ ID No:44 to sequence; The fluorescent probe of rs1383888 is SEQ IDNo:47 and SEQ ID No:48 to sequence; The fluorescent probe of rs9642723 is SEQ ID No:51 and SEQ IDNo:52 to sequence; The fluorescent probe of rs3822296 is SEQ ID No:55 and SEQ ID No:56 to sequence; The fluorescent probe of rs2160046 is SEQ ID No:59 and SEQ ID No:60 to sequence; The fluorescent probe of rs10002931 is SEQ ID No:63 and SEQ ID No:64 to sequence; The fluorescent probe of rs10032941 is SEQ ID No:67 and SEQ ID No:68 to sequence; The fluorescent probe of rs6449138 is SEQ ID No:71 and SEQ ID No:72 to sequence; The fluorescent probe of rs1048037 is SEQ ID No:75 and SEQ ID No:76 to sequence; The fluorescent probe of rs10086636 is SEQ ID No:79 and SEQ ID No:80 to sequence.
Described auxiliary diagnostic box, this test kit can also comprise the conventional enzyme of PCR reaction and reagent, as Taq enzyme, dNTP mixed solution, MgCl2 solution, deionized water etc.; Standard substance and/or reference substance can also be contained.
Specifically, the technical scheme that the present invention deals with problems comprises: (1) sets up sample storehouse and the database of unified standard: gather standard compliant blood sample with Standard operation procedure SOP (SOP), the demographic data that systematic collection is complete and clinical data.(2) genotype detection: select to accept platinum-based chemotherapy and the NSCLC patient having detailed hepatotoxicity evaluation information, utilize high-density SNP chip, find out the SNP relevant to platinum-based chemotherapy medicine hepatotoxicity within the scope of full-length genome.(3) development of platinum-based chemotherapy medicine hepatotoxicity auxiliary diagnostic box: develop SNP auxiliary diagnostic box according to the genotype distribution frequency SNP that there were significant differences in the NSCLC patient of hepatotoxicity in various degree.
The present inventor gathers standard compliant blood sample with Standard operation procedure SOP (SOP), the demographic data that systematic collection is complete, clinical data etc., and have employed Affymetrix6.0 chip and carry out genome-wide screening.
The experimental technique studied specifically mainly comprises following components:
1. study the selection of sample
(1) first admission and make a definite diagnosis through histopathology or cytology new send out NSCLC patient, the standard that its diagnosis is promulgated according to the World Health Organization is confirmed by least two Pathologis;
(2) liver function test indices is all normal before chemotherapy first;
(3) treatment based on platinum-based chemotherapy in 2 ~ 6 cycles is accepted; Get rid of before chemotherapy or chemotherapeutic period accepts the NSCLC patient of radiotherapy;
(4) detailed chemotherapy 2 cycle or the evaluation information of 3 weeks after date hepatotoxicities is had.
This research adopts 334 routine standard compliant samples to study altogether.
2. phenol-chloroform method extracts peripheral blood genomic dna, operates according to a conventional method.Usually, can obtain 20ng/ μ l ~ 50ng/ μ l DNA, purity (ultraviolet 260OD and 280OD ratio) is at 1.6-2.0.
3.Affymetrix6.0 chip detection
(1) experimenter's complete genome DNA sample is got;
(2) on Affymetrix6.0 chip (being purchased from Ang Fei company of the U.S., lower same), genome-wide screening is carried out;
(3) to detect and the difference difference of more each genotype in the NSCLC patient of hepatotoxicity in various degree.
4. diagnostic reagent box preparation method
Affymetrix6.0 chip carries out genome-wide screening and determines the genotype distribution frequency SNP that there were significant differences in the NSCLC patient of hepatotoxicity in various degree, as platinum-based chemotherapy medicine hepatotoxicity diagnosis index.Relevant SNP is there is and forms auxiliary diagnostic box (rs6681909, rs4140932, rs13131227, rs4446279, rs17053350, rs9402873, rs16878272, rs13267737, rs7008590, rs947853, rs2838566, rs1383888, rs9642723, rs3822296, rs2160046, rs10002931, rs10032941, rs6449138, rs1048037 and rs10086636) in what finally filter out with platinum-based chemotherapy medicine hepatotoxicity.Diagnostic reagent can comprise Auele Specific Primer and the specificity fluorescent probe pair of these SNP, and the reagent such as Taq enzyme, dNTP.
6. statistical analysis technique
Chi square test (for classified variable) or student t is used to check (for continuous variable) to compare demographic characteristics, smoking, organization type, the difference that by stages etc. distributes between research object group.Association analysis is carried out, ratio calculated ratio (Odds Ratio, OR) and their 95% credibility interval with the additive model in logistic regression analysis.Defining the SNP that those toxin for liver side reactions OR is less than 1 is protectiveness SNP; Defining the SNP that those toxin for liver side reactions OR is greater than 1 is dangerous SNP.
In order to study the effect of comprehensive indication for platinum-based chemotherapy medicine hepatotoxicity auxiliary diagnosis of these 20 SNP formations further, we construct a mathematical formula, consider positive and negative that each SNP and platinum-based chemotherapy medicine hepatotoxicity occur and associate situation and relation intensity.Specifically, we mark to three of each SNP kinds of genotype, wild homozygous=" 0 ", heterozygous=" 1 ", make a variation homozygous=" 2 ", with the regression coefficient under additive model during single snp analysis for weight, the situation considering each SNP determines a dangerous score value to each research object.Risk score calculation method is as follows: risk score = (0.72 x rs6681909 score) + (0.63 x rs4140932 score) + (0.63 x rs13131227 score) + (0.69 x rs4446279 score) + (0.71 x rs17053350 score) + (1.21 x rs9402873 score) + (0.67 x rs16878272 score) + (0.77 x rs13267737 score) + (0.73 x rs7008590 score) + (0.65 x rs947853 ratings) + (1.33 x rs2838566 score) + (0.78 x rs1383888 ratings) + (0.65 x rs9642723 score) + (0.67 x rs3822296 score) + (0.67 x rs2160046 score) + (0.66 x rs10002931 score) + (0.66 x rs10032941 score) + (0.66 x rs6449138 score) + (0.65 x rs1048037 score) + (0.73 x rs10086636 score), the risk score coefficient and threshold value is used for 334 cases of the whole genome association study samples.(be the regression coefficient (see table 1) of rs6681909 variant sites for rs6681909: 0.72; The scoring of rs6681909, wild homozygous=" 0 ", heterozygous=" 1 ", make a variation homozygous=" 2 ".Certain SNP is that wild homozygous, heterozygous or make a variation homozygously is determined by Instrumental results; The overall score of certain sample is the summation that this 20 SNP mark respectively, and the genotype of single SNP just calculates a pilot process of scoring, does not need to list concrete genotype.）
Statistical analysis is all completed (PLINK1.07) by special statistical analysis software.The horizontal P value of significance,statistical is set to 1.0 × 10-4, and all statistical test are two-tailed test.
Below further instruction of the present invention:
We by above-mentioned accept platinum-based chemotherapy after there is hepatotoxicity in various degree NSCLC patient carry out genome-wide screening through Affymetrix6.0 chip and obtain correlated results.
According to Affymetrix6.0 chip detection, the present inventor detects that the SNP that genotype distribution frequency there are differences in the NSCLC patient of hepatotoxicity in various degree comprises: rs6681909, rs4140932, rs13131227, rs4446279, rs17053350, rs9402873, rs16878272, rs13267737, rs7008590, rs947853, rs2838566, rs1383888, rs9642723, rs3822296, rs2160046, rs10002931, rs10032941, rs6449138, rs1048037 and rs10086636.
Logistic regression analysis result shows, these 20 SNP and platinum-based chemotherapy medicine hepatotoxicity occurrence risk also exist dose-response relationship: Hazard Factor (rs6681909, rs4140932, rs13131227, rs4446279, rs17053350, rs9402873, rs13267737, rs7008590, rs283856, rs3822296, rs2160046, rs10002931, rs10032941, rs6449138, rs1048037 and rs10086636, variation allelotype increases liver toxicity risk), protection factor (rs16878272, rs947853, rs1383888 and rs9642723, variation allelotype reduces liver toxicity risk).
The combination of these 20 SNP of further analysis is used for the effect of platinum-based chemotherapy medicine hepatotoxicity diagnosis, finds that its combination can be good at distinguishing the NSCLC patient of hepatotoxicity in various degree.
According to above-mentioned experimental result, the present inventor has prepared a kind of test kit that can be used for clinical platinum-based chemotherapy medicine hepatotoxicity auxiliary diagnosis, comprise measure above-mentioned SNP in experimenter blood specimen DNA Auele Specific Primer, specificity fluorescent probe to other detection reagent.
Specifically; the combination of these 20 SNP; or the dependent diagnostic test kit that the Auele Specific Primer of these 20 SNP and the right combination of specificity fluorescent probe are formed contributes to the diagnosis of platinum-based chemotherapy medicine hepatotoxicity; sfgd. is targetedly taked in advance to high-risk individuals; really realize individualized treatment, seem extremely important.
Beneficial effect of the present invention:
SNP mark provided by the invention is as the superiority of the mark that platinum-based chemotherapy medicine hepatotoxicity is correlated with:
(1) SNP is a kind of novel gene biomarker, be different from traditional biological mark, stable, Wicresoft, be easy to detect, to greatly improve the Sensitivity and Specificity of medical diagnosis on disease, the successful exploitation of such biomarker starts brand-new situation, for the development of other diseases biomarker is offered reference by for the diagnosis of platinum-based chemotherapy medicine hepatotoxicity and control.
(2) SNP test kit is a kind of system, comprehensively diagnostic kit, can be used for the auxiliary diagnosis of platinum-based chemotherapy medicine hepatotoxicity, contribute to reflection tumour patient to the easy trouble degree of platinum medicine hepatotoxicity, for clinician quick and precisely grasps conditions of patients, takes the control prece of more personalized to provide support in time.
(3) the present inventor adopts full-length genome chip scanning to obtain the relevant SNP spectrum of platinum medicine hepatotoxicity, and the SNP of covering is more comprehensive; The application acceleration of this strategy and ensure that SNP biomarker and the application clinically of auxiliary diagnostic box is also the reference on the development supplying method of other diseases biomarker and strategy.
The influence factor that the present invention was occurred toxic side effects by the control age etc., the application prospect of research SNP in the diagnosis of platinum-based chemotherapy medicine hepatotoxicity, sets forth the impact that SNP is in progress for platinum-based chemotherapy medicine hepatotoxicity, discloses its diagnostic value.Therefore, present invention obtains platinum-based chemotherapy medicine hepatotoxicity related SNP spectrum and Specific marker; By the development and application of SNP biomarker and auxiliary diagnostic box, clinician can be made to identify the high-risk individuals of platinum medicine hepatotoxicity better, lay the foundation for quick and precisely grasping conditions of patients and realizing individualized treatment, and for finding that the new small molecule drug targets with potential therapeutic value is offered help.
Accompanying drawing explanation
Fig. 1: the ROC curve that the NSCLC patient of hepatotoxicity occurs in display genome-wide association study or do not occur.
Display accepts hepatotoxicity group to occur to the ROC curve that hepatotoxicity group is reference does not occur in the NSCLC patient of platinum-based chemotherapy.
Embodiment
The collection of embodiment 1 sample and the arrangement of sample data
Contriver started to have collected a large amount of primary NS CLC patient blood specimens in January, 2011 from attached tumour hospital of Nanjing Medical University and The First Affiliated Hospital with Nanjing Medical University 2005 month April, by the arrangement to sample data, contriver therefrom have selected the full-length genome chip scanning sample that 334 examples meet following standard:
(1) first admission and make a definite diagnosis through histopathology or cytology new send out NSCLC patient, the standard that its diagnosis is promulgated according to the World Health Organization is confirmed by least two Pathologis;
(2) liver function test indices is all normal before chemotherapy first;
(3) treatment based on platinum-based chemotherapy in 2 ~ 6 cycles is accepted; Get rid of before chemotherapy or chemotherapeutic period accepts the NSCLC patient of radiotherapy;
(4) detailed chemotherapy 2 cycle or the evaluation information of 3 weeks after date hepatotoxicities is had.
And the system acquisition situation such as demographic data and clinical data of these samples.
The genome-wide screening of SNP in embodiment 2 peripheral blood DNA
The NSCLC peripheral blood in patients DNA above-mentioned qualified 334 examples being accepted platinum-based chemotherapy carries out Affymetrix6.0 chip detection and obtains correlated results.Concrete steps are:
1, hemolyzing reagent (i.e. lysate is added to the hemocyte be stored in 2ml cryopreservation tube, 40 deal collocation methods are as follows: after sucrose 219.72g, magnesium chloride 2.02g and triton x-100 (amresco0694) 20ml mix, 2000ml is settled to TrisHcl solution, lower same), proceed to completely after putting upside down mixing.
2, red corpuscle is removed: mend 5ml centrifuge tube to 4ml with hemolyzing reagent, put upside down mixing, centrifugal 10 minutes of 4000rpm, abandons supernatant.In precipitation, add 4ml hemolyzing reagent, again put upside down mixing cleaning once, centrifugal 10 minutes of 4000rpm, abandons supernatant.
3, extracting DNA: add 1ml extract and (contain 122.5ml0.2M sodium-chlor in every 300ml, 14.4ml0.5M ethylenediamine tetraacetic acid (EDTA), 15ml10% sodium lauryl sulphate in precipitation, 148.1ml distilled water, lower same) and 8 μ l Proteinase Ks, oscillator fully shakes mixing, 37 DEG C of water-baths are spent the night.
4, protein is removed: add the saturated phenol of 1ml and fully mix (have gentle hands shakes 15 minutes), centrifugal 10 minutes of 4000rpm, get supernatant and proceed in new 5ml centrifuge tube.In supernatant liquor, add equal-volume chloroform and primary isoamyl alcohol mixed solution (chloroform: primary isoamyl alcohol=24:1, v/v, lower same), fully after mixing (hand 15 minutes), centrifugal 10 minutes of 4000rpm, gets supernatant (being divided into the centrifuge tube of two 1.5ml).
5, DNA precipitation: the sodium-acetate 60 μ l adding 3M in supernatant liquor, then add isopyknic ice dehydrated alcohol with supernatant liquor, upper and lower jog, visible white flocculent precipitate, then with the centrifugal 10min of 12000rpm.
6, DNA washing: add ice dehydrated alcohol 1ml in precipitation, the centrifugal 10min of 12000rpm, abandon supernatant final vacuum and drain or be placed in clean dry environment evaporate to dryness.
7, concentration is measured: usually can obtain 20ng/ μ l-50ng/ μ l DNA, purity (ultraviolet 260OD and 280OD ratio) is at 1.6-2.0.
8, genome-wide screening is carried out: Affymetrix6.0 chip carries out genome-wide screening
9, data analysis and process: the genotype distribution frequency SNP that there were significant differences occurring to find in the NSCLC patient of hepatotoxicity in various degree after accepting platinum-based chemotherapy is enumerated out hereinbefore, the results are shown in Table 1.
NSCLC patient's whole-genome association result of table 1 hepatotoxicity in various degree
Embodiment 3 utilizes MELD method to analyze the hepatotoxicity of SNP and platinum-based chemotherapy medicine further
According to the above results, the present inventor is by the comparison to the NSCLC Patient genotype distribution frequency of hepatotoxicity in various degree, select the SNP of positive association, with SNP regression coefficient single in genome-wide screening sample for weight, try to achieve dangerous score value further, draw susceptibility and specificity that ROC assesses diagnosis, and then assess the diagnosis capability of these SNP to platinum-based chemotherapy medicine hepatotoxicity.The Conjoint Analysis of 20 SNP marks is found, these 20 SNP with 73.5% ROC by generation liver toxicity with there is not hepatotoxic NSCLC patient and separate, the sensitivity of best cut point is 66.7%, specific degree: 69.4%(is shown in Fig. 1).
Therefore, the present inventor demonstrates and adopts the combination of rs6681909, rs4140932, rs13131227, rs4446279, rs17053350, rs9402873, rs16878272, rs13267737, rs7008590, rs947853, rs2838566, rs1383888, rs9642723, rs3822296, rs2160046, rs10002931, rs10032941, rs6449138, rs1048037 and rs10086636 the NSCLC patient that hepatotoxicity in various degree easily occurs to be distinguished well.
Embodiment 4 is for the making of the auxiliary diagnosis SNP test kit of platinum-based chemotherapy medicine hepatotoxicity
The making of SNP test kit and operating process are based on Affymetrix6.0 chip detection technology.Test kit contains a collection of SNP Auele Specific Primer and (comprises following primer: the primer sequence of rs6681909 is SEQ ID No:1 and SEQID No:2; The primer sequence of rs4140932 is SEQ ID No:5 and SEQ ID No:6; The primer sequence of rs13131227 is SEQ ID No:9 and SEQ ID No:10; The primer sequence of rs4446279 is SEQ ID No:13 and SEQID No:14; The primer sequence of rs17053350 is SEQ ID No:17 and SEQ ID No:18; The primer sequence of rs9402873 is SEQ ID No:21 and SEQ ID No:22; The primer sequence of rs16878272 is SEQ ID No:25 and SEQ ID No:26; The primer sequence of rs13267737 is SEQ ID No:29 and SEQ ID No:30; The primer sequence of rs7008590 is SEQ ID No:33 and SEQ ID No:34; The primer sequence of rs947853 is SEQ ID No:37 and SEQ ID No:38; The primer sequence of rs2838566 is SEQ ID No:41 and SEQ IDNo:42; The primer sequence of rs1383888 is SEQ ID No:45 and SEQ ID No:46; The primer sequence of rs9642723 is SEQ ID No:49 and SEQ ID No:50; The primer sequence of rs3822296 is SEQ ID No:53 and SEQ ID No:54; The primer sequence of rs2160046 is SEQ ID No:57 and SEQ ID No:58; The primer sequence of rs10002931 is SEQ ID No:61 and SEQ ID No:62; The primer sequence of rs10032941 is SEQ IDNo:65 and SEQ ID No:66; The primer sequence of rs6449138 is SEQ ID No:69 and SEQ ID No:70; The primer sequence of rs1048037 is SEQ ID No:73 and SEQ ID No:74; The primer sequence of rs10086636 is SEQ ID No:77 and SEQ ID No:78), specificity fluorescent probe is to (comprising following fluorescent probe: the fluorescent probe of rs6681909 is SEQ ID No:3 and SEQ ID No:4 to sequence; The fluorescent probe of rs4140932 is SEQ ID No:7 and SEQ ID No:8 to sequence; The fluorescent probe of rs13131227 is SEQ ID No:11 and SEQID No:12 to sequence; The fluorescent probe of rs4446279 is SEQ ID No:15 and SEQ ID No:16 to sequence; The fluorescent probe of rs17053350 is SEQ ID No:19 and SEQ ID No:20 to sequence; The fluorescent probe of rs9402873 is SEQ ID No:23 and SEQ ID No:24 to sequence; The fluorescent probe of rs16878272 is SEQID No:27 and SEQ ID No:28 to sequence; The fluorescent probe of rs13267737 is SEQ ID No:31 and SEQ IDNo:32 to sequence; The fluorescent probe of rs7008590 is SEQ ID No:35 and SEQ ID No:36 to sequence; The fluorescent probe of rs947853 is SEQ ID No:39 and SEQ ID No:40 to sequence; The fluorescent probe of rs2838566 is SEQ ID No:43 and SEQ ID No:44 to sequence; The fluorescent probe of rs1383888 is SEQ ID No:47 and SEQ ID No:48 to sequence; The fluorescent probe of rs9642723 is SEQ ID No:51 and SEQ ID No:52 to sequence; The fluorescent probe of rs3822296 is SEQ ID No:55 and SEQ ID No:56 to sequence; The fluorescent probe of rs2160046 is SEQ ID No:59 and SEQ ID No:60 to sequence; The fluorescent probe of rs10002931 is SEQ IDNo:63 and SEQ ID No:64 to sequence; The fluorescent probe of rs10032941 is SEQ ID No:67 and SEQ IDNo:68 to sequence; The fluorescent probe of rs6449138 is SEQ ID No:71 and SEQ ID No:72 to sequence; The fluorescent probe of rs1048037 is SEQ ID No:75 and SEQ ID No:76 to sequence; The fluorescent probe of rs10086636 is SEQ ID No:79 and SEQ ID No:80 to sequence), the common agents needed for corresponding round pcr can also be had, as: dNTPs, MgCl2, distilled water, fluorescent probes etc., these common agents are all well known to those skilled in the art, can also have standard substance and contrast (as determined genotypic standard substance and blank etc.) in addition.The value of this test kit is only to need peripheral blood and does not need other tissue sample, by simplifying most with special primer and fluorescent probe detection SNP, the tumour patient toxin for liver side reaction of platinum-based chemotherapy is accepted again by the diagnosis of SNP spectrum, not only stablize, easy to detect, and accurately, greatly improve the Sensitivity and Specificity of medical diagnosis on disease, therefore this test kit is dropped into practice, can help to instruct the screening of high risk population and more effective individualized treatment.
Table 2. related SNP primer and detecting probe information
Claims (7)
1. a SNP mark relevant to platinum-based chemotherapy medicine hepatotoxicity, is characterized in that this mark is the combination of rs6681909, rs4140932, rs13131227, rs4446279, rs17053350, rs9402873, rs16878272, rs13267737, rs7008590, rs947853, rs2838566, rs1383888, rs9642723, rs3822296, rs2160046, rs10002931, rs10032941, rs6449138, rs1048037 and rs10086636.
2. require the Auele Specific Primer of the SNP mark described in 1 for test right, it is characterized in that this primer is:
The primer sequence of rs6681909 is SEQ ID No:1 and SEQ ID No:2; The primer sequence of rs4140932 is SEQ ID No:5 and SEQ ID No:6; The primer sequence of rs13131227 is SEQ ID No:9 and SEQ IDNo:10; The primer sequence of rs4446279 is SEQ ID No:13 and SEQ ID No:14; The primer sequence of rs17053350 is SEQ ID No:17 and SEQ ID No:18; The primer sequence of rs9402873 is SEQ ID No:21 and SEQ IDNo:22; The primer sequence of rs16878272 is SEQ ID No:25 and SEQ ID No:26; The primer sequence of rs13267737 is SEQ ID No:29 and SEQ ID No:30; The primer sequence of rs7008590 is SEQ ID No:33 and SEQ IDNo:34; The primer sequence of rs947853 is SEQ ID No:37 and SEQ ID No:38; The primer sequence of rs2838566 is SEQ ID No:41 and SEQ ID No:42; The primer sequence of rs1383888 is SEQ ID No:45 and SEQ IDNo:46; The primer sequence of rs9642723 is SEQ ID No:49 and SEQ ID No:50; The primer sequence of rs3822296 is SEQ ID No:53 and SEQ ID No:54; The primer sequence of rs2160046 is SEQ ID No:57 and SEQ IDNo:58; The primer sequence of rs10002931 is SEQ ID No:61 and SEQ ID No:62; The primer sequence of rs10032941 is SEQ ID No:65 and SEQ ID No:66; The primer sequence of rs6449138 is SEQ ID No:69 and SEQ IDNo:70; The primer sequence of rs1048037 is SEQ ID No:73 and SEQ ID No:74; The primer sequence of rs10086636 is SEQ ID No:77 and SEQ ID No:78.
3. require the specificity fluorescent probe pair of the SNP mark described in 1 for test right, it is characterized in that this fluorescent probe is:
The fluorescent probe of rs6681909 is SEQ ID No:3 and SEQ ID No:4 to sequence; The fluorescent probe of rs4140932 is SEQ ID No:7 and SEQ ID No:8 to sequence; The fluorescent probe of rs13131227 is SEQ IDNo:11 and SEQ ID No:12 to sequence; The fluorescent probe of rs4446279 is SEQ ID No:15 and SEQ IDNo:16 to sequence; The fluorescent probe of rs17053350 is SEQ ID No:19 and SEQ ID No:20 to sequence; The fluorescent probe of rs9402873 is SEQ ID No:23 and SEQ ID No:24 to sequence; The fluorescent probe of rs16878272 is SEQ ID No:27 and SEQ ID No:28 to sequence; The fluorescent probe of rs13267737 is SEQ ID No:31 and SEQID No:32 to sequence; The fluorescent probe of rs7008590 is SEQ ID No:35 and SEQ ID No:36 to sequence; The fluorescent probe of rs947853 is SEQ ID No:39 and SEQ ID No:40 to sequence; The fluorescent probe of rs2838566 is SEQID No:43 and SEQ ID No:44 to sequence; The fluorescent probe of rs1383888 is SEQ ID No:47 and SEQ IDNo:48 to sequence; The fluorescent probe of rs9642723 is SEQ ID No:51 and SEQ ID No:52 to sequence; The fluorescent probe of rs3822296 is SEQ ID No:55 and SEQ ID No:56 to sequence; The fluorescent probe of rs2160046 is SEQID No:59 and SEQ ID No:60 to sequence; The fluorescent probe of rs10002931 is SEQ ID No:63 and SEQ IDNo:64 to sequence; The fluorescent probe of rs10032941 is SEQ ID No:67 and SEQ ID No:68 to sequence; The fluorescent probe of rs6449138 is SEQ ID No:71 and SEQ ID No:72 to sequence; The fluorescent probe of rs1048037 is SEQID No:75 and SEQ ID No:76 to sequence; The fluorescent probe of rs10086636 is SEQ ID No:79 and SEQ IDNo:80 to sequence.
4. Auele Specific Primer according to claim 2 is preparing the application in platinum-based chemotherapy medicine hepatotoxicity auxiliary diagnostic box.
5. Auele Specific Primer according to claim 2 and specificity fluorescent probe according to claim 3 are to preparing the application in platinum-based chemotherapy medicine hepatotoxicity auxiliary diagnostic box.
6. a platinum-based chemotherapy medicine hepatotoxicity auxiliary diagnostic box, it is characterized in that this test kit contains Auele Specific Primer according to claim 2, or containing Auele Specific Primer according to claim 2 and specificity fluorescent probe pair according to claim 3, for detecting in peripheral blood DNA whether there is rs6681909, rs4140932, rs13131227, rs4446279, rs17053350, rs9402873, rs16878272, rs13267737, rs7008590, rs947853, rs2838566, rs1383888, rs9642723, rs3822296, rs2160046, rs10002931, rs10032941, rs6449138, rs1048037 and rs10086636.
7. auxiliary diagnostic box according to claim 6, is characterized in that this test kit also comprises the conventional reagent of round pcr.
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