CN108441560B - SNP marker located in CEP128 gene and related to radioactive brain injury caused by radiotherapy and application thereof - Google Patents
SNP marker located in CEP128 gene and related to radioactive brain injury caused by radiotherapy and application thereof Download PDFInfo
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Abstract
The invention disclosesCEP128The application of the SNP locus on the gene as a marker related to the onset risk of the radioactive brain injury caused by tumor radiotherapy or the application in the preparation of a kit for predicting the onset risk of the radioactive brain injury caused by tumor radiotherapy. Meanwhile, the kit for predicting the morbidity risk of the radioactive brain injury caused by tumor radiotherapy is prepared, the kit is used for detecting the SNP marker typing, and the morbidity risk of the radioactive brain injury of the nasopharyngeal carcinoma patient can be comprehensively and accurately evaluated by combining with the clinical information of the patient. The invention is applied to clinic, takes protective measures in advance aiming at high-risk patients, is beneficial to realizing individual treatment of the patients and improves the long-term life quality of the patients with nasopharyngeal carcinoma. Meanwhile, a method and a strategic reference can be provided for the risk prediction of normal tissue damage caused by other radiotherapy.
Description
Technical Field
The invention relates to the technical field of genetic engineering and oncology, in particular to a gene locusCEP128SNP markers of genes related to radioactive brain injury caused by radiotherapy and application thereof.
Background
Radiotherapy is the first choice for treatment of nasopharyngeal carcinoma, and radioactive brain injury caused by radiotherapy is one of the most serious adverse reactions in the late stage of patients with nasopharyngeal carcinoma. The adverse reactions are often irreversible, and the clinical symptoms of the adverse reactions comprise dizziness, headache, memory deterioration, cognitive dysfunction and the like, so that the quality of life of a patient is greatly influenced. Once radioactive brain injury occurs, the treatment is rather difficult, and drugs such as hormone, ganglioside or citicoline are generally taken for clinical treatment, or hyperbaric oxygen or surgical treatment. In recent years, bevacizumab and nerve growth factor are found to have certain curative effect. Most of the treatment modes can only relieve the symptoms of the patients to a certain extent, and other adverse reactions can be caused by long-term use of part of the medicines. Therefore, the prediction of the onset risk of radioactive brain injury of a nasopharyngeal carcinoma patient receiving radiotherapy is very important for taking targeted protective measures in advance for high-risk individuals to realize the individual treatment of the nasopharyngeal carcinoma.
Studies have shown that a variety of clinical factors are associated with the risk of developing radioactive brain injury, such as radiation dose, tumor staging, radiation therapy techniques, and the like. However, the occurrence and severity of radioactive brain injury still vary greatly among patients under the same clinical factors and therapeutic measures, suggesting that genetic factors may be an important cause of the occurrence of radioactive brain injury.
The current research finds that Single Nucleotide Polymorphism (SNP) is an important genetic factor causing radiation damage of normal tissues. Genetic susceptibility studies on normal tissue damage caused by radiotherapy are divided into candidate gene studies and whole genome association studies. The research of candidate genes focuses on the effects of genes on pathways such as DNA damage repair, cell cycle, inflammatory reaction and the like on early/late side reactions such as skin fibrosis, gastrointestinal mucosal reaction, erectile dysfunction, radiation esophagitis, radiation pneumonia and the like caused by radiotherapy; and the whole genome association research successfully reveals susceptibility sites which are obviously related to the acute/late adverse reactions of the breast cancer and the prostate cancer. These findings suggest that genetic factors play an important role in individual variation in radiosensitivity.
However, no genetic studies related to radioactive brain injury have been reported. If SNP loci related to radioactive brain injury caused by radiotherapy can be screened out and a corresponding diagnostic kit is developed, prediction and early intervention can be carried out on patients with high risk of radioactive brain injury, and meanwhile, the diagnostic kit can be popularized and applied to other tumors which are subjected to therapeutic or preventive radiotherapy except nasopharyngeal carcinoma, so that the life quality of tumor patients is improved.
Disclosure of Invention
The purpose of the invention isTo overcome the deficiencies of the prior art, a solution is providedCEP128SNP markers of genes related to radioactive brain injury caused by radiotherapy and a detection method thereof.
The first purpose of the invention is to provideCEP128The application of the SNP locus on the gene as a marker related to the onset risk of the radioactive brain injury caused by tumor radiotherapy or the application in the preparation of a kit for predicting the onset risk of the radioactive brain injury caused by tumor radiotherapy.
The second purpose of the invention is to provide a pair of specific amplification primers for detecting any one of the SNP sites.
It is a third object of the present invention to provide a pair of specific probes for detecting any of the SNP sites described above.
It is a fourth object of the present invention to provide a pair of specific amplification primers for detecting SNP site rs162171 as described above.
A fifth object of the present invention is to provide a pair of specific probes for detecting SNP site rs162171 as described above.
It is a sixth object of the present invention to provide a pair of specific amplification primers for detecting the SNP site rs17111237 described above.
The seventh object of the present invention is to provide a pair of specific probes for detecting the SNP site rs17111237 described above.
The eighth purpose of the present invention is to provide an application of the specific amplification primer described above and/or the specific probe described above in the preparation of a kit for predicting the risk of the onset of radioactive brain injury caused by tumor radiotherapy.
The ninth purpose of the invention is to provide a kit for predicting the risk of the radioactive brain injury caused by tumor radiotherapy.
In order to achieve the purpose, the invention is realized by the following technical scheme:
the inventor searches SNP with high specificity and sensitivity which is highly related to radioactive brain injury by separating and researching single nucleotide polymorphism in DNA of peripheral blood of nasopharyngeal darcinoma patients who receive radiotherapy and have radioactive brain injury at different time after the radiotherapy, develops a kit which is convenient for clinical application and used for predicting the morbidity risk of the radioactive brain injury caused by tumor radiotherapy, provides a theoretical basis for the prevention and control of the radioactive brain injury and provides clues for exploring and discovering new drugs with potential treatment values.
The invention is thus claimedCEP128The application of the SNP locus on the gene as a marker related to the onset risk of radioactive brain injury caused by tumor radiotherapy or the application in the preparation of a kit for predicting the onset risk of radioactive brain injury caused by tumor radiotherapy is characterized in that the SNP locus is rs162171, rs17111237, rs10133034, rs10135084, rs10141970, rs10142753, rs1016699, rs1038523, rs11625199, rs11627934, rs1197467, rs1197474, rs12050151, rs12050342, rs 505050416, rs12433134, rs12434318, rs12437260, rs162174, rs162178, rs17059, rs 186170596, rs 1711121716, rs 15020, rs 3766, rs2010847, rs 979719, rs228119, rs 222281347, rs 25255632801607, rs 17146, rs 28063274327446, rs 74327446, rs 743274327446, rs 74327446, rs 743274327446, rs 74327446, rs327446, rs 3274327446, rs 74327448, rs 3274327446, rs 327448, rs 327432743274327446, rs 3274327446, rs 74327446, rs 743274327448, rs 3274327446, rs 3274327432743274327446, rs 3274327446, rs 3274327448, rs 3274327432743274327432743274327446, rs327446, rs 3274327448, rs.
Preferably, the sample is risk scored, wherein the sample is a high risk population for radioactive brain injury when the risk score is greater than 2.177 and the sample is a low risk population for radioactive brain injury when the risk score is less than 2.177;
risk score = (0.6013 × score for T stage) + (-0.8509 × score for radiotherapeutic technique) + (0.02226 × score for age) + (score for score of 0.506 × rs162171 type) + (score for score of 0.49 × rs17111237 type);
in the formula, the score of T stage: for the clinical variables T epoch, T1= "0", T2= "1", T3= "2", T4= "3";
scoring of radiotherapy techniques: for radiotherapy techniques, conventional conformal radiotherapy = "0", intensity modulated radiotherapy = "1";
age scoring: substituting with actual age;
scoring for typing: for SNP typing, wild homozygous type = "0", heterozygous type = "1", mutant homozygous type = "2".
Preferably, the tumor is nasopharyngeal carcinoma.
A pair of specific amplification primers for detecting any one of the SNP sites.
A pair of specific probes for detecting any one of the SNP sites.
A pair of specific amplification primers for detecting the SNP locus rs162171, the nucleotide sequence of which is shown as SEQ ID No: 1 to 2.
A pair of specific probes for detecting the SNP locus rs162171, the nucleotide sequence of which is shown as SEQ ID No: 3 to 4.
A pair of specific amplification primers for detecting the SNP locus rs17111237, wherein the nucleotide sequence is shown as SEQ ID No: 5 to 6.
A pair of specific probes for detecting the SNP locus rs17111237, wherein the nucleotide sequence is shown as SEQ ID No: 7 to 8.
The application of the specific amplification primer and/or the specific probe in the preparation of the kit for predicting the onset risk of the radioactive brain injury caused by tumor radiotherapy also belongs to the protection scope of the invention.
A kit for predicting the risk of the onset of radioactive brain injury caused by tumor radiotherapy comprises components capable of carrying out typing detection on the SNP sites.
Preferably, the components comprise specific amplification primers as described above and/or specific probes as described above.
Preferably, the components include an amplification primer specific for SNP site rs 162171.
More preferably, the nucleotide sequence of the specific amplification primer is as shown in SEQ ID No: 1 to 2.
Preferably, the component comprises a probe specific for SNP site rs 162171.
More preferably, the nucleotide sequence of the specific probe is as shown in SEQ ID No: 3 to 4.
Preferably, the components comprise an amplification primer specific for SNP site rs 17111237.
More preferably, the nucleotide sequence of the specific amplification primer is as shown in SEQ ID No: 5 to 6.
Preferably, the component comprises a probe specific for SNP site rs 17111237.
More preferably, the nucleotide sequence of the specific probe is as shown in SEQ ID No: 7 to 8.
Preferably, the kit further contains PCR reaction enzymes and reagents.
Preferably, the PCR reaction enzymes and reagents include reaction enzymes, dNTP mixtures, MgCl2Solution and deionized water; standards and controls may also be included.
Preferably, the PCR reaction system is: forward primer, reverse primer and typing probe mix 0.3. mu.l, dNTP mix (10mM) 0.2. mu.l, MgCl20.4. mu.l (25mM), 1. mu.l reaction buffer, 0.1. mu.l reaction enzyme, 2. mu.l double distilled water and 1. mu.l test DNA.
Most preferably, the kit contains a nucleotide sequence of the specific amplification primer of rs162171 as shown in SEQ ID No: 1-2, the nucleotide sequence of the specific probe of rs162171 is shown as SEQ ID No: 3-4, the nucleotide sequence of the specific amplification primer of rs17111237 is shown as SEQ ID No: 5-6, the nucleotide sequence of the specific probe of rs17111237 is shown as SEQ ID No: 7 to 8, a reaction enzyme, a dNTP mixture, and MgCl2Solution and deionized water; standards and controls may also be included.
Preferably, the PCR reaction procedure of the kit is: 60 ℃, 30s, 1 cycle; at 95 ℃, 10min, 1 cycle; 45 cycles of 95 ℃, 15s and 60 ℃, 1 min; 60 ℃, 30s, 1 cycle.
Preferably, the kit is used in a method, and comprises the following steps:
s1, carrying out statistics on SNP typing of the sample, clinical variable T stage, radiotherapy technology and age for scoring.
For SNP typing, wild homozygous = "0", heterozygous = "1", mutant homozygous = "2"; for clinical variables T epoch T1= "0", T2= "1", T3= "2", T4= "3"; for radiotherapy techniques, conventional conformal radiotherapy = "0", intensity modulated radiotherapy = "1"; the score for age was substituted with actual age (continuous variable).
S2, calculating the risk score according to the scores, wherein the formula is as follows:
risk score = (0.6013 × score by T-stage) + (-0.8509 × score by radiotherapeutic technique) + (0.02226 × score by age) + (score by score of 0.506 × rs162171 type) + (score by score of 0.49 × rs17111237 type).
The sample is a high risk population for radioactive brain injury when the risk score is greater than 2.177 and a low risk population for radioactive brain injury when the risk score is less than 2.177.
Specifically, the technical solution of the present invention to solve the problem includes:
(1) establishing a unified specimen library and a database: standard procedures (SOP) were used to collect standardized blood samples and the system collected complete demographic and clinical data.
(2) Patients are subjected to regular MR follow-up and the diagnosis of radioactive brain injury is carried out according to MR images.
(3) And (3) genotype detection: for patients with complete clinical data and MR follow-up after radiotherapy, SNP chips with whole genome are utilized to search for SNP related to radioactive brain injury.
(4) For the significantly related SNPs screened out, a TaqMan (applied biosystems) genotyping platform is further adopted for detection, and the repeatability of applying the SNP to risk prediction is verified.
(5) The development of a kit for predicting the onset risk of radioactive brain injury caused by tumor radiotherapy: a kit for predicting the risk of onset of radioactive brain injury caused by tumor radiotherapy was developed based on SNPs that have significant differences in the frequency of genotype distributions in patients who progressed to radioactive brain injury and patients who did not progress to radioactive brain injury.
The inventor collects blood samples meeting the standard by a Standard Operation Program (SOP), systematically collects and visits patients meeting the inclusion standard, adopts an Illumina Human610-Quad chip to carry out genome-wide SNP scanning, and verifies screened sites by TaqMan (applied biosystems) genotyping.
Specifically, the experimental method mainly includes the following parts:
1. selection of study samples
Inclusion criteria were:
(1) nasopharyngeal carcinoma patients with definite pathological diagnosis and clinical stages;
(2) expected survival is greater than 6 months;
(3) the functional state score (KPS) is more than or equal to 70 points;
(4) age 18-80 years;
(5) has complete medical record data (medical history, physical examination, related examination and previous treatment);
(6) completing radiotherapy and periodically performing MR image review;
(7) voluntarily attend and sign an informed consent.
At the same time, patients who presented any of the following conditions were excluded:
(1) the patient did not complete all radiotherapy plans;
(2) patients who cannot be matched for follow-up due to psychological, social, household and geographic reasons;
(3) the patient has long-distance metastasis at the initial diagnosis;
(4) abnormal signals of unknown reasons exist in nasopharyngeal/craniocerebral MR images of patients before radiotherapy;
(5) patients had received head and neck radiotherapy for other diseases (except nasopharyngeal carcinoma).
1082 samples meeting the standard are taken into a discovery stage in total, and whole genome SNP scanning is carried out; the 1119 and 741 samples meeting the standard are respectively adopted to be included in two independent verification stages to carry out the verification of the model.
2. Extracting peripheral blood genome DNA by phenol-chloroform method, and performing conventional method. Generally, 20 ng/. mu.l to 50 ng/. mu.l of DNA can be obtained, and the purity (OD 260/280) is 1.6-2.0.
3. Detecting an Illumina Human610-Quad chip;
(1) taking a DNA sample of a subject;
(2) carrying out whole genome scanning on an Illumina Human610-Quad chip;
(3) detecting and comparing the difference of the incidence and the occurrence time of the radioactive brain injury of individuals carrying various genotypes.
4. TaqMan (applied biosystems) genotyping assay
(1) Taking a DNA sample of a subject;
(2) designing SNP specific amplification primers and specific probe sequences;
(3) carrying out PCR amplification reaction;
(4) judging the SNP typing result according to the fluorescence color;
(5) the difference between the incidence and the incidence time of the radioactive brain injury of individuals carrying each genotype is analyzed and verified.
5. Preparation method of kit for predicting morbidity risk of radioactive brain injury caused by tumor radiotherapy
A Human610-Quad chip is used for carrying out whole genome SNP scanning, SNP sites (rs 162171 and rs 17111237) which are obviously related to radioactive brain injury are determined and used as a radioactive brain injury risk prediction index, and a kit for predicting the onset risk of the radioactive brain injury caused by tumor radiotherapy is manufactured. The kit can comprise a specific primer and a specific fluorescent probe pair of the SNP, and reagents such as reaction enzyme, dNTP and the like.
6. Statistical analysis method
In the discovery stage, a Cox regression model is used for analyzing the correlation between the demographic factors and the clinical factors (sex, nasopharyngeal cancer affected age, clinical stage, T stage, treatment mode, radiotherapy technology and the like) and the occurrence of radioactive brain injury. Using an additive model, factors (age, T-stage, radiotherapy technique (intensity modulated radiotherapy vs conventional radiotherapy)) significantly related to the occurrence of radioactive brain injury were used as covariates, and the risk Ratio (Hazard Ratio, HR) of each SNP and their 95% confidence interval were calculated. A dangerous SNP is defined as having an HR value greater than 1, and a protective SNP is defined as having an HR value less than 1.
In order to further research the effect of the comprehensive indication formed by combining the SNP and the clinical factors on predicting the radioactive brain injury risk, a mathematical formula is constructed, and three genotypes of the SNP and the clinical factors are comprehensively considered for scoring. Wherein, for SNP typing, wild homozygous type = "0", heterozygous type = "1", mutant homozygous type = "2"; for clinical variables T epoch T1= "0", T2= "1", T3= "2", T4= "3"; for radiotherapy techniques, conventional conformal radiotherapy = "0", intensity modulated radiotherapy = "1"; the score for age was substituted with actual age (continuous variable). The risk score based on the typing of rs162171 and rs17111237 is obtained by taking the multifactor Cox regression coefficient beta as a weight during analysis according to the following formula:
risk score = (0.6013 × score by T-stage) + (-0.8509 × score by radiotherapeutic technique) + (0.02226 × score by age) + (score by score of 0.506 × rs162171 type) + (score by score of 0.49 × rs17111237 type).
The risk score obtained was compared to the optimal limit value 2.1777 and applied to all samples with follow-up times exceeding 3 years for whole genome association studies and subsequent two-stage validation.
Statistical analysis was done by analysis software R and plink (v 1.9), with the statistical significance level P value set to 0.05, and all statistical tests were two-sided.
The following is a further description of the invention:
1082 patients with nasopharyngeal carcinoma who received radiotherapy and satisfied the entry condition were subjected to a regular MR follow-up and their occurrence and time of radioactive brain damage were recorded according to the image examination results, and the time-to-event data (event data) was used as the outcome variable. Meanwhile, the 1082 nasopharyngeal carcinoma patients were scanned with Illumina Human610-Quad whole genome chip, and correlation results were obtained by whole genome correlation analysis.
Based on Illumina Human610-Quad assay, the inventors detected that the SNP most significantly associated with the occurrence of radioactive brain injury is located inCEP128Intron site rs162171 and is located atCEP128Promoter site rs 17111237. This SNP was detected on the Taqman (applied biosystems) genotyping platform with results consistent with Illumina detection. Furthermore, the two sites are well verified by two subsequent independent verification populations, in the two independent verification stages, the two SNP sites and the radioactive brain injury also show obvious correlation, and the p value of the statistical test is less than 0.05.
The multifactor Cox regression finds that the SNP locus has a dose-response relationship (dose-response) with the occurrence risk of radioactive brain injury, and the occurrence risk of radioactive brain injury is increased along with the increase of the number of variant alleles.
Further analyzing the effect of the SNP in combination with clinical indexes (T stage and radiotherapy technology) and age on predicting the risk of radioactive brain injury, and finding that the model can well distinguish nasopharyngeal carcinoma patients with and without radioactive brain injury.
According to the experimental result, the invention prepares a kit for clinical radioactive brain injury risk assessment, which comprises specific amplification primers, probes and other detection reagents for determining the SNP in the DNA of a blood sample of a subject. The kit is used for detecting the rs162171 and rs17111237 loci, and simultaneously combines patient information (T stage, radiotherapy technology and age), so that the risk prediction and evaluation of radioactive brain injury of a patient are facilitated, and targeted protective measures such as preventive medication and the like can be taken for high-risk individuals in advance, so that individual treatment of the patient is really realized.
In addition, according to the description of the "SNPinfo" database,CEP128the other SNP sites on the gene are highly linked with the two sites of rs162171 and rs17111237 (R)2>0.8), equivalent substitution can be carried out on the two sites of rs162171 and rs17111237, the occurrence of radioactive brain injury is obviously related, and the tumor can be well predictedThe risk of radioactive brain damage in the patient, the other SNP sites are as follows:
rs10133034、rs10135084、rs10141970、rs10142753、rs1016699、rs1038523、rs11625199、rs11627934、rs1197467、rs1197474、rs12050151、rs12050342、rs12050416、rs12433134、rs12434318、rs12437260、rs162174、rs162178、rs17059、rs170596、rs17111246、rs17111256、rs17615020、rs1863766、rs2010847、rs2059719、rs228119、rs228122、rs2288347、rs2556607、rs2556611、rs2619661、rs28850656、rs2888032、rs327423、rs327424、rs327428、rs327429、rs327434、rs327435、rs327440、rs327442、rs327443、rs327446、rs327452、rs327454、rs327457、rs327462、rs327463、rs327467、rs327470、rs327472、rs328206、rs328212、rs328213、rs328258、rs4899780、rs4903949、rs6574608、rs6574609、rs6574610、rs7140452、rs7156572、rs7157845、rs7158936、rs722906、rs7493314、rs759919、rs768359、rs8008366、rs8011273、rs8016507、rs9323694。
compared with the prior art, the invention has the following beneficial effects:
(1) the SNP is a novel gene biomarker which is different from the traditional biomarker, has the advantages of stability, minimal invasion and easy detection, and provides a more efficient and convenient assessment method for predicting the morbidity risk of the radioactive brain injury due to high specificity and high sensitivity.
(2) The kit for predicting the morbidity risk of the radioactive brain injury caused by tumor radiotherapy is a systematic and comprehensive diagnostic kit, can be used for evaluating the risk of the radioactive brain injury of a patient, helps a clinician quickly and accurately evaluate the genetic susceptibility of the radioactive brain injury of the patient, and provides support for timely taking a preventive treatment scheme.
(3) A strict verification and evaluation system is adopted, whole genome association research of a large sample is combined with population verification of two independent stages, and finally a high-sensitivity and high-specificity model is obtained through quantitative risk scoring to ensure that the model can be effectively applied clinically.
In summary, the present inventionThe provided kit and risk prediction model provide a yet undisclosed localization associated with radioactive brain injuryCEP128The SNP marker combination on the gene can be combined with clinical information of patients to more comprehensively and accurately evaluate the risk of the nasopharyngeal carcinoma patients suffering from radioactive brain injury. The invention is applied to clinic, takes protective measures in advance aiming at high-risk patients, is beneficial to realizing individual treatment of the patients and improves the long-term life quality of the patients with nasopharyngeal carcinoma. Meanwhile, a method and a strategic reference can be provided for the risk prediction of normal tissue damage caused by other radiotherapy.
Drawings
FIG. 1 is a graph showing the combination of clinical factors (age, T staging, radiotherapeutic technique) andCEP128and (4) constructing an ROC curve of a model for predicting whether radioactive brain injury occurs at sites (rs 162171 and rs 17111237) on the gene.
Detailed Description
The invention is described in further detail below with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Example 1 screening of SNP markers
1. Sample collection and data collation
In 2002-2010, a large number of blood samples were collected from the biological sample library of the Zhongshan university tumor hospital, and 2942 samples meeting the following criteria were selected for genome-wide chip scanning and single SNP Taqman genotyping by collating sample information:
inclusion criteria were:
(1) nasopharyngeal carcinoma patients with definite pathological diagnosis and clinical stages;
(2) expected survival is greater than 6 months;
(3) the functional state score (KPS) is more than or equal to 70 points;
(4) age 18-80 years;
(5) has complete medical record data (medical history, physical examination, related examination and previous treatment);
(6) completing radiotherapy and periodically performing MR image review;
(7) voluntarily attend and sign an informed consent.
At the same time, patients who presented any of the following conditions were excluded:
(1) the patient did not complete all radiotherapy plans;
(2) patients who cannot be matched for follow-up due to psychological, social, household and geographic reasons;
(3) the patient has long-distance metastasis at the initial diagnosis;
(4) abnormal signals of unknown reasons exist in nasopharyngeal/craniocerebral MR images of patients before radiotherapy;
(5) patients had received head and neck radiotherapy for other diseases (except nasopharyngeal carcinoma).
2. Whole genome scanning of peripheral blood DNA
In the discovery stage, with 1082 samples meeting the above conditions, 243 nasopharyngeal carcinoma patients with radioactive brain injury and 839 nasopharyngeal carcinoma patients without radioactive brain injury by the last MR follow-up are obtained by regular MR follow-up (follow-up time to 2016, 12 and 31 days). And detecting the two groups of people by an Illumina Human610 Quad chip to obtain related results. The method comprises the following specific steps:
(1) 3mL of peripheral venous blood is collected by an EDTA anticoagulation tube, and plasma is sucked out after centrifugation at 3500r/min for 10 minutes.
(2) Removing red blood cells: adding equal volume of erythrocyte lysate (10 mmol/L Tris pH7.6; 5 mmol/L MgCl) to the remaining blood components2(ii) a 10mmol/L NaCl), turning upside down and mixing well, centrifuging at 4000r/min for 10min, and discarding the supernatant. Then 5mL of erythrocyte lysate is added, the mixture is fully mixed by turning upside down, and the supernatant is discarded after centrifugation for 10 minutes at 4000 r/min.
(3) Lysis of nucleated cells: 1mL of leukocyte lysate (50 mmol/L Tris HCl pH8.0; 50mmol/L EDTA disodium salt; 10mmol/L NaCl; 1% sodium dodecyl sulfate (w/v)) was added, 10g of RNase 10g of proteinase K was added, followed by shaking thoroughly on a shaker, mixing well in a water bath at 65 ℃ for 30 minutes, and mixing well 1 time with shaking every 5 minutes.
(4) Removing proteins: adding 1mL of saturated phenol, fully shaking and uniformly mixing on an oscillator, centrifuging at 12000r/min for 10 minutes, and transferring the upper-layer water phase to a new EP tube; equal volume of chloroform/isoamyl alcohol (24: 1, v/v) was added, mixed well on a shaker, centrifuged at 12000r/min for 10 minutes and the upper aqueous phase was transferred to a new EP tube.
(5) DNA precipitation: adding 1/10 volumes of 3M sodium acetate and equal volume of precooled isopropanol, shaking up and down gently to mix uniformly to obtain white flocculent precipitate, and then centrifuging for 5 minutes at 12000 r/min.
(6) And (3) DNA rinsing: the supernatant was discarded, 1ml of 75% ethanol was added thereto, the mixture was inverted upside down, and centrifuged at 12000r/min for 5 minutes.
(7) And 6, repeating the step.
(8) Drying the DNA: the supernatant was discarded, the cap was opened in a clean environment to allow ethanol to evaporate sufficiently, and an appropriate amount (50. mu.L) of TE buffer was added to dissolve the DNA.
(9) And (3) measuring the concentration: DNA concentration and purity were measured using a NanoDrop 2000. Generally, 20 ng/. mu.l to 100 ng/. mu.l of DNA can be obtained, and the purity (OD 260/280) is 1.6-2.0.
(10) Performing whole genome scanning on an Illumina Human610 Quad chip;
(11) data analysis and processing:
and analyzing the data by using a Cox proportional risk model, and correcting three factors related to radioactive brain injury, namely T stage, radiotherapy technology and age on the assumption that the risk effect of the SNP meets an additive model.
3. Analysis results
Analyzed to obtainCEP128The rs162171 and rs17111237 sites on the gene are obviously related to the occurrence of radioactive brain injury, and the results are shown in table 1.
Table 1: whole genome analysis results of 1082 nasopharyngeal carcinoma patients by Cox regression
Example 2 TaqMan (applied biosystems) genotyping assay for Single SNPs
Sites rs162171 and rs17111237 related to radioactive brain injury found by whole genome scanning in example 1 were tested in two validation populations, including 1119 and 741 nasopharyngeal carcinoma patients, including 177 and 261 patients with radioactive brain injury. Then the samples are detected by using Taqman genotyping,
1. the method comprises the following specific steps:
(1) 3mL of peripheral venous blood is collected by an EDTA anticoagulation tube, and plasma is sucked out after centrifugation at 3500r/min for 10 minutes.
(2) Removing red blood cells: adding equal volume of erythrocyte lysate (10 mmol/L Tris pH7.6; 5 mmol/L MgCl) to the remaining blood components2(ii) a 10mmol/L NaCl), turning upside down and mixing well, centrifuging at 4000r/min for 10min, and discarding the supernatant. Then 5mL of erythrocyte lysate is added, the mixture is fully mixed by turning upside down, and the supernatant is discarded after centrifugation for 10 minutes at 4000 r/min.
(3) Lysis of nucleated cells: 1mL of leukocyte lysate (50 mmol/L Tris HCl pH8.0; 50mmol/L EDTA disodium salt; 10mmol/L NaCl; 1% sodium dodecyl sulfate (w/v)) was added, 10g of RNase 10g of proteinase K was added, followed by shaking thoroughly on a shaker, mixing well in a water bath at 65 ℃ for 30 minutes, and mixing well 1 time with shaking every 5 minutes.
(4) Removing proteins: adding 1mL of saturated phenol, fully shaking and uniformly mixing on an oscillator, centrifuging at 12000r/min for 10 minutes, and transferring the upper-layer water phase to a new EP tube; equal volume of chloroform/isoamyl alcohol (24: 1, v/v) was added, mixed well on a shaker, centrifuged at 12000r/min for 10 minutes and the upper aqueous phase was transferred to a new EP tube.
(5) DNA precipitation: adding 1/10 volumes of 3M sodium acetate and equal volume of precooled isopropanol, shaking up and down gently to mix uniformly to obtain white flocculent precipitate, and then centrifuging for 5 minutes at 12000 r/min.
(6) And (3) DNA rinsing: the supernatant was discarded, 1ml of 75% ethanol was added thereto, the mixture was inverted upside down, and centrifuged at 12000r/min for 5 minutes.
(7) And 6, repeating the step.
(8) Drying the DNA: the supernatant was discarded, the cap was opened in a clean environment to allow ethanol to evaporate sufficiently, and an appropriate amount (50. mu.L) of TE buffer was added to dissolve the DNA.
(9) And (3) measuring the concentration: DNA concentration and purity were measured using a NanoDrop 2000. Generally, 20 ng/. mu.l to 100 ng/. mu.l of DNA can be obtained, and the purity (OD 260/280) is 1.6-2.0.
(10) Detection was performed on a Taqman (applied biosystems) genotyping platform. The correlation between radioactive brain injury caused by nasopharyngeal carcinoma radiotherapy and the whole genome scanningCEP128SNPs on a gene are designed with specific amplification primers and specific probe sequences.
Using 384 well plates for loading, each 5 u l reaction system including: forward primer, reverse primer and typing probe mix 0.3. mu.l, dNTP mix (10mM) 0.2. mu.l, MgCl20.4. mu.l (25mM), 1. mu.l reaction buffer, 0.1. mu.l reaction enzyme, 2. mu.l double distilled water and 1. mu.l test DNA.
The amplification system was as follows: 60 ℃, 30s, 1 cycle; at 95 ℃, 10min, 1 cycle; 45 cycles of 95 ℃, 15s and 60 ℃, 1 min; 60 ℃, 30s, 1 cycle.
The apparatus used was an ABI7900 type PCR apparatus. The detection result is derived by using QuantStaudio Real-Time PCR Software v1.3 Software.
2. Further analyzing the relation between SNP and radioactive brain injury by using a risk scoring method
According to the results, the genetic risk scoring method using the multi-factor Cox regression coefficient beta is adopted, the multi-factor Cox regression coefficient beta obtained in example 1 is substituted into a formula, and the risk estimation is performed on the sample in the verification stage to verify the effect of the model, wherein the risk estimation comprises the sample with radioactive brain damage and the nasopharyngeal carcinoma patients who visit for three years and have no radioactive brain damage.
The risk score formula is:
risk score = (0.6013 × score by T-stage) + (-0.8509 × score by radiotherapeutic technique) + (0.02226 × score by age) + (score by score of 0.506 × rs162171 type) + (score by score of 0.49 × rs17111237 type).
Wherein, for SNP typing, wild homozygous type = "0", heterozygous type = "1", mutant homozygous type = "2"; for clinical variables T epoch T1= "0", T2= "1", T3= "2", T4= "3"; for radiotherapy techniques, conventional conformal radiotherapy = "0", intensity modulated radiotherapy = "1"; the score for age was substituted with actual age (continuous variable).
The sample is a high risk population for radioactive brain injury when the risk score is greater than 2.177 and a low risk population for radioactive brain injury when the risk score is less than 2.177.
The specific statistical results are shown in table 2.
Table 2: model effect of radioactive brain injury risk scoring
The results of the two samples were combined and an ROC curve was plotted for the risk score to assess the predicted sensitivity and specificity, which distinguishes samples with radioactive brain injury from those without radioactive brain injury by an area under the curve (AUC) of 71%, with an optimal cut-off specificity of 64.17% and a sensitivity of 65.06% (see fig. 1).
Thus proving the adoption ofCEP128The SNP loci rs162171 and rs17111237 combined with clinical factors can well predict the radioactive brain injury risk of nasopharyngeal carcinoma patients.
Example 3 kit for predicting the risk of onset of radiation brain injury caused by tumor radiotherapy
The manufacturing and operation process of the kit for predicting the onset risk of radioactive brain injury caused by tumor radiotherapy is based on a Taqman (applied biosystems) genotyping detection technology.
1. The kit contains SNP specific amplification primers (the nucleotide sequences of the rs162171 and rs17111237 specific amplification primers are respectively shown as SEQ ID No. 1-2 and SEQ ID No. 3-4), and specific probes (the nucleotide sequences of the rs162171 and rs17111237 specific probes are respectively shown as SEQ ID No. 5-6 and SEQ ID No. 7-8)Shown). Common reagents required by the corresponding PCR technology are also included, such as: dNTP mixture, MgCl2Double distilled water, reaction enzyme and reaction buffer; in addition, the kit also comprises a label and a control (a standard substance for determining the genotype and a blank control).
Specific primer and specific probe information are shown in table 3.
TABLE 3 SNP specific primers and specific Probe information:
2. PCR system of kit
Forward primer, reverse primer and typing probe mix 0.3. mu.l, dNTP mix (10mM) 0.2. mu.l, MgCl20.4. mu.l (25mM), 1. mu.l reaction buffer, 0.1. mu.l reaction enzyme, 2. mu.l double distilled water and 1. mu.l test DNA.
3. PCR amplification system
60 ℃, 30s, 1 cycle; at 95 ℃, 10min, 1 cycle; 45 cycles of 95 ℃, 15s and 60 ℃, 1 min; 60 ℃, 30s, 1 cycle.
4. Criteria for determining risk of onset
First, the SNP typing of the sample, the clinical variable T stage, the radiotherapy technology and the age are counted and scored.
For SNP typing, wild homozygous = "0", heterozygous = "1", mutant homozygous = "2"; for clinical variables T epoch T1= "0", T2= "1", T3= "2", T4= "3"; for radiotherapy techniques, conventional conformal radiotherapy = "0", intensity modulated radiotherapy = "1"; the score for age was substituted with actual age (continuous variable).
Then, calculating the risk score according to the scores of the items, wherein the formula is as follows:
risk score = (0.6013 × score by T-stage) + (-0.8509 × score by radiotherapeutic technique) + (0.02226 × score by age) + (score by score of 0.506 × rs162171 type) + (score by score of 0.49 × rs17111237 type).
The sample is a high risk population for radioactive brain injury when the risk score is greater than 2.177 and a low risk population for radioactive brain injury when the risk score is less than 2.177.
In addition, according to the description of the "SNPinfo" database,CEP128the other SNP sites on the gene are highly linked with the two sites of rs162171 and rs17111237 (R)2>0.8), equivalent substitutions can be carried out on two sites, namely rs162171 and rs17111237, which are obviously related to the occurrence of radioactive brain injury, and the radioactive brain injury risk of tumor patients can be well predicted, and the other SNP sites are as follows:
rs10133034、rs10135084、rs10141970、rs10142753、rs1016699、rs1038523、rs11625199、rs11627934、rs1197467、rs1197474、rs12050151、rs12050342、rs12050416、rs12433134、rs12434318、rs12437260、rs162174、rs162178、rs17059、rs170596、rs17111246、rs17111256、rs17615020、rs1863766、rs2010847、rs2059719、rs228119、rs228122、rs2288347、rs2556607、rs2556611、rs2619661、rs28850656、rs2888032、rs327423、rs327424、rs327428、rs327429、rs327434、rs327435、rs327440、rs327442、rs327443、rs327446、rs327452、rs327454、rs327457、rs327462、rs327463、rs327467、rs327470、rs327472、rs328206、rs328212、rs328213、rs328258、rs4899780、rs4903949、rs6574608、rs6574609、rs6574610、rs7140452、rs7156572、rs7157845、rs7158936、rs722906、rs7493314、rs759919、rs768359、rs8008366、rs8011273、rs8016507、rs9323694。
sequence listing
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Claims (3)
- The application of the SNP locus combination on the CEP128 gene in the preparation of a kit for predicting the onset risk of radioactive brain injury caused by tumor radiotherapy is characterized in that the SNP locus combination is rs162171 and rs 17111237.
- 2. The use of claim 1, wherein the sample is risk scored, wherein the sample is a high risk population for radioactive brain injury when the risk score is greater than 2.177, and wherein the sample is a low risk population for radioactive brain injury when the risk score is less than 2.177;risk score ═ score (0.6013 × T stage score) + (-score for 0.8509 × radiotherapeutic technique) + (0.02226 × age score) + (score for score of 0.506 × rs162171 type) + (score for score of 0.49 × rs17111237 type);in the formula, the score of T stage: for clinical variables T staging, T1 is "0", T2 is "1", T3 is "2", T4 is "3";scoring of radiotherapy techniques: for radiotherapy techniques, traditional conformal radiotherapy is "0", intensity modulated radiotherapy is "1";age scoring: substituting with actual age;scoring for typing: for SNP typing, the wild homozygous type is "0", the heterozygous type is "1", and the mutant homozygous type is "2".
- 3. Use of specific amplification primers for detecting the combination of SNP sites of claim 1 and specific probes for detecting the combination of SNP sites of claim 1 in the preparation of a kit for predicting the risk of the onset of radioactive brain injury caused by tumor radiotherapy.
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| CN104745710B (en) * | 2015-04-16 | 2017-10-20 | 上海洛施生物科技有限公司 | A kind of SNP mark related to primary hepatoma auxiliary diagnosis and its application |
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| Radiation-induced brain injury: A review;Michael Robbins等;《Frontiers in Oncology》;20120126;第1-36页 * |
| ss24103632;佚名;《dbSNP》;20090413;第1-4页 * |
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