CN111286540A - Use of POLN gene as molecular marker for predicting radiation sensitivity - Google Patents

Abstract

The invention belongs to the technical field of biological detection, and relates to an application of a POLN gene as a molecular marker for predicting radiation sensitivity. By using the POLN gene of the invention as a molecular marker for predicting radiation sensitivity, the radiation sensitivity can be predicted by whether the rs2022302 locus of the POLN gene is mutated with high efficiency and low cost.

Description

Use of POLN gene as molecular marker for predicting radiation sensitivity

Technical Field

The invention belongs to the technical field of biological detection, and relates to an application of a POLN gene as a molecular marker for predicting radiation sensitivity.

Background

Currently, radiation therapy remains the mainstay of modern cancer therapy, and approximately 50% of cancer patients require radiation therapy. However, patients receiving the same dose of radiation therapy exhibit different radiotoxicity: few have no obvious toxic effects, most have mild or moderate toxic reactions clinically, while few cause severe normal tissue complications and may even be life threatening. Therefore, there is a need to find a molecular marker associated with radiosensitivity for predicting whether a patient is a radiosensitive (or radioresistant) individual.

The prediction of radiation sensitivity is to be able to tailor the radiation treatment regimen to individual patients to improve prognosis, so that on the one hand the radiation dose can be reduced to reduce toxicity to sensitive individuals, and on the other hand the radiation dose can be increased to allow more radiation resistant patients to be effectively treated. This will maximize control of the tumor while minimizing damage to the patient's normal tissues. In addition, about 20 thousands of radiation workers exist in China, the radiation workers need to receive professional irradiation for a long time during daily work, and prediction of radiation sensitivity of the radiation workers is helpful for preventing and reducing professional injuries.

The locus POLN rs2022302 is located on chromosome 4, 2174006, the allele is a > G, and the gene in which the locus is located encodes a member of the type a family of DNA polymerases. The encoded proteins play a role in DNA repair and homologous recombination.

Single nucleotide polymorphism markers (SNPs) are "third generation DNA genetic markers", and 300 ten thousand of them exist in the human genome, and are considered as genetic markers with the best application prospect. SNPs can truly reflect genetic differences and are associated with radiation-induced toxicity in normal tissues.

Disclosure of Invention

The invention aims to provide the application of the POLN gene as a molecular marker for predicting radiation sensitivity so as to predict the radiation sensitivity through high efficiency and low cost of whether the rs2022302 site of the POLN gene is mutated.

To achieve this object, in a basic embodiment, the present invention provides the use of a POLN gene as a molecular marker for predicting radiation sensitivity, i.e., the use of a POLN gene as a molecular marker for preparing a detection kit for predicting radiation sensitivity.

In a preferred embodiment, the invention provides the use of the mutant site of the POLN gene rs2022302 as a molecular marker for predicting radiation sensitivity, i.e. the use of the mutant site of the POLN gene rs2022302 as a molecular marker for preparing a detection kit for predicting radiation sensitivity.

The sequence of the POLN gene of the invention is shown in SEQ ID NO.1, the mutation site is rs2022302 site (mutation from A to G) of the POLN gene, and the sequence of the mutated POLN gene is shown in SEQ ID NO. 2.

The invention has the advantages that by using the POLN gene of the invention as a molecular marker for predicting radiation sensitivity, the radiation sensitivity can be predicted by whether the rs2022302 locus of the POLN gene is mutated with high efficiency and low cost.

Detailed Description

The following examples further illustrate the practice of the present invention, but the embodiments of the present invention are not limited to the following examples.

Example 1:

1. preliminary screening of molecular markers for predicting radiosensitivity by using whole exon capture sequencing technology

1) Sample acquisition and irradiation and chromosome aberration analysis

Collecting peripheral blood of healthy adult male of 20-30 years old, and administering 0, 2Gy⁶⁰And (4) irradiating Co gamma rays. And (3) carrying out chromosome aberration analysis on the 2Gy gamma ray irradiation sample, and dividing the population into a sensitive group, a general group and a resistant group. After the irradiation, the blood sample is cultured for 52h, and then the chromosome is harvested and sliced. Each sample was analyzed for 200 metaphase phases.

2) Peripheral blood genome DNA extraction

Genomic DNA from 0Gy irradiated samples was extracted. The whole blood genome DNA extraction is carried out by adopting a blood genome DNA extraction kit of Beijing Tianzhu Biochemical technology Co., Ltd according to a product specification, and the specific steps are shown in the specification. The quantitative detection A260/280 of the sampled nucleic acid is between 1.70 and 1.90, the quality meets the experiment requirements, and the subsequent experiment can be carried out.

3) Whole exon capture sequencing

Sequencing data is firstly subjected to data filtration to remove low-quality data, and clear Reads are obtained. The sequencing needs to reach the clean Reads rate of more than 90%, the clean base rate of more than 20G, the clean base rate of more than 90%, and the Q20 rate of more than 98%, and the experimental sample meets the requirements.

4) Biological information analysis

Clear Reads were aligned to the reference genome and differential SNP sites were screened. Reference genome version: GRCh37(hg19), ftp:// ftp.1000genes.ebi.ac.uk/vol 1/ftp/technical/reference/human _ g1k _ v37. fasta.gz. The locus of POLN rs2022302 was selected by bioinformatic analysis, as shown in Table 1.

TABLE 1 preliminary screening of sites predictive of radiosensitivity

SNP site	Gene	SNP site position	Alleles
				rs2022302	POLN	chr4：2174006	A/G

2. Experimental verification of primary screening sites by matrix-assisted laser desorption ionization mass spectrometry

1) The method utilizes a blood genome DNA extraction kit (non-centrifugal column type; catalog number: DP319) human whole blood genome (blood from healthy male volunteers between 20-30 years old) DNA extraction was performed according to the product instructions.

2) The amplification primer pair of SEQ ID NO.3 and SEQ ID NO.4 is adopted, and a specific reaction system (5 mu l of the reaction system comprises 0.95 mu l H)₂O、0.625μl PCR Buffer(10×)、0.325μl MgCl₂PCR reaction (25mM), 1. mu.l dNTP (2.5mM), 1. mu.l primer, 0.1. mu.l HotstarTaq (5U/. mu.L)) was performed according to the following reaction program: 15min at 94 ℃; [94 ℃, 20sec, 56 ℃, 30sec ]]45 cycles; 72 ℃ for 4 min. The reaction product was stored at 4 ℃.

3) Using an SAP reaction solution (2. mu.l SAP reaction solution included 1.53. mu.l H)₂O, 0.17. mu.l SAP Buffer (10X), 0.3. mu.l SAP enzyme (1U/. mu.L)) were applied to the reaction product of step 2) according to the following procedure: at 37 ℃ for 40 min; 85 ℃ for 5 min. The treated product was stored at 4 ℃.

4) Carrying out extension reaction on the treated product in the step 3) by adopting an extension primer of SEQ ID NO.5,

2 μ l reaction included 0.755 μ l H₂O, 0.2. mu.l of iPLex Buffer (10X), 0.2. mu.l of iPLEX termination mix, 0.041. mu.l of iPLex enzyme, 0.804. mu.l of primer.

The reaction procedure is as follows: 30s at 94 ℃; 5 cycles of [94 ℃, 5s, (52 ℃, 5s, 80 ℃, 5s) ]40 cycles; 72 ℃ for 3 min. The extension product was stored at 4 ℃.

5) Purifying the extension product of step 4). 6mg of resin was uniformly applied to cover the 384 well plate and left for 20 min. The 384 well plate containing the extension product of step 4) was centrifuged at 1000rpm for 1min, 25. mu.L of deionized water was added to each well, inverted on the resin plate, and then the resin plate was snapped on the 384 well plate in the inverted position, and the resin was dropped into the 384 well plate by tapping, and the membrane was sealed. The 384 well plate was inverted for 20 minutes with the long axis of the 384 well plate as the axis, and centrifuged at 3500rpm for 5 minutes for use.

6) Detecting the genotype of the gene locus: the sample treated in step 5) was transferred to a MassARRAYPectroCHIP chip (SAMSUNG, MassArray. TM. Nanodispenser) and put into a mass spectrometer (SEQUENOM, MassARRAY compact System) for detection, and the results are shown in Table 2.

Table 2 experimental verification results

3. Crowd verification of experimental verification sites by matrix-assisted laser desorption ionization mass spectrometry

1) Sample acquisition and irradiation and chromosome aberration analysis

Collecting peripheral blood of healthy adult male of 20-30 years old, and administering 0, 2Gy⁶⁰And (4) irradiating Co gamma rays. And (3) carrying out chromosome aberration analysis on the 2Gy gamma ray irradiation sample, and dividing the population into a sensitive group, a general group and a resistant group. After culturing for 52h, the chromosomes are harvested and sliced. Each sample was analyzed for 200 metaphase phases.

2) Peripheral blood genome DNA extraction

Genomic DNA of 0Gy irradiated samples of susceptible and non-susceptible groups was extracted. The whole blood genome DNA extraction is carried out by adopting a blood genome DNA extraction kit of Beijing Tianzhu Biochemical technology Co., Ltd according to a product specification, and the specific steps are shown in the specification. The quantitative detection A260/280 of the sampled nucleic acid is between 1.70 and 1.90, the quality meets the experiment requirements, and the subsequent experiment can be carried out.

3) Mass spectrometry detection

And performing population verification on the sample, and further verifying the site.

4) Statistical analysis

The rs2022302 site of the POLN gene was tested by the Hardy-Weinberg equilibrium test, P >0.05, indicating that the samples are population representative and the results are shown in table 3.

Correlation of the locus rs2022302 of the POLN gene with radiosensitivity under different genetic models was analyzed using logistic regression. Under the co-dominant model, AG genotypes were significantly different in the sensitive group from the general group (p 0.010, OR 13.980, 95% CI 2.090-93.360); the sensitive group differed significantly from the general group in the dominant model (p 0.010, OR 8.030, 95% CI 1.620-39.880) and the results are shown in table 4.

TABLE 3 Hardy-Weinberg equilibrium test results

TABLE 4 results of logistic regression analysis

Genotype(s)	P	OR	95％CI
				AA	-	1	-
AG	0.010	13.980	2.090-93.360
				GG	0.970	0.920	0.020-39.810
TT VS CT+CC	0.010	8.030	1.620-39.880

Therefore, the rs2022302 locus of the POLN gene is screened as a molecular marker for predicting radiation sensitivity. The AG genotype is a genotype for predicting radiation sensitivity and can be used as an index for predicting radiation sensitivity of people.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is intended to include such modifications and variations. The foregoing examples or embodiments are merely illustrative of the present invention, which may be embodied in other specific forms or in other specific forms without departing from the spirit or essential characteristics thereof. The described embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. The scope of the invention should be indicated by the appended claims, and any changes that are equivalent to the intent and scope of the claims should be construed to be included therein.

Sequence listing

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Claims (2)

1. Use of a POLN gene as a molecular marker for predicting radiation sensitivity.
2. Use of the rs2022302 mutation site of a POLN gene as a molecular marker for predicting radiation sensitivity.