CN111172271A - Application of UIMC1 gene as molecular marker for judging susceptibility of radiation damage - Google Patents
Application of UIMC1 gene as molecular marker for judging susceptibility of radiation damage Download PDFInfo
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- CN111172271A CN111172271A CN202010051740.3A CN202010051740A CN111172271A CN 111172271 A CN111172271 A CN 111172271A CN 202010051740 A CN202010051740 A CN 202010051740A CN 111172271 A CN111172271 A CN 111172271A
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Abstract
The invention belongs to the technical field of biological detection, and relates to an application of UIMC1 gene as a molecular marker for judging susceptibility to radiation damage. By using the UIMC1 gene provided by the invention as a molecular marker for judging susceptibility to radiation damage, the radiation damage can be detected by whether the site rs1700490 of the UIMC1 gene is mutated with high efficiency and low cost.
Description
Technical Field
The invention belongs to the technical field of biological detection, and relates to an application of UIMC1 gene as a molecular marker for judging susceptibility to radiation damage.
Background
At present, about 20 thousands of radiation workers exist in China. Radiation workers are required to receive occupational irradiation for a long time during daily work, and the injury of accumulated irradiation to susceptible people is obvious, so that an effective method is needed for judging whether the personnel meet the post requirements or not so as to reduce occupational injury.
In addition, radiation therapy is one of the main therapeutic approaches to malignancy, and over 50% of cancer patients require radiation therapy. However, even with the same radiation treatment regimen, the degree of radiation damage varies from patient to patient, and the cause of this is mainly related to the individual differences in susceptibility to radiation damage: under the same treatment condition, people susceptible to radiation damage may cause serious adverse reactions, and people not susceptible to radiation damage may have a risk of poor treatment effect.
Therefore, the method can accurately distinguish the person susceptible to radiation damage, is favorable for the post adaptability judgment of the radiation worker, and is also favorable for customizing a clinical personalized treatment scheme or optimizing the treatment scheme.
The UIMC1 rs1700490 locus is located on chromosome 5 and the gene at this locus encodes a nucleoprotein, also known as RAP80 protein, which interacts with Brca1 (breast cancer 1) in the complex to identify and repair different types of DNA damage. RAP80 responds to DNA damage induced by ionizing radiation and is phosphorylated at Ser 205. Another study showed that uv-light can induce translocation of RAP80 to DNA lesions co-localized with γ -H2AX for damage repair.
Susceptibility refers to the different tendency of different individuals to become predisposed to disease under the influence of the external environment due to genetic differences. At present, a great deal of research proves that different individuals have great difference in response to ionizing radiation, and the effect of susceptibility factors of the individuals on the occurrence of radiation damage is not negligible. The problem of susceptibility to radiation damage among individuals of different genetic backgrounds was mentioned earlier in publications ICRP60 and 79, and there is a high-risk subpopulation of people who are highly sensitive to ionizing radiation damage, the presence of which increases the uncertainty of the risk of radiation carcinogenesis. With the support of the eu 5 th framework, the research group found that phenotypic changes with early effects of radiation of more than 70% were attributed to intrinsic differences among individuals. Accordingly, the research group believes that inherent differences in individual susceptibility to radiation can be identified by Single Nucleotide Polymorphism (SNP) analysis methods.
Disclosure of Invention
The invention aims to provide application of the UIMC1 gene as a molecular marker for judging susceptibility to radiation damage, so that radiation damage can be detected through high mutation efficiency and low cost of the rs1700490 site of the UIMC1 gene.
To achieve this, in a basic embodiment, the present invention provides the use of the UIMC1 gene as a molecular marker for susceptibility determination of radiation damage.
In a preferred embodiment, the invention provides the use of the rs1700490 mutation site of the UIMC1 gene as a molecular marker for determining susceptibility to radiation damage.
In a preferred embodiment, the invention provides the use of the UIMC1 gene as a molecular marker for determining susceptibility to radiation damage, wherein the radiation damage is a local or systemic tissue-organ adverse reaction (e.g., nausea, vomiting, anorexia, leukopenia, and/or red skin itching, etc.).
The sequence of the UIMC1 gene is shown as SEQ ID NO.1, the mutation site is rs1700490 site (mutation from A to G) of the UIMC1 gene, and the sequence of the mutated UIMC1 gene is shown as SEQ ID NO. 2.
The invention has the beneficial effects that by using the use of the UIMC1 gene as a molecular marker for judging the susceptibility to radiation damage, the radiation damage can be detected by whether the mutation of the rs1700490 site of the UIMC1 gene is high in efficiency and low in 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 judging susceptibility to radiation damage by using full 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 susceptible group, a general group and an insensitive 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 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) 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. By utilizing biological information analysis, the UIMC1 rs1700490 locus which is the difference locus between the susceptible group and the non-susceptible group is screened out, and the site is shown in the table 1.
TABLE 1 preliminary screening of radiation injury susceptible sites
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 (derived from 1) of step 1) 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) The extension product from step 4) was purified (6 mg of resin was uniformly covered on 384 well plates 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, centrifuged at 3500rpm for 5 minutes, and then prepared).
6) Detecting the genotype of the gene locus: transferring the sample treated in step 5) to MassARRAYSpectroCHIP chip (MassArray)TMNanodispenser, SAMSUNG), was put into a mass spectrometer (massarrycompact System, SEQUENOM), and the results were 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 susceptible group, a general group and an insensitive 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
Respectively testing the susceptible group and the non-susceptible group at rs1700490 locus of the UIMC1 gene by using Hardy-Weinberg equilibrium test, wherein X2 of the susceptible group is 0.521, and P is more than 0.05; the non-susceptible group X2, 0.586 with P >0.05, indicates that the sample is representative of a population, and the results are shown in table 3.
The differences of wild genotypes and mutant genotypes of rs1700490 sites of the UIMC1 genes of the susceptible group and the non-susceptible group are compared by utilizing one-factor variance analysis, wherein X2 is 4.081, P is less than or equal to 0.05, the differences of the sites between the two groups are obvious, and the results are shown in Table 4.
The difference of rs1700490 locus alleles of the UIMC1 genes of the susceptible group and the non-susceptible group is compared by utilizing one-factor variance analysis, wherein X2 is 3.831, P is less than or equal to 0.05, the locus has obvious difference between the two groups, and the result is shown in Table 5.
And comparing the difference of each genotype of rs1700490 locus of UIMC1 gene of the susceptible group and the non-susceptible group by using logistic regression analysis, wherein the GA type is 6 times of the risk of the GG type, and the AA type is 0.875 time of the risk of the GG type.
TABLE 3 Hardy-Weinberg equilibrium test results
TABLE 4 test results for wild and mutant genotypes
TABLE 5 test results for wild and mutant genotypes
Therefore, the rs1700490 site of the UIMC1 gene is screened to serve as a molecular marker for judging the susceptibility to radiation damage, and the GA type is a genotype for judging and detecting the susceptibility to radiation damage and can serve as an index for judging and detecting the susceptibility 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
<110> China institute for radiation protection
Use of <120> UIMC1 gene as molecular marker for judging susceptibility to radiation damage
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<211>1001
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<400>1
gatttttcat ttaccctctt tttcctttcc tttactatcc attgtccaaa agagtgattt 60
aaaataaatg aatacacgct gagtttaaac attattattg ttttttcata tcttcaatca 120
agctactctg atctttggtg atgcttactg gtattttaga gatagactag tttttgtctg 180
actgtttata tgacagataa taatgccaat atatcacatt tttgttctta cagtttttat 240
tggttctaga tcttatgttt tcccttttgt actgaaaagg atcttctaat gttttatgtg 300
catatttata acaatcacca attcctcaga ggcctctctt agccataggg gaggtagaag 360
agagagaaat cactactttt agtggcagcc acactgatga tatttcttgt tttgcaacag 420
gaaccaaagg aggaaaatgg gttgcagaaa acgaagacaa aacagtcgaa tagagcaaag 480
tgtttggcca aaagaaaaat cgcacgtatg ttttcatttt gttgataatg gtttgggaat 540
tgtaatagtc atagaactga tacattttgg actagttgat tatgttgcat tcttaggtct 600
ctgatagatt caagaaaagt tgtgaattta tcagtttttg ttgctattgt tgtaaggatt 660
ggaacaataa cttttttcag tttcctgtat cttcagtgga agccagaagt ggtcattctg 720
aatctttaaa aaaatttttt taaatttatt ttttttttag agacaggatc tcactctgtc 780
acccaggctt aagtgtaggg gtatggtatg atcatagcta actgcagcct tgaactcatt 840
ggctcaagtg attctcctgc ctcagcttcc caactatgcc ccacgacgcc tggctaattt 900
taattttttt tttaacacag ggtcctgctg tgctgtccag gctcatctca aactcctggc 960
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gatttttcat ttaccctctt tttcctttcc tttactatcc attgtccaaa agagtgattt 60
aaaataaatg aatacacgct gagtttaaac attattattg ttttttcata tcttcaatca 120
agctactctg atctttggtg atgcttactg gtattttaga gatagactag tttttgtctg 180
actgtttata tgacagataa taatgccaat atatcacatt tttgttctta cagtttttat 240
tggttctaga tcttatgttt tcccttttgt actgaaaagg atcttctaat gttttatgtg 300
catatttata acaatcacca attcctcaga ggcctctctt agccataggg gaggtagaag 360
agagagaaat cactactttt agtggcagcc acactgatga tatttcttgt tttgcaacag 420
gaaccaaagg aggaaaatgg gttgcagaaa acgaagacaa aacagtcgaa tagagcaaag 480
tgtttggcca aaagaaaaat tgcacgtatg ttttcatttt gttgataatg gtttgggaat 540
tgtaatagtc atagaactga tacattttgg actagttgat tatgttgcat tcttaggtct 600
ctgatagatt caagaaaagt tgtgaattta tcagtttttg ttgctattgt tgtaaggatt 660
ggaacaataa cttttttcag tttcctgtat cttcagtgga agccagaagt ggtcattctg 720
aatctttaaa aaaatttttt taaatttatt ttttttttag agacaggatc tcactctgtc 780
acccaggctt aagtgtaggg gtatggtatg atcatagcta actgcagcct tgaactcatt 840
ggctcaagtg attctcctgc ctcagcttcc caactatgcc ccacgacgcc tggctaattt 900
taattttttt tttaacacag ggtcctgctg tgctgtccag gctcatctca aactcctggc 960
ctcaagcaat cctccagctt gggcctccca gtgttgggat t 1001
<210>3
<211>31
<212>DNA
<213> human (Homo sapiens)
<400>3
acgttggatg gactattaca attcccaaac c 31
<210>4
<211>30
<212>DNA
<213> human (Homo sapiens)
<400>4
acgttggatg cagtcgaata gagcaaagtg 30
<210>5
<211>24
<212>DNA
<213> human (Homo sapiens)
<400>5
gcaacaaaat gaaaacatac gtgc 24
Claims (3)
- Use of the UIMC1 gene as a molecular marker for the determination of susceptibility to radiation damage.
- Use of the rs1700490 mutation site of the UIMC1 gene as a molecular marker for determining susceptibility to radiation damage.
- 3. Use according to claim 1 or 2, characterized in that: the radiation injury is local or systemic tissue organ adverse reaction.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112251506A (en) * | 2020-07-23 | 2021-01-22 | 中国辐射防护研究院 | UIMC1 gene mutation site detection kit based on Taqman probe method and application thereof |
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| WO2012167112A2 (en) * | 2011-06-01 | 2012-12-06 | Illumina, Inc. | Gastric cancer biomarkers |
| WO2014039958A1 (en) * | 2012-09-10 | 2014-03-13 | Nestec Sa | Immune function biomarkers |
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2020
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012167112A2 (en) * | 2011-06-01 | 2012-12-06 | Illumina, Inc. | Gastric cancer biomarkers |
| WO2014039958A1 (en) * | 2012-09-10 | 2014-03-13 | Nestec Sa | Immune function biomarkers |
Non-Patent Citations (3)
| Title |
|---|
| PETRA DUSATKOVA等: "Genesis of two most prevalent PROP1 gene variants causing combined pituitary hormone deficiency in 21 populations", 《EUROPEAN JOURNAL OF HUMAN GENETICS》 * |
| SCHMUTZ,J.等: "Homo sapiens chromosome 5 genomic scaffold, GRCh38.p13 Primary Assembly HSCHR5_CTG5", 《GENBANK》 * |
| 罗相相: "全基因组外显子测序在肝细胞癌转移研究中的应用", 《万方中国学位论文全文数据库》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112251506A (en) * | 2020-07-23 | 2021-01-22 | 中国辐射防护研究院 | UIMC1 gene mutation site detection kit based on Taqman probe method and application thereof |
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