CN116536417B - Application of SNP rs9790196 as target in developing kit for screening plateau pulmonary edema susceptible population - Google Patents
Application of SNP rs9790196 as target in developing kit for screening plateau pulmonary edema susceptible population Download PDFInfo
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Abstract
The application discloses application of SNP rs9790196 as a target in developing a kit for screening susceptible crowd of plateau pulmonary edema. The application provides application of a substance for detecting genotype of a subject based on SNP rs9790196 in preparation of a product; the function of the product is as follows (a) or (b): (a) screening a subject for altitude pulmonary edema susceptibility; (b) assessing the risk of developing altitude pulmonary edema in the subject. The application also protects the application of SNP rs9790196 as a detection target in developing products; the function of the product is as follows (a) or (b): (a) screening a subject for altitude pulmonary edema susceptibility; (b) assessing the risk of developing altitude pulmonary edema in the subject. The application can be used for avoiding or reducing the occurrence of the pulmonary edema of the plateau and has great application and popularization values for the plateau areas.
Description
Technical Field
The application belongs to the field of gene detection, and relates to application of SNP rs9790196 as a target in developing a kit for screening susceptible crowd of plateau pulmonary edema.
Background
Altitude pulmonary edema (High altitude pulmonary edema, HAPE) is an acute severe altitude disease occurring in the altitude environment (typically altitude > 3000 m), and is manifested by dyspnea, cough, pink cough or white foam sputum, general debilitation, etc. at rest. Most of the people who cannot adapt to the low-oxygen environment after entering the plateau for the first time or returning to the plateau, the disease is urgent in onset, rapid in progress and high in mortality rate, and is the most common cause of death caused by high altitude.
In recent years, the research shows that HAPE disease has race specificity and family and individual genetic susceptibility tendency, and is comprehensively influenced by factors such as heredity, environment and the like. Genomic studies have found that multiple Single Nucleotide Polymorphism Sites (SNPs) are associated with the risk of developing HAPE, suggesting that susceptible gene polymorphism sites can be an important molecular marker for HAPE, providing more medical detection basis for disease prevention, diagnosis, disease severity assessment and treatment strategies.
Disclosure of Invention
The application aims to solve the technical problems of screening a susceptible individual of plateau pulmonary edema or predicting the susceptibility of a subject to the plateau pulmonary edema or screening an individual carrying a susceptible gene of human plateau pulmonary edema or evaluating the risk of the plateau pulmonary edema.
The application provides application of a substance for detecting genotype of a subject based on SNP rs9790196 in preparation of a product; the function of the product is as follows (a) or (b):
(a) Screening a susceptible individual for altitude pulmonary edema;
(b) The subjects were evaluated for risk of developing altitude pulmonary edema.
Illustratively, the substance for detecting a genotype of a subject based on SNP rs9790196 is a primer set. Specifically, the primer group consists of three primers, which are respectively a single-stranded DNA molecule shown in a sequence 1 of a sequence table, a single-stranded DNA molecule shown in a sequence 2 of the sequence table and a single-stranded DNA molecule shown in a sequence 3 of the sequence table.
Illustratively, the substance for detecting a genotype of a subject based on SNP rs9790196 is a primer pair. Specifically, the primer pair consists of two primers, namely a single-stranded DNA molecule shown in a sequence 4 of a sequence table and a single-stranded DNA molecule shown in a sequence 5 of the sequence table.
The GG genotype subjects are susceptible individuals to altitude pulmonary edema.
GG genotype subjects are at higher risk for developing altitude pulmonary edema than GA genotype subjects or AA genotype subjects.
Based on SNP rs9790196, the genotype of the subject is GG genotype, GA genotype or AA genotype.
The application also protects a product comprising a substance for detecting a genotype of a subject based on SNP rs 9790196; the function of the product is as follows (a) or (b):
(a) Screening a susceptible individual for altitude pulmonary edema;
(b) The subjects were evaluated for risk of developing altitude pulmonary edema.
Illustratively, the substance for detecting a genotype of a subject based on SNP rs9790196 is a primer set. Specifically, the primer group consists of three primers, which are respectively a single-stranded DNA molecule shown in a sequence 1 of a sequence table, a single-stranded DNA molecule shown in a sequence 2 of the sequence table and a single-stranded DNA molecule shown in a sequence 3 of the sequence table.
Illustratively, the substance for detecting a genotype of a subject based on SNP rs9790196 is a primer pair. Specifically, the primer pair consists of two primers, namely a single-stranded DNA molecule shown in a sequence 4 of a sequence table and a single-stranded DNA molecule shown in a sequence 5 of the sequence table.
The GG genotype subjects are susceptible individuals to altitude pulmonary edema.
GG genotype subjects are at higher risk for developing altitude pulmonary edema than GA genotype subjects or AA genotype subjects.
Based on SNP rs9790196, the genotype of the subject is GG genotype, GA genotype or AA genotype.
The application also protects the application of SNP rs9790196 as a detection target in developing products; the function of the product is as follows (a) or (b):
(a) Screening a susceptible individual for altitude pulmonary edema;
(b) The subjects were evaluated for risk of developing altitude pulmonary edema.
SNP rs9790196 is located in human genome DNA, and the position corresponds to nucleotide 301 in sequence 6 of the sequence table, and is represented by R. R represents G or A.
Any of the above products may be a kit.
Any of the above subjects may be chinese.
Any of the above subjects may be chinese Han.
Any of the above subjects may be chinese han males.
Any of the above subjects may be chinese in plain areas.
Any of the above subjects may be chinese han nationality in plain.
Any of the above subjects may be chinese han nationality males in plain areas.
Any of the above subjects may be chinese from plain to plateau.
Any of the above subjects may be chinese han population from plain to plateau.
Any of the above subjects may be chinese han males who reach the plateau region from the plain region.
Any of the above subjects may be chinese predicted to go to a plateau region from a plain region.
Any of the above subjects may be chinese han population from plain areas predicted to go to plateau areas.
Any of the above subjects may be chinese han males who are predicted to go to plateau from plain areas.
Any of the above plateau regions may be Tibet plateau regions.
Any of the above plateau regions may be a pizza region of Tibet plateau.
Any of the above plateau regions may be an archway region, a jojo region, a cumin region, a conding region or a new bridge region of the Qinghai-Tibet plateau.
The substance for detecting a genotype of a subject based on SNP rs9790196 described above may be a substance detected based on any of the following techniques: DNA sequencing, restriction enzyme fragment length polymorphism, single-stranded conformational polymorphism, denaturing high performance liquid chromatography, SNP chip, etc. SNP chips may include chips based on nucleic acid hybridization reactions, chips based on single base extension reactions, chips based on allele-specific primer extension reactions, chips based on "one-step" reactions, chips based on primer ligation reactions, chips based on restriction enzyme reactions, chips based on protein DNA binding reactions, chips based on fluorescent molecule DNA binding reactions, and the like.
The substance for detecting a genotype of a subject based on SNP rs9790196 described above includes a primer set. The primer group consists of three primers, wherein one primer is a single-base extension primer, and the other two primers are used for amplifying DNA fragments including SNP rs 9790196. The two amplification primers have no special requirement on sequence, so long as the genomic DNA fragments including SNP rs9790196 can be amplified. The single base extension primer may be designed based on the upstream or downstream of SNP rs9790196 in the human genome (excluding the SNP site), and the nucleotide extended by the single base extension primer corresponds to SNP rs9790196 in the human genome, i.e., the 3' -terminal nucleotide of the single base extension primer corresponds to the adjacent nucleotide of SNP rs9790196 in the human genome (i.e., the previous nucleotide of SNP rs9790196 or the subsequent nucleotide of SNP rs 9790196).
The substances for detecting the genotype of the subject based on SNP rs9790196 as described above may also include PCR reagents, DNA sequencing reagents, DNA sequencers and the like.
The substance for detecting a genotype of a subject based on SNP rs9790196 described above includes a primer pair. The primer pair consists of two primers and is used for amplifying the DNA fragment including SNP rs 9790196. The two primers have no special requirement on sequence, so long as the genome DNA fragments including SNP rs9790196 can be amplified.
SNP rs9790196 can be used for screening plateau pulmonary edema susceptible individuals, predicting susceptibility of a person to plateau pulmonary edema, screening individuals carrying a human plateau pulmonary edema susceptible gene, and evaluating risk of developing plateau pulmonary edema. In practical application, in order to improve the accuracy, the substance for detecting the SNP rs9790196 genotype can be combined with other substances (such as substances for detecting other single nucleotide polymorphisms or genotypes related to the altitude pulmonary edema) to prepare the product.
The application discovers that the distribution frequency of the genotype based on SNP rs9790196 in the patient population with the altitude pulmonary edema and the normal human population is obviously different, and the genotype frequency of the GG genotype in the patient population with the altitude pulmonary edema is obviously higher than that of the GG genotype in the normal human population. In examples 3 and 4, the present application is exemplified by different population samples, respectively, illustrating genotype distribution frequency data in a patient population with altitude pulmonary edema and in a normal population. The application can be used for avoiding or reducing the occurrence of the pulmonary edema of the plateau and has great application and popularization values for the plateau areas.
Detailed Description
The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the application in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified. p-value: personAnd (6) test. OR: ratio of ratios.
Ethical statement: each subject signed an informed consent, and the study was approved by the medical ethics committee of the general hospitals of the release army and the general hospitals of the Tibetan army area. Patients with altitude pulmonary edema are all patients diagnosed by hospitals, and the diagnosis standard is as follows: naming, parting and diagnosis standard of the highland diseases in China; journal of plateau medicine, 2010, volume 20, phase 1; 9-11.
Example 1 screening for SNPs associated with risk of Condition of altitude pulmonary edema
The matched population consisted of a plateau pulmonary edema patient population and a corresponding normal population.
And establishing a plurality of groups of pairing groups, and performing a large number of whole genome sequencing and sequence comparison analysis to obtain a large number of differential SNP. The allele frequency and genotype frequency of each differential SNP in the plateau pulmonary edema patient population and corresponding normal population were sequentially validated for association analysis.
SNP rs9790196, i.e., SNP number rs9790196 in NCBI, is specifically located at position 133920377 of human chromosome 3 (GRCh38.p14), and the polymorphic form is G/A. In the human genome DNA, SNP rs9790196 and the peripheral nucleotide thereof are shown as sequence 6 of a sequence table (SNP rs9790196 is the 301 th nucleotide in sequence 6 of the sequence table, and is expressed by R).
No clinical diagnostic use of SNP rs9790196 has been found in the prior art.
The inventors found through a great deal of work that SNP rs9790196 is associated with a risk of altitude pulmonary edema, and that GG genotype subjects have a higher risk of altitude pulmonary edema.
Example 2, methods of establishing a genotype of a test subject based on SNP rs9790196
1. Taking peripheral blood of a subject, and extracting genomic DNA.
2. And (3) taking the genomic DNA extracted in the step (1) as a template, and adopting a specific primer pair to carry out PCR amplification.
The specific primer pair consists of a primer F1 and a primer R1.
Primer F1 (sequence 1 of the sequence table): 5'-ACGTTGGATGCTGAGGACCACAGAGGGAAA-3';
primer R1 (sequence 2 of the sequence table): 5'-ACGTTGGATGTTGCTTGGGATTCACTGAGTT-3'.
PCR amplification was performed in 384 well plates, one reaction system per well.
Composition of PCR amplified reaction system (5. Mu.L): 0.5. Mu.L of 10 XPCR buffer, 0.4. Mu.L of 25mM MgCl 2 The volume was made up with 0.1. Mu.L of 25mM dNTP mix, genomic DNA, hotStart Taq DNA polymerase, primer F1, primer R1, and ultrapure water. In a 5 mu L system, the content of genome DNA is 20-50ng, hotStarTaq enzyme content was 0.5U, primer F1 content was 0.5pmol, and primer R1 content was 0.5pmol.
Reaction procedure for PCR amplification: 94 ℃ for 4 minutes; 94℃for 20 seconds, 56℃for 30 seconds, 72℃for 1 minute, 45 cycles; 3 minutes at 72 ℃; maintained at 4 ℃.
3. Alkaline phosphatase treatment (for the purpose of removing free dNTPs in the system) was performed.
Taking 384-well plates which finish the step 2, preparing a reaction system and then carrying out reaction.
Composition of the reaction system (7. Mu.L): 5. Mu.L of the product solution obtained in step 2, 0.3. Mu.L of SAP, 0.17. Mu.L of 10 XSAD buffer, and 1.53. Mu.L of ultrapure water.
SAP (shrimp alkaline phosphatase ): the specification of the product is 1.7U/. Mu.l; agena company, cat No. 10002.1. The 10 XSP buffer is a kit of SAP.
Reaction conditions: 40 minutes at 37 ℃; 5 minutes at 85 ℃; maintained at 4 ℃.
4. Single base extension was performed.
Taking 384-well plates which finish the step 3, preparing a reaction system and then carrying out single-base extension reaction.
Composition of single base extension reaction System (10. Mu.L): 7. Mu.L of the product solution obtained in step 3, 0.3. Mu.L of single base extension reaction enzyme, 0.17. Mu.L of 10 Xsingle base extension reaction buffer, 1. Mu.L of single base extension primer, and 1.53. Mu.L of ultrapure water.
Single base extension reaction enzyme: the specification of the product is 1.7U/. Mu.l; agena company, cat# 1432. The 10 times single base extension reaction buffer is a matched buffer of single base extension reaction enzyme.
Single base extension primer (sequence 3 of sequence table): 5'-GTGGAAATGAAGGAAAATG-3'.
The reaction procedure for single base extension is as follows:
(1) 94 ℃ for 30 seconds;
(2) 94 ℃ for 5 seconds;
(3) 5 cycles at 52℃for 5 seconds and 80℃for 5 seconds;
(4) 94 ℃ for 5 seconds, 52 ℃ for 5 seconds, 80 ℃ for 5 seconds, 40 cycles;
(5) 3 minutes at 72 ℃;
(6) maintained at 4 ℃.
5. And (5) purifying the resin.
Taking 384-well plate with step 4, adding 16 μl of water into each well, adding Clean Resin (Sequencom Co., U.S.) into each well, sealing, rotating vertically at low speed for 30 min to make the Resin fully contact with the reactant, centrifuging to make the Resin sink into the bottom of the well, and collecting supernatant as the extension product after Resin purification.
6. And (5) chip sample application.
The MassARRAYNanodispenser RS Spotter (SEQUENOM) was started and the resin purified extension product was transferred to 384 well SpectroCHIP (Sequenom) chip (SEQUENOM).
7. And (5) mass spectrum detection.
The spotted SpectroCHIP chip was analyzed using MALDI-TOF, and the detection results were typed using TYPER 4.0 software (sequenom) and outputted.
Example 3, analysis of susceptibility to SNP rs9790196 and altitude pulmonary edema
Peripheral blood samples were obtained from the general hospitals in the Tibetan military area and collected from month 9 in 2018 to month 9 in 2022. 84 peripheral blood samples were obtained from 84 patients with altitude pulmonary edema and 160 peripheral blood samples were obtained from 160 normal persons (non-altitude pulmonary edema patients). 84 cases of patients with altitude pulmonary edema and 160 cases of normal people are Chinese male Han population unrelated to blood source, and all live in plain areas (for more than 1 year) for a long time and take peripheral blood after reaching Lassa (altitude 3658 m). All had no history of smoking and drinking, no autoimmune related diseases (e.g., vitiligo, psoriasis, type I diabetes, etc.), and no prior history of altitude travel.
The procedure was as in example 2. The genotype of the subject based on SNP rs9790196 was detected.
Of 84 cases of plateau pulmonary edema patients, 50 cases (genotype frequency 59.5%) of individuals with genotype GG, 32 cases (genotype frequency 38.1%) of individuals with genotype GA, and 2 cases (genotype frequency 2.4%) of individuals with genotype AA. Of 160 normal individuals, 59 individuals with genotype GG (genotype frequency of 36.9%), 82 individuals with genotype GA (genotype frequency of 51.2%), and 19 individuals with genotype AA (genotype frequency of 11.9%) were used.
The test data were processed using R4.0.5 statistical software. After correction by Logistic regression analysis, SNP rs9790196 had a significance P value of 0.00024 and or of-0.83 with the population. The results demonstrate that SNP rs9790196 is a SNP associated with altitude pulmonary edema, with subjects of genotype GG being at higher risk of developing HAPE than those of genotype GA and those of genotype AA.
Example 4 analysis of susceptibility to SNP rs9790196 and altitude pulmonary edema
Peripheral blood samples were obtained from the general hospitals in the Tibetan military area and collected from month 9 in 2018 to month 9 in 2022. 157 peripheral blood samples were obtained from 157 cases of altitude pulmonary edema patients, and 233 peripheral blood samples were obtained from 233 cases of normal persons (non-altitude pulmonary edema patients). 157 cases of plateau pulmonary edema patients and 233 cases of normal people are both blood-related unrelated Chinese male Han population, and live in plain areas (for over 1 year) for a long period of time and take peripheral blood after reaching Lhasa (altitude 3658 m). All had no history of smoking and drinking, no autoimmune related diseases (e.g., vitiligo, psoriasis, type I diabetes, etc.), and no prior history of altitude travel.
1. Taking peripheral blood of a subject, and extracting genomic DNA.
2. And (3) taking the genomic DNA extracted in the step (1) as a template, and adopting a specific primer pair to carry out PCR amplification.
The specific primer pair consists of a primer F2 and a primer R2.
Primer F2 (sequence 4 of the sequence listing): 5'-GGAGTCTCAGCAGTGAAATGG-3';
primer R2 (sequence 5 of the sequence listing): 5'-CAGTCCATTTGTGCCATTGA-3'.
3. After the step 2 is completed, the PCR amplification product is recovered and sequenced, and the genotype of the subject based on SNP rs9790196 is obtained according to the sequencing result.
Of 157 cases of plateau pulmonary edema patients, 87 cases (genotype frequency 55.4%) of individuals with genotype GG, 60 cases (genotype frequency 38.2%) of individuals with genotype GA, and 10 cases (genotype frequency 6.4%) of individuals with genotype AA. Out of 233 normal individuals, 96 individuals with genotype GG (genotype frequency of 41.2%), 105 individuals with genotype GA (genotype frequency of 45.1%), and 32 individuals with genotype AA (genotype frequency of 13.7%).
The test data were processed using R4.0.5 statistical software. After correction by Logistic regression analysis, SNP rs9790196 had a significance P value <0.05 (p=0.001) with the population, OR-0.52. Consistent with the results of example 3, subjects with genotype GG were at higher risk of developing HAPE than those with genotype GA and those with genotype AA.
The results of this example further demonstrate that SNP rs9790196 is a risk site associated with plateau pulmonary edema and can be used to screen plateau pulmonary edema susceptible individuals, predict human susceptibility to plateau pulmonary edema, screen individuals carrying a human plateau pulmonary edema susceptible gene, and evaluate the risk of developing plateau pulmonary edema.
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Claims (3)
1. Use of a substance for detecting a genotype of a subject based on SNP rs9790196 in the preparation of a product; the function of the product is as follows (a) or (b):
(a) Screening a susceptible individual for altitude pulmonary edema;
(b) The subjects were evaluated for risk of developing altitude pulmonary edema.
2. The use according to claim 1, wherein: the substance for detecting the genotype of the subject based on SNP rs9790196 is a primer group, and consists of three primers, namely a single-stranded DNA molecule shown in a sequence 1 of a sequence table, a single-stranded DNA molecule shown in a sequence 2 of the sequence table and a single-stranded DNA molecule shown in a sequence 3 of the sequence table.
3. The use according to claim 1, wherein: the substance for detecting the genotype of the subject based on SNP rs9790196 is a primer pair, and consists of two primers, namely a single-stranded DNA molecule shown in a sequence 4 of a sequence table and a single-stranded DNA molecule shown in a sequence 5 of the sequence table.
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| CN114292909A (en) * | 2022-03-10 | 2022-04-08 | 中国人民解放军总医院 | Application of SNP rs241970 as target in development of kit for screening plateau pulmonary edema susceptible population |
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| CN114292909A (en) * | 2022-03-10 | 2022-04-08 | 中国人民解放军总医院 | Application of SNP rs241970 as target in development of kit for screening plateau pulmonary edema susceptible population |
Non-Patent Citations (4)
| Title |
|---|
| Association between genetic polymorphism of telomere-associated gene ACYP2 and the risk of HAPE among the Chinese Han population A Case–control study;Linhao Zhu等;《Medicine》;第96卷(第13期);第1-5页 * |
| Association Between the Polymorphism of Steroid Hormone Metabolism Genes and High-Altitude Pulmonary Edema in the Chinese Han Population;Hui Gao等;《International Journal of General Medicine》;第15卷;第787-794页 * |
| rs9790196;佚名;《Genbank》;第1-7页 * |
| 中国汉族高原肺水肿易感基因的全基因组关联研究;杨应忠等;《遗传 HEREDITAS》;第35卷(第11期);第1291-1299页 * |
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