CN116574803A - Application of SNP rs1730586 as target in developing kit for screening plateau pulmonary edema susceptible population - Google Patents
Application of SNP rs1730586 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 rs1730586 as a target in developing a kit for screening susceptible crowd of plateau pulmonary edema. The application provides application of SNP rs1730586 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. Subjects carrying allele T are at higher risk of developing HAPE than subjects not carrying allele T. 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 rs1730586 as a target in developing a kit for screening susceptible crowd of plateau pulmonary edema.
Background
There are various types of pulmonary diseases caused by the hypoxic environment of the plateau, and plateau pulmonary edema (High altitude pulmonary edema, HAPE) is one of the common plateau pulmonary diseases. Plateau pulmonary oedema is usually a idiopathic disease caused by elevated pulmonary arterial pressure, increased pulmonary capillary volume, altered permeability, and exudation of fluid from the blood vessels to the pulmonary interstitium and alveoli due to the low pressure and low oxygen environment following entry from plain (altitude >3000 meters). HAPE clinically has a series of symptoms such as headache, dyspnea, cough, limited movement and the like, and the disease is rapidly developed, so that the life safety of people entering a plateau is seriously threatened.
The race specificity of HAPE onset and susceptibility to familial and individual predisposition suggest that both environmental and genetic factors can affect HAPE onset, while the susceptibility of the body to environmental factors is also affected by genetic factors. In recent years, genetic factors have been increasingly emphasized in the pathogenesis of HAPE, and some literature reporter polymorphisms are closely related to HAPE susceptibility. Genomic polymorphism analysis is widely used to assess risk of disease and may be used as a genetic marker for susceptibility to disease, as a means of assessing severity of disease, and to provide effective treatment for clinical use.
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 SNP rs1730586 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.
The application also protects a product comprising a substance for detecting an allele carried by the SNP rs1730586 locus in the genomic DNA of a subject; 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.
Subjects carrying allele T at SNP rs1730586 locus are plateau pulmonary edema susceptible individuals.
Subjects carrying allele T at SNP rs1730586 locus are at higher risk of developing HAPE than subjects not carrying allele T.
Based on SNP rs1730586, the genotype of the subject is TT or TC or CC.
Illustratively, the substance for detecting an allele carried by the SNP rs1730586 locus in the genomic DNA of the subject 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 used to detect the allele carried by the SNP rs1730586 locus in the genomic DNA of the subject 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 application also protects the application of the substance for detecting the allele carried by SNP rs1730586 locus in the genomic DNA of the subject in preparing 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.
Subjects carrying allele T at SNP rs1730586 locus are plateau pulmonary edema susceptible individuals.
Subjects carrying allele T at SNP rs1730586 locus are at higher risk of developing HAPE than subjects not carrying allele T.
Based on SNP rs1730586, the genotype of the subject is TT or TC or CC.
Illustratively, the substance for detecting an allele carried by the SNP rs1730586 locus in the genomic DNA of the subject 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 used to detect the allele carried by the SNP rs1730586 locus in the genomic DNA of the subject 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.
SNP rs1730586 is located in human genome DNA, and the position corresponds to nucleotide 301 in sequence 6 of the sequence table, and is denoted by Y. Y represents T or C.
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 ari region, a Ganmai region, a stove hall region, a conding region or a new bridge region of Qinghai-Tibet plateau.
The substance for detecting an allele carried by the SNP rs1730586 locus in the genomic DNA of a subject as described above may be a substance detected based on any one 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.
Any of the above materials for detecting alleles carried at SNP rs1730586 locus in genomic DNA of a subject include primer sets. 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 1730586. The two amplification primers have no special requirement on sequence, so long as the genomic DNA fragments including SNP rs1730586 can be amplified. The single base extension primer may be designed based on the upstream or downstream of SNP rs1730586 in the human genome (excluding the SNP site), and the nucleotide extended by the single base extension primer corresponds to SNP rs1730586 in the human genome, i.e., the 3' -terminal nucleotide of the single base extension primer corresponds to the adjacent nucleotide of SNP rs1730586 in the human genome (i.e., the previous nucleotide of SNP rs1730586 or the latter nucleotide of rs 1730586).
Any of the above materials used to detect alleles carried by SNP rs1730586 locus in genomic DNA of a subject may also include PCR reagents, DNA sequencing reagents, DNA sequencers, and the like.
Any of the above materials for detecting alleles carried at SNP rs1730586 locus in genomic DNA of a subject include primer pairs. The primer pair consists of two primers and is used for amplifying the DNA fragment including SNP rs 1730586. The two primers have no special requirement on sequence, so long as the genome DNA fragments including SNP rs1730586 can be amplified.
SNP rs1730586 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 applications, in order to improve the accuracy, the product can also be prepared by combining a substance for detecting the allele carried by the SNP rs1730586 locus in the genomic DNA of the subject with other substances (such as a substance for detecting other single nucleotide polymorphisms related to altitude pulmonary edema).
The application discovers that subjects carrying allele T are susceptible individuals to altitude pulmonary edema based on SNP rs1730586, and the risk of HAPE occurrence of subjects carrying allele T is higher than those not carrying allele T. In examples 3 and 4, the present application is exemplified by different population samples, respectively, illustrating genotype distribution frequency data and allele carrying frequencies in a patient population with altitude pulmonary edema and 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 used in the following examplesReagents, etc., unless otherwise specified, are commercially available. 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 release army. 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 rs1730586, i.e.SNP number rs1730586 in NCBI, is specifically located at position 36154352 of human chromosome 2 (GRCh38.p14), and the polymorphic form is T/C. In the human genome DNA, SNP rs1730586 and the peripheral nucleotide thereof are shown as sequence 6 of a sequence table (SNP rs1730586 is the 301 th nucleotide in sequence 6 of the sequence table and is expressed by Y).
No clinical diagnostic use of SNP rs1730586 has been found in the prior art.
The inventors found through extensive work as described above that SNP rs1730586 has a correlation with the risk of developing altitude pulmonary edema, and that subjects carrying allele T have a higher risk of developing altitude pulmonary edema.
Example 2, methods of establishing a genotype of a test subject based on SNP rs1730586
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'-ACGTTGGATGATGCCCATCTGCCTACTGAA-3';
primer R1 (sequence 2 of the sequence table): 5'-ACGTTGGATGAGGCTGGTACATCTGGTGCT-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 amount of genomic DNA was 20-50ng, the amount of HotStar Taq enzyme was 0.5U, the amount of primer F1 was 0.5pmol, and the amount of primer R1 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'-GTGGATTCATCCATTCACTCA-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 rs1730586 and altitude pulmonary edema
Peripheral blood samples were obtained from the tibetan release army general hospital and collected from month 9 in 2018 to month 9 in 2019. 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 rs1730586 was detected.
Of 84 cases of plateau pulmonary edema patients, 21 cases of individuals with genotype TT, 42 cases of individuals with genotype TC, 21 cases of individuals with genotype CC, that is, 63 cases of individuals carrying T allele (frequency: 75%), 21 cases of individuals not carrying T allele (frequency: 25%). Of 160 normal individuals, 26 individuals with genotype TT, 77 individuals with genotype TC, 57 individuals with genotype CC, i.e., 103 individuals with T allele (frequency 64.4%), 57 individuals without T allele (frequency 35.6%), were used.
The test data were processed using R4.0.5 statistical software. After correction by Logistic regression analysis, SNP rs1730586 had a significance P value of 0.024 and or of 0.46 with the population. The results demonstrate that SNP rs1730586 is a SNP associated with altitude pulmonary edema, and that subjects carrying allele T are at higher risk of developing HAPE than subjects not carrying allele T.
Example 4 analysis of susceptibility to SNP rs1730586 and altitude pulmonary edema
Peripheral blood samples were obtained from the tibetan release army general hospital and collected from month 9 in 2018 to month 9 in 2019. 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'-CTTCATTTCAACCCGGAGAC-3';
primer R2 (sequence 5 of the sequence listing): 5'-AGAGGATGGAATTGGCTGTG-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 rs1730586 is obtained according to the sequencing result.
Of 157 cases of plateau pulmonary edema patients, 47 cases of individuals with genotype TT, 80 cases of individuals with genotype TC, 30 cases of individuals with genotype CC, namely 127 cases of individuals carrying T allele (frequency of 80.9%), and 30 cases of individuals not carrying T allele (frequency of 19.1%). Out of 233 normal individuals, 42 individuals with genotype TT, 112 individuals with genotype TC, 79 individuals with genotype CC, namely 154 individuals with T allele (frequency 66.1%), 79 individuals without T allele (frequency 33.9%).
The test data were processed using R4.0.5 statistical software. After correction by Logistic regression analysis, SNP rs1730586 had a significance P value of <0.05 (p= 0.00013444) with the population, OR of 0.57. Consistent with the results of example 3, subjects carrying allele T were at higher risk of developing HAPE than subjects not carrying allele T.
The results of this example further demonstrate that SNP rs1730586 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 (7)
- Application of SNP rs1730586 as 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.
- 2. A product comprising a substance for detecting an allele carried by a SNP rs1730586 locus in genomic DNA of a subject; 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.
- 3. The product of claim 2, wherein: the substance for detecting the allele carried by SNP rs1730586 locus in the genome DNA of the subject is a primer group, and consists of three primers, namely a single-stranded DNA molecule shown in sequence 1 of a sequence table, a single-stranded DNA molecule shown in sequence 2 of the sequence table and a single-stranded DNA molecule shown in sequence 3 of the sequence table.
- 4. The product of claim 2, wherein: the substance for detecting the allele carried by SNP rs1730586 locus in the genome DNA of the subject 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.
- 5. Use of a substance for detecting an allele carried by a SNP rs1730586 locus in genomic DNA of a subject 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.
- 6. The use according to claim 5, wherein: the substance for detecting the allele carried by SNP rs1730586 locus in the genome DNA of the subject is a primer group, and consists of three primers, namely a single-stranded DNA molecule shown in sequence 1 of a sequence table, a single-stranded DNA molecule shown in sequence 2 of the sequence table and a single-stranded DNA molecule shown in sequence 3 of the sequence table.
- 7. The use according to claim 5, wherein: the substance for detecting the allele carried by SNP rs1730586 locus in the genome DNA of the subject 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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| Publication number | Priority date | Publication date | Assignee | Title |
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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 (2)
| Title |
|---|
| "RS1730586", 《UCSC》 * |
| LINING SI等: "MIR17HG polymorphisms contribute to high-altitude pulmonary edema susceptibility in the Chinese population", 《SCI REP》, vol. 14, no. 12, pages 1 - 10 * |
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