CN114292910B

CN114292910B - Kit for screening plateau pneumochysis susceptible population developed based on SNP rs11620394 and application thereof

Info

Publication number: CN114292910B
Application number: CN202210231946.3A
Authority: CN
Inventors: 王艳; 侯磊; 刘柳
Original assignee: Chinese PLA General Hospital
Current assignee: Chinese PLA General Hospital
Priority date: 2022-03-10
Filing date: 2022-03-10
Publication date: 2022-05-17
Anticipated expiration: 2042-03-10
Also published as: CN114292910A

Abstract

The invention discloses a kit for screening plateau pneumochysis susceptible population developed based on SNP rs11620394 and application thereof. The invention provides an application of a substance for detecting the genotype of a subject based on SNP rs11620394 in preparing a product; the function of the product is as follows (a) or (b): (a) screening susceptible individuals of plateau pulmonary edema; (b) the subject is assessed for risk of developing high altitude pulmonary edema. The invention also protects the application of the SNP rs11620394 as a detection target in developing products; the function of the product is as follows (a) or (b): (a) screening susceptible individuals of plateau pulmonary edema; (b) the subject is assessed for risk of developing high altitude pulmonary edema. The invention can be used for avoiding or reducing the occurrence of the plateau pulmonary edema and has great application and popularization values for plateau areas.

Description

Kit for screening plateau pneumochysis susceptible population developed based on SNP rs11620394 and application thereof

Technical Field

The invention belongs to the field of gene detection, and relates to a kit for screening plateau pneumochysis susceptible population developed based on SNP rs11620394 and application thereof.

Background

High Altitude Pulmonary Edema (HAPE) means that the patient feels hypodynamia or reduced mobility when the patient reaches the plateau (generally, the altitude is more than 3000 m, and a small amount is less than 3000 m), and the patient suffers from dyspnea, chest distress, chest oppression, cough, white or pink sputum foam at rest. The plateau pulmonary edema is a plateau idiopathic disease, is mostly seen in high altitude areas with the height of more than 3000 m, and is mainly characterized by acute onset, great harm and high fatality rate.

In recent years, a series of studies have been made on the occurrence of HAPE at home and abroad, and the disease has a remarkable tendency of genetic susceptibility and is a result of combined action of heredity and environment. The incidence of disease in hypoxic environment is closely related to individual susceptibility genes, so the susceptibility genes may be an important molecular marker for preventing and diagnosing diseases, evaluating the severity of diseases and providing an effective treatment method.

Disclosure of Invention

The invention aims to solve the technical problem of how to screen individuals susceptible to high altitude pulmonary edema or predict the susceptibility of a subject to high altitude pulmonary edema or screen individuals carrying human genes susceptible to high altitude pulmonary edema or evaluate the risk of high altitude pulmonary edema.

The invention provides a product comprising a substance for detecting the genotype of a subject based on SNP rs 11620394; the function of the product is as follows (a) or (b):

(a) screening susceptible individuals of plateau pulmonary edema;

(b) the subject is assessed for risk of developing high altitude pulmonary edema.

The TT genotype subject is susceptible to high altitude pulmonary edema.

Subjects with TT genotype are more likely to develop hyperplastic pulmonary edema than subjects with GT genotype or subjects with GG genotype.

Illustratively, the substance for detecting the genotype of the subject based on SNP rs11620394 is a primer set. Specifically, the primer group 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.

Illustratively, the substance for detecting the genotype of the subject based on SNP rs11620394 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 invention also protects the application of the SNP rs11620394 as a detection target in developing products; the function of the product is as follows (a) or (b):

(a) screening susceptible individuals of plateau pulmonary edema;

The invention also provides the application of the substance for detecting the genotype of the subject based on the SNP rs11620394 in preparing products; the function of the product is as follows (a) or (b):

(a) screening susceptible individuals of plateau pulmonary edema;

The TT genotype subject is susceptible to high altitude pulmonary edema.

SNP rs11620394 is located in human genomic DNA at a position corresponding to nucleotide 301 in sequence 6 of the sequence listing and is denoted by K.

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 nationality.

Any of the above subjects may be chinese han-nationality men.

Any of the subjects described above may be Chinese in plain.

Any of the above subjects may be chinese han population in plain region.

Any of the above subjects may be chinese han men in plain regions.

Any of the subjects described above may be Chinese from a plain area to a plateau area.

Any of the subjects described above may be chinese han population arriving at plateau from plain.

Any of the above subjects may be a chinese han male who arrives at the plateau from the plain region.

Any of the subjects described above may be Chinese expected to go to plateau from plain.

Any of the subjects described above may be chinese Han nationality predicted to go to plateau from plain areas.

Any of the above subjects may be chinese men expected to go to plateau from plain.

Any one of the above plateau regions may be a Tibet plateau region.

Any one of the above plateau regions may be the rasa region of tibetan plateau.

Any of the above substances for detecting the genotype of the subject based on the SNP rs11620394 may be a substance for detecting the genotype of the subject based on the SNP rs11620394 based on any of the following techniques: DNA sequencing, restriction enzyme fragment length polymorphism, single-strand conformation polymorphism, denaturing high performance liquid chromatography, SNP chip, etc. The SNP chip may include a chip based on a nucleic acid hybridization reaction, a chip based on a single base extension reaction, a chip based on an allele-specific primer extension reaction, a chip based on a "one-step" reaction, a chip based on a primer ligation reaction, a chip based on a restriction enzyme reaction, a chip based on a protein DNA binding reaction, a chip based on a fluorescent molecule DNA binding reaction, and the like.

Any of the above materials for detecting the genotype of the subject based on SNP rs11620394 include 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 a DNA fragment comprising the SNP rs 11620394. The two amplification primers have no special requirements on the sequence as long as the genomic DNA fragment including the SNP rs11620394 can be amplified. The single base extension primer can be designed according to the upstream or downstream (excluding the SNP locus) of the SNP rs11620394 in the human genome, and the extended nucleotide of the single base extension primer corresponds to the SNP rs11620394 in the human genome, namely, the 3' terminal nucleotide of the single base extension primer corresponds to the adjacent nucleotide of the SNP rs11620394 in the human genome (namely, the former nucleotide of the SNP rs11620394 or the latter nucleotide of the rs 11620394).

Any of the above materials for detecting the genotype of the subject based on the SNP rs11620394 may further include PCR reagents, DNA sequencing reagents, DNA sequencers, and the like.

Any of the above materials for detecting the genotype of the subject based on SNP rs11620394 include a primer pair. The primer pair consists of two primers and is used for amplifying a DNA fragment comprising the SNP rs 11620394. The two primers have no special requirements on the sequence as long as the genomic DNA fragment including the SNP rs11620394 can be amplified.

The SNP rs11620394 can be used for screening individuals susceptible to the high altitude pulmonary edema, predicting the susceptibility of people to the high altitude pulmonary edema, screening individuals carrying genes susceptible to the high altitude pulmonary edema of people and evaluating the risk of the high altitude pulmonary edema. In practical application, in order to improve the accuracy, the substance for detecting the SNP rs11620394 genotype can be combined with other substances (for example, substances for detecting other single nucleotide polymorphisms or genotypes related to high altitude pulmonary edema) to prepare a product.

The invention discovers that the distribution frequency of the SNP rs 11620394-based genotype in a high altitude pulmonary edema patient population and a normal human population is obviously different, and the genotype frequency of the TT genotype in the high altitude pulmonary edema patient population is obviously higher than that of the TT genotype in the normal human population. In example 3 and example 4, the genotype distribution frequency data in the patient population with high altitude pulmonary edema and the normal population are exemplarily shown by taking different population samples as examples. The invention can be used for avoiding or reducing the occurrence of the plateau pulmonary edema and has great application and popularization values for plateau areas.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. p-value: person

test. OR: ratio of ratios.

Ethical statement: each subject signed an informed consent, which was approved by the seventh medical center of the liberty military and the medical ethics committee of the general hospital of the tibetan liberty military. The plateau pulmonary edema patients are all patients diagnosed in hospitals, and the diagnosis standard is shown in documents: naming, typing and diagnosing standards of the altitude diseases in China; volume 20, stage 1 of the journal of plateau medicine 2010; 9-11.

Example 1 screening for SNPs associated with risk of high altitude pulmonary edema

The matched population consists of a population of patients with high altitude pulmonary edema and a corresponding normal population.

Establishing a plurality of groups of paired groups, performing a large amount of whole genome sequencing and sequence comparison analysis work, and obtaining a large amount of differential SNPs. The allele frequencies and genotype frequencies of each differential SNP in the plateau pneumochysis patient population and the corresponding normal population were verified in turn for association analysis.

SNP rs11620394, the SNP numbered rs11620394 in NCBI, is located in an intron of MTUS2 gene, specifically at position 29141411 (GRCh38. p13) of human chromosome 13, and has a polymorphism of T/G. In human genome DNA, SNP rs11620394 and its peripheral nucleotides are shown as sequence 6 in the sequence table (SNP rs11620394 is the 301 rd nucleotide in sequence 6 in the sequence table, and is shown as K).

Any clinical diagnostic use of SNP rs11620394 has not been found in the prior art.

Through a great deal of work, the inventor finds that SNP rs11620394 has correlation with the risk of the high altitude pulmonary edema, and a TT genotype subject has higher risk of the high altitude pulmonary edema.

Example 2 method for establishing SNP rs 11620394-based genotype of test subject

1. The peripheral blood of the subject was collected and genomic DNA was extracted.

2. And (3) performing PCR amplification by using the genomic DNA extracted in the step (1) as a template and adopting a specific primer pair.

The specific primer pair consists of a primer F1 and a primer R1.

Primer F1 (SEQ ID NO: 1 of the sequence Listing): 5'-ACGTTGGATGCAGCATCTCTAGGCTGTAAC-3', respectively;

primer R1 (SEQ ID NO: 2 of the sequence Listing): 5'-ACGTTGGATGTCCTGAAGGTAAGCCTTAAC-3' are provided.

PCR amplification was performed in 384-well plates, one reaction system per well.

Composition of reaction system for PCR amplification (5. mu.L): 0.5. mu.L of 10 XPCR buffer, 0.4. mu.L of 25mM MgCl₂The solution, 0.1. mu.L of 25mM dNTP mix, genomic DNA, HotStart Taq DNA polymerase, primer F1, primer R1, and the volume was made up with ultrapure water. In the 5. mu.L system, the content of genomic DNA was 20 to 50ng, the content of HotStar Taq enzyme was 0.5U, the content of primer F1 was 0.5pmol, and the content of primer R1 was 0.5 pmol.

Reaction procedure for PCR amplification: 4 minutes at 94 ℃; at 94 ℃ for 20 seconds, at 56 ℃ for 30 seconds, at 72 ℃ for 1 minute, for 45 cycles; 3 minutes at 72 ℃; keeping at 4 ℃.

3. Alkaline phosphatase treatment was performed (in order to remove free dNTPs from the system).

Taking the 384-well plate which completes the step 2, preparing a reaction system and then carrying out reaction.

Composition of reaction system (7. mu.L): mu.L of the product solution obtained in step 2, 0.3. mu.L of SAP, 0.17. mu.L of 10 XSAP buffer, and 1.53. mu.L of ultrapure water.

SAP (shrimp alkaline phosphatase ): the product specification is 1.7U/mu L; agena corporation, cat # 10002.1. The 10 × SAP buffer is the matched buffer of SAP.

Reaction conditions are as follows: 40 minutes at 37 ℃; 5 minutes at 85 ℃; maintaining the temperature at 4 ℃.

4. Single base extension was performed.

Taking the 384-well plate which completes the step 3, preparing a reaction system and then carrying out single-base extension reaction.

Composition of reaction System for Single-base extension (10. mu.L): 7. mu.L of the product solution obtained in step 3, 0.3. mu.L of the single-base extension reaction enzyme, 0.17. mu.L of the 10 Xsingle-base extension reaction buffer, 1. mu.L of the single-base extension primer, and 1.53. mu.L of ultrapure water.

Single base extension reaction enzyme: the product specification is 1.7U/mu L; agena, cat. No. 1432. The 10 Xsingle-base extension reaction buffer is the matched buffer of the single-base extension reaction enzyme.

Single base extension primer (sequence 3 of sequence listing): 5'-GTAACTTCTGCCCGAA-3' are provided.

The reaction procedure for single base extension is as follows:

firstly, 94 ℃ for 30 seconds;

② 94 ℃ for 5 seconds;

③ 5 cycles of 5 seconds at 52 ℃ and 5 seconds at 80 ℃;

fourthly, 40 cycles of 5 seconds at 94 ℃, 5 seconds at 52 ℃ and 5 seconds at 80 ℃;

fifthly, the temperature is 72 ℃ for 3 minutes;

maintaining at 4 deg.c.

5. Resin purification is performed.

Taking the 384-well plate which completes the step 4, adding 16 μ l of water into each well, then adding Clean Resin (Sequenom corporation, USA) into each well, sealing the membrane, vertically rotating at a low speed for 30 minutes to enable the Resin to be fully contacted with the reactant, then centrifuging to enable the Resin to sink to the bottom of the well, and obtaining the supernatant which is the extension product after the Resin is purified.

6. And (4) spotting the chip.

The MassARRAYANodeipenser RS1000 Spotter (SEQUENOM) was started and the resin-purified extension product was transferred to a 384-well SpectroCHIP (Sequenom) chip (SEQUENOM).

7. And (4) detecting by mass spectrometry.

The spotted SpectroCHIP chip is analyzed by MALDI-TOF, and the detection result is typed by TYPER 4.0 software (sequenom) and output.

Example 3 analysis of SNP rs11620394 and susceptibility to high altitude pulmonary edema

Peripheral blood samples were obtained from the general hospital of the liberty military, tibet, and collected between 2018 and 2019, months 9. 101 peripheral blood samples were obtained from 101 high altitude pulmonary edema patients, and 69 peripheral blood samples were obtained from 69 normal persons (non-high altitude pulmonary edema patients). 101 patients with high altitude pulmonary edema and 69 normal people were both Han nationalities of Chinese male unrelated in blood margin, both lived in plain areas for a long time (long term means more than 1 year) and received peripheral blood after arriving at Lhasa (altitude 3658 m). All people had no history of smoking and drinking, and no autoimmune related diseases (e.g. vitiligo, psoriasis, type I diabetes etc.) and no history of high altitude trips before. The age range of 101 patients with high altitude pulmonary edema was 15-55 years. 69 normal individuals ranged in age from 18-31 years.

The procedure was followed as in example 2. Detecting the genotype of the subject based on SNP rs 11620394.

Of 101 patients with high altitude pulmonary edema, 64 individuals with genotype TT (genotype frequency of 63.4%), 35 individuals with genotype GT (genotype frequency of 34.7%), and 2 individuals with genotype GG (genotype frequency of 2.0%) were observed. 69 of normal persons, 30 individuals with genotype TT (genotype frequency of 43.5%), 35 individuals with genotype GT (genotype frequency of 50.7%), and 4 individuals with genotype GG (genotype frequency of 5.8%).

The test data are processed and counted by R4.0.5 statistical software. After correction by Logistic regression analysis, the significant P value of SNP rs11620394 and the population is 0.004, and OR is 2.222. The results indicate that SNP rs11620394 is a SNP associated with high altitude pulmonary edema, and subjects with genotype TT are at higher risk of HAPE than subjects with genotype GT and subjects with genotype GG.

Example 4 analysis of SNP rs11620394 and susceptibility to high altitude pulmonary edema

Peripheral blood samples were obtained from the general hospital of the liberty military, tibet, and collected between 2018 and 2019, months 9. 61 peripheral blood samples were obtained from 61 patients with high altitude pulmonary edema, and 71 peripheral blood samples were obtained from 71 normal persons (non-high altitude pulmonary edema patients). 61 patients with high altitude pulmonary edema and 71 normal people were Han nationalities of Chinese male unrelated in blood margin, both lived in plain areas for a long time (long term means more than 1 year) and received peripheral blood after arriving at Lhasa (altitude 3658 m). All people had no history of smoking and drinking, and no autoimmune related diseases (e.g. vitiligo, psoriasis, type I diabetes etc.) and no history of high altitude trips before. The age range of 61 patients with high altitude pulmonary edema was 10-60 years. The age range of 71 normal persons is 18-31 years.

The specific primer pair consists of a primer F2 and a primer R2.

Primer F2 (SEQ ID NO: 4 of the sequence Listing): 5'-TACCAAACTTTACCCACT-3', respectively;

primer R2 (SEQ ID NO: 5 of the sequence Listing): 5'-CTTCTATGACCCATCTCC-3' are provided.

3. And (3) after the step 2 is completed, recovering and sequencing the PCR amplification product, and obtaining the genotype of the subject based on the SNP rs11620394 according to the sequencing result.

Of 61 patients with high altitude pulmonary edema, 42 individuals with genotype TT (genotype frequency of 68.9%), 18 individuals with genotype GT (genotype frequency of 29.5%), and 1 individual with genotype GG (genotype frequency of 1.6%) were identified. Of the 71 normal persons, 37 individuals with genotype TT (genotype frequency of 52.1%), 27 individuals with genotype GT (genotype frequency of 38.0%), and 7 individuals with genotype GG (genotype frequency of 9.9%) were identified.

The test data are processed and counted by R4.0.5 statistical software. After correction by Logistic regression analysis, the significance P value of SNP rs11620394 and population is <0.05, OR is 2.326. Consistent with the results of example 3, subjects with genotype TT are at higher risk of developing HAPE than subjects with genotype GT and subjects with genotype GG.

The results of this example further illustrate that SNP rs11620394 is a risk site related to high altitude pulmonary edema, and can be used to screen individuals susceptible to high altitude pulmonary edema, predict susceptibility of a person to high altitude pulmonary edema, screen individuals carrying genes susceptible to high altitude pulmonary edema, and evaluate risk of high altitude pulmonary edema.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

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Claims

1. The use of a substance for detecting the genotype of a subject based on SNP rs11620394 in the preparation of a product; the function of the product is as follows (a) or (b):

(a) screening susceptible individuals of plateau pulmonary edema;

(b) assessing the risk of the subject for developing high altitude pulmonary edema;

the substance for detecting the genotype of the subject based on the SNP rs11620394 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.

2. Use of a substance for detecting the genotype of a subject based on SNP rs11620394 in the manufacture of a product; the function of the product is as follows (a) or (b):

(a) screening susceptible individuals of plateau pulmonary edema;

the substance for detecting the genotype of the subject based on the SNP rs11620394 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.