Computer-readable storage medium and computer device for screening susceptible persons for acute mountain sickness

Abstract

The present invention discloses a computer-readable storage medium and a computer device for screening susceptible persons of acute mountain sickness. The present invention provides a computer device, which includes a memory, a processor and a computer program stored in the memory, characterized in that: the processor executes the computer program to implement the following steps: s1, data reception: receiving information on whether the subject's SNP rs117075665 site carries allele G, and obtaining sample data; s2, result output: outputting result information from the computer based on the sample data, and the result information is whether the subject is an individual susceptible to acute mountain sickness. The computer device and computer-readable storage medium provided by the present invention can be used to avoid or reduce the occurrence of acute mountain sickness.

Description

Technical Field

[0001] The invention belongs to the field of medical care informatics and relates to a computer-readable storage medium and a computer device for screening susceptible persons of acute mountain sickness. Background Art

[0002] Acute mountain sickness (AMS) refers to a clinical syndrome caused by rapid exposure to high altitude environment (altitude over 2500m), with typical manifestations of severe headache, dizziness, fatigue, vomiting, etc. caused by hypoxia. It usually occurs 4-12 hours after reaching the high altitude environment and lasts for about a week. The incidence of AMS is high, as high as 30%-90%. AMS is very harmful. In mild cases, the working ability is significantly reduced. In severe cases, further deterioration may develop into high-altitude pulmonary edema and high-altitude cerebral edema, increasing the risk of death.

[0003] The pathogenesis of AMS is complex, involving differences in physiological reactions, changes in biochemical metabolism, abnormal pathological morphology, changes in psychological behavior, and molecular biological mechanisms. Among them, molecular regulatory mechanisms play a key role. In high-altitude environments, oxygen content is low and climatic conditions are harsh. The human body needs to adapt to these changes by changing gene expression. Gene expression levels are significantly upregulated in low-oxygen environments, thereby improving the body's ability to adapt to low-oxygen environments by promoting erythropoiesis and increasing vascular density. Studies have found that changes in specific single nucleotide polymorphisms (SNPs) of genes are associated with the risk of AMS, involving disorders in multiple biological links and multiple susceptibility genes. Therefore, systematically exploring the pathogenesis of AMS from the perspective of susceptibility genes and finding possible prevention and treatment targets are of great practical significance for maintaining the life and health of people exposed to high altitudes. Summary of the invention

[0004] The technical problem to be solved by the present invention is how to screen individuals susceptible to acute mountain sickness or how to predict the susceptibility of subjects to acute mountain sickness.

[0005] The object of the present invention is to provide a computer-readable storage medium and a computer device for screening persons susceptible to acute mountain sickness.

[0006] The present invention provides a computer device, comprising a memory, a processor and a computer program stored in the memory, wherein the processor executes the computer program to implement the following steps:

[0007] s1. Data reception: receiving information on whether the subject's SNP rs117075665 site carries allele G, and obtaining sample data;

[0008] s2. Result output: Outputting result information from a computer based on the sample data, wherein the result information indicates whether the subject is susceptible to acute mountain sickness.

[0009] The logical judgment rule for outputting the result information from the computer based on the sample data is: if the subject carries allele G at the SNPrs117075665 site, the subject is an individual susceptible to acute mountain sickness; if the subject does not carry allele G at the SNPrs117075665 site, the subject is not susceptible to acute mountain sickness.

[0010] The present invention also provides a computer program product, including a computer program, characterized in that: when the computer program is executed by a processor, the above-mentioned steps s1 and s2 are implemented.

[0011] The present invention further provides a computer-readable storage medium on which a computer program is stored, wherein the computer program implements the above-mentioned steps s1 and s2 when executed by a processor.

[0012] The present invention also provides a device for screening individuals susceptible to acute mountain sickness, characterized in that the device comprises the following modules:

[0013] M1, data receiving module: used to receive information about whether the subject's SNP rs117075665 site carries allele G, and obtain sample data;

[0014] M2. Result output module: used to output result information from a computer based on the sample data, wherein the result information is whether the subject is an individual susceptible to acute mountain sickness.

[0015] The logical judgment rule for outputting the result information from the computer based on the sample data is: if the subject carries allele G at the SNPrs117075665 site, the subject is an individual susceptible to acute mountain sickness; if the subject does not carry allele G at the SNPrs117075665 site, the subject is not susceptible to acute mountain sickness.

[0016] The present invention also provides a method for screening individuals susceptible to acute mountain sickness, characterized in that:

[0017] The method comprises the following steps:

[0018] Receive information about whether the subject's SNP rs117075665 site carries allele G, and obtain sample data;

[0019] Result information is output from the computer based on the sample data, wherein the result information indicates whether the subject is an individual susceptible to acute mountain sickness.

[0020] The logical judgment rule for outputting the result information from the computer based on the sample data is: if the subject carries allele G at the SNPrs117075665 site, the subject is an individual susceptible to acute mountain sickness; if the subject does not carry allele G at the SNPrs117075665 site, the subject is not susceptible to acute mountain sickness.

[0021] The present invention also provides a device, which is a device for comparing the susceptibility of subjects to acute mountain sickness, characterized in that the device comprises the following modules:

[0022] M3, data receiving module: used to receive information about whether the subject's SNP rs117075665 site carries allele G, and obtain sample data;

[0023] M4. Result output module: used to output result information from a computer based on the sample data, wherein the result information is that the susceptibility to acute mountain sickness of subjects carrying allele G at the SNP rs117075665 site is higher than the susceptibility to acute mountain sickness of subjects not carrying allele G at the SNP rs117075665 site.

[0024] The present invention also provides a method for comparing the susceptibility of subjects to acute mountain sickness, characterized in that:

[0025] The method comprises the following steps:

[0026] Receive information about whether the subject's SNP rs117075665 site carries allele G, and obtain sample data;

[0027] Result information is output from a computer based on the sample data, wherein the result information indicates that the susceptibility to acute mountain sickness of subjects carrying the G allele at the SNP rs117075665 site is higher than the susceptibility to acute mountain sickness of subjects not carrying the G allele at the SNP rs117075665 site.

[0028] Specifically, the subjects are two or more persons.

[0029] The present invention also provides the use of a primer set or a primer pair in the preparation of a product. The present invention also provides a product, which includes a primer set or a primer pair. The function of the product is to screen individuals susceptible to acute mountain sickness or to compare the susceptibility of subjects to acute mountain sickness. The primer set consists of three primers, which are the single-stranded DNA molecule shown in SEQ ID NO: 1, the single-stranded DNA molecule shown in SEQ ID NO: 2, and the single-stranded DNA molecule shown in SEQ ID NO: 3. The primer pair consists of two primers, which are the single-stranded DNA molecule shown in SEQ ID NO: 4 and the single-stranded DNA molecule shown in SEQ ID NO: 5. If the subject carries allele G at the SNP rs117075665 site, the subject is an individual susceptible to acute mountain sickness; if the subject does not carry allele G at the SNP rs117075665 site, the subject is not susceptible to acute mountain sickness. The susceptibility to acute mountain sickness of the subject carrying the G allele at the SNP rs117075665 site is higher than the susceptibility to acute mountain sickness of the subject whose result information is that the SNP rs117075665 site does not carry the G allele.

[0030] Specifically, based on SNP rs117075665, the subject's genotype is GG or GA or AA.

[0031] SNP rs117075665 is located in human genomic DNA, and its position corresponds to the 301st nucleotide in sequence 6 of the sequence list, and is represented by R. R represents G or A.

[0032] Any of the above subjects may be Chinese.

[0033] Any of the above-mentioned subjects may be Chinese Han nationality.

[0034] Any of the above-mentioned subjects may be Chinese Han males.

[0035] Any of the above subjects may be Chinese living in plain areas.

[0036] Any of the above-mentioned subjects may be Chinese Han people living in plain areas.

[0037] Any of the above-mentioned subjects may be Chinese Han males living in plain areas.

[0038] Any of the above subjects may be Chinese who have arrived in plateau areas from plain areas.

[0039] Any of the above-mentioned subjects may be Chinese Han people who move from plain areas to plateau areas.

[0040] Any of the above-mentioned subjects may be Chinese Han males who move from plain areas to plateau areas.

[0041] Any of the above subjects may be Chinese who plan to go to highland areas from plain areas.

[0042] Any of the above-mentioned subjects may be Chinese Han people who are expected to move from a plain area to a plateau area.

[0043] Any of the above-mentioned subjects may be Chinese Han males who are expected to move from plain areas to plateau areas.

[0044] Any of the above-mentioned plateau areas may be the Qinghai-Tibet Plateau area.

[0045] Any of the plateau areas mentioned above may be the Lhasa area of ​​the Qinghai-Tibet Plateau.

[0046] Any of the above-mentioned plateau areas may be the Ali region, Luhuo region, Ganzi region, Kangding region or Xinduqiao region of the Qinghai-Tibet Plateau.

[0047] The inventors of the present invention found that based on SNP rs117075665, subjects carrying allele G are susceptible to acute mountain sickness, and the risk of AMS in subjects carrying allele G is higher than that in subjects not carrying allele G. In Example 3 and Example 4, the present invention uses samples of different populations as examples to exemplarily show the genotype distribution frequency data in the acute mountain sickness patient group and the normal population. Based on the above findings, the inventors of the present invention developed the above-mentioned computer device and computer-readable storage medium.

[0048] The computer device and computer-readable storage medium provided by the present invention can be used to avoid or reduce the occurrence of acute mountain sickness. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 A computer flow chart. DETAILED DESCRIPTION

[0050] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0051] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0052] p-value:Person test. lnBeta=OR, OR: odds ratio.

[0053] Ethics statement: Each subject signed an informed consent form, and this study was approved by the Medical Ethics Committee of the PLA General Hospital. The 2018 version of the Louis Lake Score was used to assess acute mountain sickness (AMS), and all patients with AMS had a score of ≥3.

[0054] Example 1: Screening for SNPs associated with the risk of acute mountain sickness

[0055] The matched groups consisted of patients with acute mountain sickness and corresponding normal people.

[0056] Multiple paired groups were established, and a large number of whole genome sequencing and sequence comparison analysis were performed to obtain a large number of differential SNPs. The allele frequency and genotype frequency of each differential SNP in the acute mountain sickness patient group and the corresponding normal population were verified in turn for association analysis.

[0057] SNP rs117075665, i.e., the SNP numbered rs117075665 in NCBI, is specifically located at position 38453746 of human chromosome 6 (GRCh38.p14), and the polymorphic form is A / G. In human genomic DNA, SNP rs117075665 and its surrounding nucleotides are shown in sequence 6 of the sequence listing (SNP rs117075665 is the 301st nucleotide in sequence 6 of the sequence listing, represented by R).

[0058] No clinical diagnostic use of SNP rs117075665 has been found in the prior art.

[0059] The inventors found through the aforementioned extensive work that SNP rs117075665 is correlated with the risk of acute mountain sickness, and subjects carrying the G allele have a higher risk of acute mountain sickness.

[0060] Example 2: Establishment of a method for detecting the genotype of a subject based on SNP rs117075665

[0061] 1. Take peripheral blood from the subjects and extract genomic DNA.

[0062] 2. Using the genomic DNA extracted in step 1 as a template, perform PCR amplification using specific primer pairs.

[0063] The specific primer pair consists of primer F1 and primer R1.

[0064] Primer F1 (SEQ ID NO: 1): 5′-ACGTTGGATGTGAGGACAGCCTGACAACTG-3′;

[0065] Primer R1 (SEQ ID NO: 2): 5'-ACGTTGGATGACGACCTGGTGTGGTGTGTA-3'.

[0066] PCR amplification was performed in a 384-well plate, with one reaction system in each well.

[0067] The composition of the PCR amplification reaction system (5 μL): 0.5 μL 10× PCR buffer (Agena Bioscience, catalog number 11327), 0.4 μL 25 mM MgCl2 solution, 0.1 μL 25 mM dNTP mix, genomic DNA, HotStart Taq DNA polymerase, primer F1, primer R1, and the volume is made up with ultrapure water. In the 5 μL system, the content of genomic DNA is 20-50 ng, the content of HotStart Taq DNA polymerase is 0.5 U, the content of primer F1 is 0.5 pmol, and the content of primer R1 is 0.5 pmol.

[0068] The reaction program of PCR amplification was as follows: 94°C for 4 minutes; 94°C for 20 seconds, 56°C for 30 seconds, 72°C for 1 minute, 45 cycles; 72°C for 3 minutes; and 4°C hold.

[0069] 3. Perform alkaline phosphatase treatment (to remove free dNTPs in the system).

[0070] Take the 384-well plate that has completed step 2, prepare the reaction system and then carry out the reaction.

[0071] The composition of the reaction system (7 μL): 5 μL of the product solution obtained in step 2, 0.3 μL of SAP, 0.17 μL of SAPbuffer, and 1.53 μL of ultrapure water. The SAP content in the reaction system was 0.073 U / μL.

[0072] SAP (shrimp alkaline phosphatase), SAP buffer, iPlex Enzyme (single base extension reaction enzyme), and iPlex Buffer Plus (single base extension reaction buffer) are all components of the kit. Commercial information of the kit: Agena Bioscience, catalog number 10165.

[0073] Reaction conditions: 37°C for 40 minutes; 85°C for 5 minutes; maintain at 4°C.

[0074] 4. Perform single base extension.

[0075] Take the 384-well plate that has completed step 3, prepare the reaction system, and then perform a single base extension reaction.

[0076] The composition of the single base extension reaction system (10 μL): 7 μL of the product solution obtained in step 3, 0.3 μL of the single base extension reaction enzyme, 0.17 μL of the single base extension reaction buffer, 1 μL of the single base extension primer, and 1.53 μL of ultrapure water. In the single base extension reaction system, the content of the single base extension primer is 5 μmol, and the content of the single base extension reaction enzyme is 0.142 U / μL.

[0077] Single base extension primer (SEQ ID NO: 3): 5'-GTGGAATGTCCCTCTGCCT-3'.

[0078] The reaction procedure for single base extension is as follows:

[0079] ①94℃ 30 seconds;

[0080] ②94℃ 5 seconds;

[0081] ③52℃ 5 seconds, 80℃ 5 seconds, 5 cycles;

[0082] ④94℃ 5 seconds, 52℃ 5 seconds, 80℃ 5 seconds, 40 cycles;

[0083] ⑤72℃ 3 minutes;

[0084] ⑥Maintain at 4℃.

[0085] 5. Perform resin purification.

[0086] Take the 384-well plate that has completed step 4, add 16 μl of water to each well, then add Clean Resin resin (Sequenom, USA) to each well, seal the film, rotate vertically at low speed for 30 minutes to allow the resin to fully contact with the reactants, then centrifuge to allow the resin to sink to the bottom of the well. The supernatant is the extension product after resin purification.

[0087] 6. Chip spotting.

[0088] The MassARRAY Nanodispenser RS1000 sample dispensing device (SEQUENOM) was started to transfer the extension product after resin purification to a 384-well SpectroCHIP (Sequenom) chip (SEQUENOM).

[0089] 7. Mass spectrometry detection.

[0090] The spotted SpectroCHIP chip was analyzed using MALDI-TOF, and the detection results were typed and output using TYPER 4.0 software (sequenom).

[0091] Example 3: Analysis of SNP rs117075665 and Susceptibility to Acute Mountain Sickness

[0092] Peripheral blood samples were obtained from Lhasa, Ali, Luhuo, Ganzi, Kangding and Xinduqiao from September 2020 to September 2023. 50 peripheral blood samples were obtained from 50 patients with acute mountain sickness, and another 232 peripheral blood samples were obtained from 232 normal people (non-acute mountain sickness patients). The 50 patients with acute mountain sickness and the 232 normal people were all unrelated Chinese Han males who had lived in the plains for a long time (long-term means more than 1 year) and had peripheral blood drawn after arriving in Lhasa, Ali, Luhuo, Ganzi, Kangding or Xinduqiao (above 2500m above sea level). All of them had no history of smoking and drinking, and no autoimmune-related diseases (such as vitiligo, psoriasis, type 1 diabetes, etc.), and no history of high altitude travel before.

[0093] The operation was performed according to the method of Example 2. The genotype of the subject based on SNP rs117075665 was detected.

[0094] Among the 50 patients with acute mountain sickness, there was 1 individual with genotype GG, 6 individuals with genotype GA, and 43 individuals with genotype AA, that is, 7 individuals carried the G allele (frequency was 14%), and 43 individuals did not carry the G allele (frequency was 86%). Among the 232 normal people, there were 0 individuals with genotype GG, 14 individuals with genotype GA, and 218 individuals with genotype AA, that is, 14 individuals carried the G allele (frequency was 6.0%), and 218 individuals did not carry the G allele (frequency was 94.0%).

[0095] The experimental data were processed and counted using R 4.0.5 statistical software. After correction by the generalized linear mixed model test, the significant P value of SNP rs117075665 and the population was <0.05 (p=0.0028), and Beta was 1.256. The results showed that SNPrs117075665 is a SNP associated with acute mountain sickness, and the risk of AMS in subjects carrying allele G is higher than that in subjects not carrying allele G.

[0096] Example 4: Analysis of SNP rs117075665 and Susceptibility to Acute Mountain Sickness

[0097] Peripheral blood samples were obtained from Lhasa, Ali, Luhuo, Ganzi, Kangding and Xinduqiao from September 2020 to September 2023. 43 peripheral blood samples were obtained from 43 patients with acute mountain sickness, and another 284 peripheral blood samples were obtained from 284 normal people (non-acute mountain sickness patients). The 43 patients with acute mountain sickness and the 284 normal people were all unrelated Chinese Han males who had lived in the plains for a long time (long-term means more than 1 year) and had peripheral blood drawn after arriving in Lhasa, Ali, Luhuo, Ganzi, Kangding or Xinduqiao (above 2500m above sea level). All of them had no history of smoking and drinking, and no autoimmune-related diseases (such as vitiligo, psoriasis, type 1 diabetes, etc.), and no history of plateau travel before.

[0098] 1. Take peripheral blood from the subjects and extract genomic DNA.

[0099] 2. Using the genomic DNA extracted in step 1 as a template, perform PCR amplification using specific primer pairs.

[0100] The specific primer pair consists of primer F2 and primer R2.

[0101] Primer F2 (SEQ ID NO: 4): 5'-GGACTCCTCTACAGCATTCCAT-3';

[0102] Primer R2 (SEQ ID NO: 5): 5'-GTCTTCAACATTCGCCCACT-3'.

[0103] 3. After completing step 2, the PCR amplification products were recovered and sequenced, and the genotype of the subject based on SNPrs117075665 was obtained according to the sequencing results.

[0104] Among the 43 patients with acute mountain sickness, there were 0 individuals with genotype GG, 11 individuals with genotype GA, and 32 individuals with genotype AA, that is, 11 individuals carried the G allele (frequency was 25.58%), and 32 individuals did not carry the G allele (frequency was 74.42%). Among the 284 normal people, there were 0 individuals with genotype GG, 34 individuals with genotype GA, and 250 individuals with genotype AA, that is, 34 individuals carried the G allele (frequency was 11.97%), and 250 individuals did not carry the G allele (frequency was 88.03%).

[0105] The experimental data were processed and analyzed using R 4.0.5 statistical software. After correction by the generalized linear mixed model test, the significant P value of SNP rs117075665 and the population was <0.05 (p=0.043), and Beta was 0.564. Consistent with the results of Example 3, the risk of AMS in subjects carrying allele G was higher than that in subjects not carrying allele G.

[0106] The results of this example further illustrate that SNP rs117075665 is a risk site associated with acute mountain sickness, and can be used to screen individuals susceptible to acute mountain sickness, predict human susceptibility to acute mountain sickness, screen individuals carrying human acute mountain sickness susceptibility genes, and evaluate the risk of acute mountain sickness.

[0107] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A device for comparing the susceptibility of subjects to acute mountain sickness, characterized in that: The device comprises the following modules: M3, data receiving module: used to receive information about whether the subject's SNP rs117075665 site carries allele G, and obtain sample data; the subject is more than two people; M4. Result output module: used to output result information from a computer based on the sample data, wherein the result information is that the susceptibility to acute mountain sickness of subjects carrying allele G at the SNP rs117075665 site is higher than the susceptibility to acute mountain sickness of subjects not carrying allele G at the SNP rs117075665 site.

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