CN116590407A - Application of SNP rs375372143 as target in developing plateau pulmonary edema screening kit - Google Patents
Application of SNP rs375372143 as target in developing plateau pulmonary edema screening kit Download PDFInfo
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Abstract
The application discloses application of SNP rs375372143 as a target in developing a plateau pulmonary edema screening kit. The application provides application of SNP rs375372143 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 provides application of a substance for detecting alleles carried by SNP rs375372143 locus in genomic DNA of a subject in preparing 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 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 rs375372143 serving as a target in developing a plateau pulmonary edema screening kit, namely application of SNP rs375372143 serving as a target in developing a kit for screening plateau pulmonary edema susceptible people.
Background
Plateau pulmonary edema (High altitude pulmonary edema, HAPE) is a non-cardiac accelerated osmotic pulmonary edema, which occurs in people reaching an altitude of 2500-3000 meters within 2-4 days and is characterized by elevated pulmonary artery pressure, dyspnea, cough and impaired exercise capacity. In the late stage, body temperature may rise to 38.5 ℃, and foam pink sputum, cyanosis, and tachycardia may occur. Altitude pulmonary edema is the cause of death of most overhead diseases and is a potentially pathogenic disease.
HAPE is considered a multifactorial disease, and its development is not only related to the manner of ascent, the rate of speed and the highest altitude reached, but also the susceptibility of the individual. In susceptible individuals, hypoxic pulmonary vasoconstriction can lead to elevated pulmonary arterial pressure and leakage of pulmonary capillaries. In recent years, genomic studies have found that many single nucleotide polymorphism sites (Single nucleotide polymorphism, SNPs) are associated with risk of developing HAPE. Therefore, the susceptibility gene locus can be used as a molecular marker for diagnosis and prognosis of HAPE, and has important biological significance in clinical research.
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 rs375372143 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 provides a product comprising a substance for detecting an allele carried by the SNP rs375372143 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.
SNP rs375372143 locus carries allele T 17 Is a subject susceptible to altitude pulmonary edema.
SNP rs375372143 locus carries allele T 17 The risk of developing HAPE is higher in subjects than in subjects not carrying allele T 17 Is a subject of (a).
Based on SNP rs375372143, the genotype of the subject is T 17 /T 17 Genotype or T 18 /T 17 Genotype or T 18 /T 18 Genotype.
Illustratively, the substance for detecting an allele carried by the SNP rs375372143 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 rs375372143 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 provides application of a substance for detecting alleles carried by SNP rs375372143 locus in genomic DNA of a 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.
SNP rs375372143 locus carries allele T 17 Is a subject susceptible to altitude pulmonary edema.
SNP rs375372143 locus carries allele T 17 Is of subject occurrence of (2)HAPE is at higher risk than not carrying allele T 17 Is a subject of (a).
Based on SNP rs375372143, the genotype of the subject is T 17 /T 17 Genotype or T 18 /T 17 Genotype or T 18 /T 18 Genotype.
Illustratively, the substance for detecting an allele carried by the SNP rs375372143 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 rs375372143 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 rs375372143 is located in human genomic DNA, T is present 18 Allelic form and T 17 Allelic form, T 18 The allelic form is shown as sequence 6 of the sequence table (corresponding to 301 th to 318 th positions in the sequence 6), T 17 The allelic form is shown as a sequence 7 of a sequence table (corresponding to the 301 th to 317 th positions in the sequence 7).
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 an allele carried by the SNP rs375372143 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 rs375372143 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 375372143. The two amplification primers have no special requirement on sequence, so long as the genomic DNA fragments including SNP rs375372143 can be amplified. The single base extension primer may be designed based on the upstream or downstream of SNP rs375372143 in the human genome (excluding the SNP site), and the nucleotide extended by the single base extension primer corresponds to SNP rs375372143 in the human genome, i.e., the 3' -terminal nucleotide of the single base extension primer corresponds to the adjacent nucleotide of SNP rs375372143 in the human genome (i.e., the previous nucleotide of SNP rs375372143 or the latter nucleotide of rs 375372143).
Any of the above materials used to detect alleles carried by SNP rs375372143 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 rs375372143 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 375372143. The two primers have no special requirement on sequence, so long as the genome DNA fragments including SNP rs375372143 can be amplified.
SNP rs375372143 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 rs375372143 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 the SNP rs 375372143-based gene carries an allele T 17 Is a subject susceptible to altitude pulmonary edema, carrying allele T 17 The risk of developing HAPE is higher in subjects than in subjects not carrying allele T 17 Is a subject of (a). 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 to avoid or reduce the occurrence of altitude pulmonary edema, for altitudeThe region has great application and popularization value.
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 rs375372143, i.e.SNP No. rs375372143 in NCBI, is located in one intron of the GPR141 gene, specifically at position 37691247-37691264 of human chromosome 7 (GRCh 38. P14), polymorphic form T 18 /T 17 . Human genomeDNA: when polymorphic form is T 18 When the SNP rs375372143 and the peripheral nucleotide thereof are shown as a sequence 6 of a sequence table; when polymorphic form is T 17 And when the SNP rs375372143 and the peripheral nucleotide thereof are shown as a sequence 7 of a sequence table.
No clinical diagnostic use of SNP rs375372143 has been found in the prior art.
The inventor discovers through a great deal of work that SNP rs375372143 has correlation with the risk of altitude pulmonary edema and carries allele T 17 With a higher risk of altitude pulmonary edema.
Example 2, methods of establishing a genotype of a test subject based on SNP rs375372143
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'-ACGTTGGATGCCTGCTCACGTTTGGTTTCT-3';
primer R1 (sequence 2 of the sequence table): 5'-ACGTTGGATGACCACTCACCAGCCTTACCA-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'-GTGGCCTTTTTTTTTTTTTTTTT-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 rs375372143 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 rs375372143 was detected.
In 84 cases of altitude pulmonary edema, T 18 /T 18 Genotype of 28 individuals, T 18 /T 17 Genotype of 39 individuals, T 17 /T 17 Genotype of individuals is 17, i.e. carrying allele T 17 Individuals with 56 cases (66.7% duty cycle) did not carry allele T 17 28 cases (33.3% duty cycle). Among 160 normal people, T 18 /T 18 Genotype individuals were 67, T 18 /T 17 80 genotype individuals, T 17 /T 17 Genotype of individuals is 13, i.e. carrying allele T 17 93 individuals (58.1% duty cycle) did not carry allele T 17 Is 67 cases (duty ratio)41.9%).
The test data were processed using R4.0.5 statistical software. After correction by Logistic regression analysis, SNP rs375372143 had a significance P value of 0.025 and or of 0.49 with the population. The results indicate that SNP rs375372143 is a SNP associated with altitude pulmonary edema, carrying allele T 17 The risk of developing HAPE is higher in subjects than in subjects not carrying allele T 17 Is a subject of (a).
Example 4 analysis of susceptibility to SNP rs375372143 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'-TGCTCTGGTGTTGGGTCATA-3';
primer R2 (sequence 5 of the sequence listing): 5'-GCAAAACGACAATCATCACG-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 rs375372143 is obtained according to the sequencing result.
In 157 cases of altitude pulmonary edema, T 18 /T 18 Genotype of 55 individuals, T 18 /T 17 Genotype of 77 individuals, T 17 /T 17 Individuals of genotype were 25, i.e. carrying allelesT 17 102 individuals (65.0% duty cycle) did not carry allele T 17 55 individuals (genotype frequency 35.0%). In 233 normal people, T 18 /T 18 Genotype of 126 individuals, T 18 /T 17 Genotype of 89 cases of individuals, T 17 /T 17 18 individuals of genotype, i.e. carrying allele T 17 Individuals with 107 cases (45.9% duty cycle) did not carry allele T 17 126 individuals (genotype frequency 54.1%).
The test data were processed using R4.0.5 statistical software. After correction by Logistic regression analysis, the significance P value of SNP rs375372143 and population<0.05 (p=0.00003), OR is-0.67. Consistent with the results of example 3, carrying allele T 17 The risk of developing HAPE is higher in subjects than in subjects not carrying allele T 17 Is a subject of (a).
The results of this example further demonstrate that SNP rs375372143 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)
1.SNP rs375372143 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 rs375372143 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 rs375372143 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 rs375372143 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 rs375372143 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 rs375372143 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 rs375372143 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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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111676283A (en) * | 2020-05-19 | 2020-09-18 | 中国人民解放军总医院第七医学中心 | Application of mitochondrial DNA single nucleotide polymorphism related to occurrence of high altitude pulmonary edema |
| 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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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111676283A (en) * | 2020-05-19 | 2020-09-18 | 中国人民解放军总医院第七医学中心 | Application of mitochondrial DNA single nucleotide polymorphism related to occurrence of high altitude pulmonary edema |
| 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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| "rs375372143", 《UCSC》, pages 1 - 2 * |
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