CN114657242A - Application of GPR33 gene in assessment of marneffei Talaromyces susceptible population - Google Patents
Application of GPR33 gene in assessment of marneffei Talaromyces susceptible population Download PDFInfo
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- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention relates to application of a GPR33 gene in a biological sample as a biomarker in developing and/or preparing products for evaluating Marneffeil fungus susceptible population, and relates to the technical field of biotechnology and medicine. The invention provides application of a GPR33 gene in a biological sample as a biomarker in developing and/or preparing products for evaluating Marneffei panulifera susceptible population, and provides the biomarker for evaluating HIV-negative Marneffei panulifera susceptible population.
Description
Technical Field
The invention relates to the technical field of biotechnology and medicine, in particular to application of GPR33 gene in assessment of Marneffei panulifera susceptible population.
Background
The Marneffei pannieri infection is an endemic mycosis seriously threatening the life of a patient, susceptible people of the Marneffei pannieri infection gradually transfer from HIV positive to HIV negative patients, and the incidence rate of the Marneffei pannieri infection is fastest in HIV negative pulmonary fungal infection, however, the susceptibility factors and the incidence mechanism of the HIV negative Marneffei pannieri infection are uncertain, the Marneffei pannieri infection is frequently misdiagnosed, missed diagnosed, and the mortality rate and the recurrence rate are high. Understanding its pathogenesis is critical to the rapid diagnosis and effective treatment of the disease.
Marneffei's disease is common in some patients with cellular immune deficiencies. In recent years, more and more potential immunodeficiency is found to be related to the susceptibility of Marneffeta, wherein the immunodeficiency caused by gene mutation is more remarkable, and the gene mutation comprises STAT1, CARD9, STAT3 and the like.
Disclosure of Invention
Aiming at the problems, the invention provides an application of GPR33 gene in a biological sample as a biomarker in developing and/or preparing a product for evaluating Marneffei panulifera susceptible population, and provides the biomarker for evaluating HIV negative Marneffei panulifera susceptible population.
In order to achieve the purpose, the invention provides application of a GPR33 gene in a biological sample as a biomarker in developing and/or preparing a product for evaluating Marneffeta nivea susceptible population.
In the process of research, the inventor finds that the gain-stop variant c.c418t on the GPR33 gene in patients with marneffei pananiasis may cause premature termination of protein translation and change the 3D structure of the protein, so that whether a person providing a biological sample is a susceptible person of marneffei pananiasis can be judged by detecting the sequence of the GPR33 gene in the biological sample.
In one embodiment, the biomarker is GPR33 gene c.c. c418t mutation.
The invention also provides a product for evaluating the predisposing population of Marneffei pannieri, which comprises the following components: an agent for detecting the GPR33 gene in a biological sample.
In one embodiment, the reagent for detecting a GPR33 gene in a biological sample comprises: the kit comprises a reagent for extracting nucleic acid in a biological sample, a PCR amplification reagent, a PCR product purification reagent and a PCR product sequencing reagent.
In one embodiment, the product is PCR amplified using the following primer pairs:
rs1047034-F：GTCTGGTTGATATGGGAGT(SEQ ID NO:1)；
rs1047034-R：CAAGGCAGTGATCTGTTTC(SEQ ID NO:2)；
rs1292324632-F：GCCTTCCTCTTCTCCATCGA(SEQ ID NO:3)；
rs1292324632-R：GCTGGCCTCGTCAATTTCAT(SEQ ID NO:4)；
rs17097921-F：AAGTCACCTTTCCTTTACG(SEQ ID NO:5)；
rs17097921-R：TATGGGTGCTAAGATTCAA(SEQ ID NO:6)；
rs62011763-F：TCTCCATGAGCCTCAGTGTC(SEQ ID NO:7)；
rs62011763-R：GCCGCCTCTCCTGTTTTAAC(SEQ ID NO:8)；
rs62011763-NF：GGAAAGAGGTCTGTGCCA(SEQ ID NO:9)；
rs 62011763-NR: TCATCCTGTTGCCGAAGC (SEQ ID NO: 10). In one embodiment, the product is subjected to PCR product sequencing using the following primer pairs:
rs1047034-R1：GAGTTTTATGCTGCTATTCT(SEQ ID NO:11)；
rs1292324632-R：GCTGGCCTCGTCAATTTCAT(SEQ ID NO:12)；
rs17097921-R1：CTCCCAACTTCAAGACAATCA(SEQ ID NO:13)；
rs 62011763-R1: AACATCTGATTGTTTTCCA (SEQ ID NO: 14). In one embodiment, the biological sample is peripheral blood.
The invention also provides a system for evaluating the susceptible population of marneffei pachyrhizus, comprising:
an analysis device: the method is used for detecting GPR33 genes in a biological sample of a subject to be evaluated, and inputting the GPR33 genes into an evaluation model for analysis;
an output device: for outputting the analysis result.
In one embodiment, the method of analyzing comprises: and when the GPR33 gene c.C418T mutation exists in the biological sample, the object to be evaluated is considered to be a Marneffei pannieri susceptible population.
The invention also provides a method for evaluating the population susceptible to Marneffei basket fungus, which comprises the following steps: peripheral blood was collected, nucleic acid was extracted, and the GPR33 gene was detected.
Compared with the prior art, the invention has the following beneficial effects:
the GPR33 gene in the biological sample is used as a biomarker in developing and/or preparing products for evaluating Marneffei panulifera susceptible population, the biomarker is provided for evaluating the susceptible population of HIV-negative Marneffei panulifera, a reliable basis is provided for researching pathogenesis of HIV-negative Marneffei panulifera, and the biomarker can be used for evaluating the susceptible population of HIV-negative Marneffei panulifera and used as a treatment target in the future.
Drawings
FIG. 1 is a flow chart of the selection of the biomarker GPR33 gene in example 1, wherein a of FIG. 1 is a patient selection flow chart, b of FIG. 1 is the recruitment of 53 HIV negative TM infected patients, their blood samples were collected and whole genome sequenced, and the combined call module of GATK (best practice) was applied to the pre-treatment reads, c of FIG. 1 is the eight-constraint filtered variants, d of FIG. 1 is a chromosome outline of the gene carrying the four mutants;
FIG. 2 is an agarose gel electrophoresis of rs1047034 in experimental example, M is 5000, 3000, 2000, 1000, 750, 500, 250, 100bp from top to bottom;
FIG. 3 is the agarose gel electrophoresis of rs1292324632+ rs1310028733 in the experimental example, M is 5000, 3000, 2000, 1000, 750, 500, 250, 100bp from top to bottom;
FIG. 4 is the agarose gel electrophoresis of rs17097921 of experimental example, M is 5000, 3000, 2000, 1000, 750, 500, 250, 100bp from top to bottom;
FIG. 5 shows the amino acid 3D map of GPR33 in example 1 and the Sanger sequencing peak map of GPR33 gene in the experimental example, wherein a in FIG. 5 is the wild type of GPR33(UniPort ID: Q49SQ1) with seven alpha-helices, in particular alpha-helices3And alpha4Disulfide bond formation, b of fig. 5 is a mutant of GPR33(R140X) in which the structure after the mutation site is drawn by a single line, c of fig. 5 is a close-up view of mutation site 140R with the dotted line being a hydrogen bond, d of fig. 5 is a heterozygous mutation c.c418t on GPR33 confirmed by sanger sequencing, and e and f of fig. 5 are homozygous mutations c.c418t on GPR33 confirmed by sanger sequencing.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
Reagents, materials, equipment sources:
qiagen FlexiGene DNA kit (Qiagen), Qiagen MiRNeasy kit (Qiagen), SuperScript VILO cDNA Synthesis kit (Thermo Fisher Scientific), BGI 2 xSuperPCR Mix (with dye)5ML (5/package, Beijing Liuhe Hua Dai Genet Co., Ltd.), BGI D2000 Plus DNA Ladder (1250 ul/package, Beijing Liuhe Hua Dai Genet Co., Ltd.), whole blood/saliva/oral genome extraction kit by magnetic bead method (100 times/cassette, Wuhanna Biotech Ltd.), agarose (100 g/flask, Sun horse), magnetic beads (10 ML/flask, Shuomei).
Reagents, materials and equipment used in the embodiment are all commercially available sources unless otherwise specified; unless otherwise specified, all the experimental methods are routine in the art.
Example 1
Screening to obtain a biomarker GPR33 gene.
The flow chart of the biomarker GPR33 gene obtained by screening is shown in figure 1.
1. 53 patients with HIV negative marneffei staphylium infection were admitted according to the inclusion exclusion criteria.
Between month 1 of 2018 and month 1 of 2021, 58 HIV-negative marneffei basket-infected patients were enrolled from 7 subcenter nationwide by a prospective multicenter cohort study according to the inclusion criteria below.
Inclusion criteria were: (1) laboratory examination definitively indicated HIV negative; (2) there are clinical or imaging manifestations of marneffei staphylinii infection; (3) malneffeta sp is isolated from the following specimens or has corresponding pathological changes: sputum, airway secretions, alveolar lavage, lung, pleural effusion, bone marrow smears, skin, lymph nodes and the like, including (i) acid-Schiff or Wright's staining with circular or elliptical shapes and obvious transverse septa; ② separating the pathogen from the culture; pathological examination shows that TM infection is accompanied with pyogenic granuloma, central necrosis and a large amount of mononuclear macrophage infiltration; or metagenomic next generation sequencing (mNGS) shows that TM and symptoms and signs are improved after antifungal treatment.
According to the following exclusion criteria: patients with HIV positive or autoimmune disease, cancer or immunodeficiency; or has received immunosuppressive drugs within the past 3 months.
Finally 53 patients were enrolled (as shown in a in fig. 1). The study was conducted according to the principles of the declaration of helsinki. The study was approved by the ethical committee of the first subsidiary hospital of Guangzhou medical university (reference number 2019026) and informed written consent was obtained from the participants.
The study was conducted according to the principles of the declaration of helsinki. The study was approved by the ethical committee of the first subsidiary hospital of Guangzhou medical university (reference number 2019026) and informed written consent was obtained from the participants
2. Sample collection and transport.
(1) Materials: ethylenediaminetetraacetic acid (EDTA) anticoagulant tube.
(2) The experimental method comprises the following steps: after the skin of 53 HIV-negative patients with Marneffei staphylium was sterilized, 5ml of venous blood was collected from the cubital vein into an anticoagulation vacuum blood collection tube. The samples were stored between 2 ℃ and 8 ℃ until ready for shipment to the standardization and approval laboratory of Guangzhou gold area diagnostics group, Inc. (Guangzhou, China). All samples were stored at-80 ℃ for DNA extraction.
3. DNA extraction and sequencing.
(1) Materials: qiagen FlexiGene DNA kit, Qiagen MiRNeasy kit, SuperScript VILO cDNA synthesis kit, Agilent 2100, BGISEQ-50/MGISEQ-2000 platform.
(2) Experimental methods.
Firstly, extracting genome DNA: genomic DNA was extracted from EDTA blood using Qiagen FlexiGene DNA kit.
Reverse transcription Polymerase Chain Reaction (PCR): RNA was extracted from freshly isolated PBMCs using Qiagen MiRNeasy kit.
Construction of cDNA library: reverse transcription into cDNA was performed using SuperScript VILO cDNA Synthesis kit. DNA libraries were constructed using DNA fragmentation, end repair, linker ligation and PCR amplification. Agilent 2100 was used for quality control of DNA libraries.
Sequencing: qualified libraries were pooled and DNA Nanospheres (DNBs) were prepared and sequenced using the BGISEQ-50/MGISEQ-2000 platform.
4. And (4) gene research.
(1) A method.
Sequencing read alignments of human reference genome assembly GRCh38 by Burrows-Wheeler-alignment (bwa) tool; PCR replicates were excluded using Picard software. The GATK best practice found small variations, with 175,537,409 high quality variations remaining after applying quality control of VQSR. In the present invention, the inventors focused on the identified 1,302,281 SNPs and 271,749 indels.
Mutants of interest were further annotated using ANNOVAR. The allele frequencies therein were annotated using the following database: 1000 genome project (1KGP), exome syndication alliance (ExAC), genome syndication database (gnomaD), NHLBI-ESP project with 6500 exomes (ESP6500) and Chinese metabolic analysis project (Chinese map).
First, variations affecting genes known to have immune function are of concern. An immune gene combination containing 1793 genes was planned from the ImmPort database (https:// pubmed. ncbi. nlm. nih. gov/29485622 /). It is further required that all patients in the present invention share the mutants of the combinatorial selection. On the other hand, all mutants were hard filtered to identify new mutants on genes with or without known immune function. During the hard filtration process, the inventors retained mutants that met the following constraints: exon or splicing mutant; a nonsense or missense mutant; ③ there is no overlap with the repeats or segment repeats; sharing by all samples; 1000GP middle allele frequency is less than or equal to 0.01% or is not reported; sixthly, the allele frequency in the gnomAD is less than or equal to 0.01 percent or is not reported; seventhly, the ESP6500 allele frequency is less than or equal to 0.01 percent or is not reported; the allele frequency of ChinaMAP is less than or equal to 0.01 percent or is not reported. The constraints (c) are used here to preserve variants that are most likely to affect gene function. The limitations from (v) to (v) aim at identifying rare or novel variations associated with the disease, especially in the chinese population.
The variation remaining after filtration is considered to have pathogenic potential and requires further manual investigation. The pathogenicity of the four mutants was evaluated using InterVar software and ClinVar's clinical report according to the American society for medical genetics and guidelines of the society for molecular pathology (ACMG/AMP). Ten combinations of harm prediction algorithms (e.g., SIFT, PolyPhen2, and LRT) were performed on potentially rational mutants and the potential impact of variants on protein function was further investigated by evolution rate analysis (GERP) scores and Combination Annotation Dependent Depletion (CADD) scores.
(2) And (6) obtaining the result.
Filtering by 8 conditions to finally obtain 4 mutants distributed in 3 chromosomes (as shown in b-d in figure 1).
② identifying possible pathogenic mutants in the immune gene combination.
From the above immune genome, 302 variants without benign reports were identified from the ClinVar or the intersvar of 251 immune-related genes. These variants included three insertion deletions, two stop gain variants, one start loss variant, and 296 non-synonymous variants. (biological relationship of three indels on MCC, TRHDE, ISCU). The stop gain variant on SLC22a24 and the start loss variant on DENND3 had negative PhyloP scores of-0.076 and-2.88, respectively, indicating that these two variants are located at a rapidly evolving position rather than a conserved position and are therefore less likely to be pathogenic. On the other hand, the GERP score for the stop gain variant c.c418t on GPR33 gene was 3.15, indicating that the site of this variant underwent 3.15 fewer mutations compared to the neutral rate of evolution. By examining the topology of the GPR33 protein gene, the inventors discovered a transmembrane domain consisting of seven α -helices, which is responsible for signal transduction across cell membranes. Further investigation of the 3D structure revealed the presence of disulfide bonds between the disulfide bonds, linking the polypeptides as covalent bonds (as shown in a-c in figure 5). However, the mutant p.r140x is located at the core of seven α -helices, losing the α -helices and the disulfide bonds between them. Furthermore, ARG-140 shows strong interaction with and through the formation of multiple hydrogen bonds, which are lost with the formation of the stop gain mutation R140X. By predicting the function of this variant, the stop gain variant c.c418t on GPR33 gene may lead to premature termination of protein translation and change of the 3D structure of the protein.
Examples of the experiments
GPR33 gene mutations were verified by Sanger sequencing.
1. Materials: BGI 2xSuper PCR Mix (with dye), BGI D2000 Plus DNA Ladder, whole blood/saliva/oral genome extraction kit by magnetic bead method, agarose, magnetic beads, and peripheral blood mononuclear cell cryopreserved specimens of 29 patients.
2. Experimental methods.
(1) Nucleic acid extraction: the operation was performed with reference to the instructions for nucleic acid extraction.
(2) And (4) PCR amplification.
Primer information: based on the detection information, the reference sequence was searched, and Primer design was performed using Primer Premier 5 software, and the Primer information is shown in the following table.
TABLE 1 primer information
Amplification System and conditions: as shown in the table below.
TABLE 2 PCR amplification reaction System and conditions
(3) And (5) electrophoretic identification.
The method comprises the following steps: mu.L of the PCR product was subjected to 1.0% agarose gel detection, and the band property was observed.
As a result: as shown in fig. 2-4.
(4) And (5) purifying a PCR product.
And (3) purifying the PCR product according to the operation of a magnetic bead purification standard operation flow, adsorbing DNA in a high-salt low-pH solution by utilizing the principle that magnetic beads can adsorb or release substances with charges, and releasing DNA in a low-salt high-pH solution, so that the aim of separating and purifying the DNA product is fulfilled.
(5) And (5) sequencing.
And (4) performing on-machine detection on the purified PCR product.
(6) And (6) analyzing the data.
And after the data is downloaded from the system, the data is renamed and classified, then the data is uploaded to the system, and after the data is downloaded from the system, phred \ phrap software is used for carrying out SNP analysis and exporting analysis results.
3. And (5) experimental results.
Of the 29 patients, 26 patients showed homozygous mutation in the GPR33 gene and 3 patients showed heterozygous mutation in the GPR33 gene, and the results are shown in d-f in FIG. 5.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
<110> Guangzhou medical university affiliated first hospital
GUANGZHOU INSTITUTE OF RESPIRATORY HEALTH
Application of <120> GPR33 gene in assessment of marneffei strain susceptible population
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Claims (10)
1. Application of GPR33 gene in biological samples as biomarker in development and/or preparation of products for evaluating Marneffei pannieri susceptible population.
2. The use of claim 1, wherein the biomarker is GPR33 gene c.c. c418t mutation.
3. A product for assessing a population susceptible to marneffei comprising: an agent for detecting the GPR33 gene in a biological sample.
4. The product of claim 3, wherein the reagents for detecting the GPR33 gene in a biological sample comprise: the kit comprises a reagent for extracting nucleic acid in a biological sample, a PCR amplification reagent, a PCR product purification reagent and a PCR product sequencing reagent.
5. The product of claim 4, wherein the product is PCR amplified using the following primer pairs:
rs1047034-F：GTCTGGTTGATATGGGAGT(SEQ ID NO:1)；
rs1047034-R：CAAGGCAGTGATCTGTTTC(SEQ ID NO:2)；
rs1292324632-F：GCCTTCCTCTTCTCCATCGA(SEQ ID NO:3)；
rs1292324632-R：GCTGGCCTCGTCAATTTCAT(SEQ ID NO:4)；
rs17097921-F：AAGTCACCTTTCCTTTACG(SEQ ID NO:5)；
rs17097921-R：TATGGGTGCTAAGATTCAA(SEQ ID NO:6)；
rs62011763-F：TCTCCATGAGCCTCAGTGTC(SEQ ID NO:7)；
rs62011763-R：GCCGCCTCTCCTGTTTTAAC(SEQ ID NO:8)；
rs62011763-NF：GGAAAGAGGTCTGTGCCA(SEQ ID NO:9)；
rs62011763-NR：TCATCCTGTTGCCGAAGC(SEQ ID NO:10)。
6. the product of claim 4, wherein the product is used for PCR product sequencing using the following primer pairs:
rs1047034-R1：GAGTTTTATGCTGCTATTCT(SEQ ID NO:11)；
rs1292324632-R：GCTGGCCTCGTCAATTTCAT(SEQ ID NO:12)；
rs17097921-R1：CTCCCAACTTCAAGACAATCA(SEQ ID NO:13)；
rs62011763-R1：AACATCTGATTGTTTTCCA(SEQ ID NO:14)。
7. the product according to any one of claims 3 to 6, wherein the biological sample is peripheral blood.
8. A system for assessing a population susceptible to marneffei, the system comprising:
an analysis device: the method is used for detecting GPR33 gene in a biological sample of a subject to be evaluated and inputting the GPR33 gene into an evaluation model for analysis;
an output device: for outputting the above analysis results.
9. The system of claim 8, wherein the method of analyzing is: when the GPR33 gene c.C418T mutation exists in the biological sample, the object to be evaluated is considered to be a Marneffeta susceptive population.
10. A method for evaluating a population susceptible to Marneffei staphylium, the method comprising the steps of: peripheral blood was collected, nucleic acid was extracted, and the GPR33 gene was detected.
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| CN114717353A (en) * | 2022-03-14 | 2022-07-08 | 广州医科大学附属第一医院 | Biomarker for auxiliary diagnosis of HIV negative Marneffei panuliferus disease and application thereof |
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| CN114717353B (en) * | 2022-03-14 | 2022-12-06 | 广州医科大学附属第一医院 | Biomarker for auxiliary diagnosis of HIV negative Marneffei panuliferus disease and application thereof |
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