JP2022183646A - Method and kit for detecting genetic factor leading to serious conditions of covid-19 - Google Patents

Abstract

To provide novel methods and kits for detecting a genetic factor leading to serious conditions of COVID-19.SOLUTION: A method for detecting a genetic factor leading to serious conditions of COVID-19 includes detecting one or more single nucleotide polymorphisms in the region from the interleukin-17A gene locus to the interleukin-17F gene locus in a subject-derived DNA-containing sample. A kit for detecting a genetic factor leading to serious conditions of COVID-19 includes one or more nucleic acid probes or primers to detect one or more single nucleotide polymorphisms in the region from the interleukin-17A gene locus to the interleukin-17F gene locus.SELECTED DRAWING: None

Description

The present invention relates to methods and kits for detecting genetic factors of COVID-19 severity.

As of April 2021, the number of people infected with the new coronavirus infection (COVID-19) caused by SARS coronavirus-2 (SARS-CoV-2) in Japan has exceeded 530,000, and the number of deaths is 9. , more than 600 people.

A genome-wide association study (GWAS)-based genomic analysis is being conducted in an international collaboration with the aim of identifying genetic factors involved in the onset and severity of COVID-19. The COVID-19 Host Genetics Initiative (COVID-19hg) is a multi-center collaborative research institute established for the above purpose, involving 50 research institutes or groups from around the world. COVID-19hg reports international meta-GWAS results in a release dated 18 January 2021. The Japanese Coronavirus Task Force is also participating in COVID-19hg, and the international meta-GWAS lists 572 Japanese COVID-19 patients (of whom 155 are severe COVID-19 patients) and 1,705 Includes healthy subjects.

In the latest international meta-GWAS comparing COVID-19 patients with healthy subjects, LZTFL1 (leucine zipper transcription Factor like 1) gene, CCHCR1 (Coiled-coil alpha-herical rod protein 1) gene, FOXP4-AS1 (Forkhead box 4 protein -antisense RNA 1) gene, ABO gene, TMEM65 (transmembrane protein 65) gene, OAS (2', 5'-oligoadenylate synthase) family gene, KANSL1 (KAT8 regulatory NSL complex subunit 1) gene, DPP9 (Dipeptidyl peptidase 9) genes, and the IFNAR2 (Interferon alpha and beta receptor subunit 2) gene. In addition, seven genes, LZTFL1 gene, CCHCR1 gene, FOXP4-AS1 gene, VSTM2A (V-set and transmembrane domain containing 2A) gene, OAS family gene, TAC4 (tachykinin 4) gene, DPP9 gene, and IFNAR2 gene, are healthy significantly associated with COVID-19 critically ill patients compared to those with severe disease. However, no significant single nucleotide polymorphisms (SNPs) were found in the international meta-GWAS of COVID-19 severe versus COVID-19 mild patients. Furthermore, it has been reported that there is a core haplotype in the 3p21.31 gene cluster containing the LZTFL1 gene, which is associated with exacerbation of COVID-19, and that the core haplotype was born in Neanderthals and inherited by Homo sapiens ( For example, see Non-Patent Document 1, etc.).

Zeberg H et al, “The major genetic risk factor for severe COVID-19 is inherited from Neanderthals.”, Nature, Vol. 587, pp. 610-612, 2020.

Among COVID-19 patients, there are a certain number of severely ill patients who require oxygen inhalation and endotracheal intubation, but the genetic factors of the aggravation of COVID-19 in Japanese have not been clarified. In addition, no genetic factors satisfying the genome-wide significance level were detected in the international meta-GWAS, which compared COVID-19 severe patients with COVID-19 mild patients.

The present invention has been made in view of the above circumstances, and provides novel methods and kits for detecting genetic factors of exacerbation of COVID-19.

That is, the present invention includes the following aspects.
(1) A method for detecting a genetic factor of aggravation of COVID-19, comprising:
A method comprising detecting one or more single nucleotide polymorphisms present in a region from the interleukin-17A locus to the interleukin-17F locus in a DNA-containing sample derived from a subject.
(2) The method according to (1), wherein the single nucleotide polymorphism is one or more selected from the group consisting of accession numbers rs11962054, rs13192563, rs13192246, and rs9474169 in the NCBI SNP Database.
(3) A single nucleotide polymorphism at one or more loci selected from the group consisting of forkhead box protein 4-antisense RNA 1 locus and Interferon alpha and beta receptor subunit 2 loci in the subject-derived DNA-containing sample The method of (1) or (2), further comprising detecting.
(4) A kit for detecting genetic factors of aggravation of COVID-19,
A kit comprising one or more nucleic acid probes or primers that detect one or more single nucleotide polymorphisms present in the region from the interleukin-17A locus to the interleukin-17F locus.
(5) The kit according to (4), wherein the single nucleotide polymorphism is one or more selected from the group consisting of accession numbers rs11962054, rs13192563, rs13192246, and rs9474169 in the NCBI SNP Database.
(6) One or more nucleic acid probes or primers that detect single nucleotide polymorphisms at one or more loci selected from the group consisting of forkhead box protein 4-antisense RNA 1 loci and Interferon alpha and beta receptor subunit 2 loci The kit according to (4) or (5), further comprising

According to the method and kit of the above aspects, the genetic factor of the exacerbation of COVID-19 in a subject can be easily and reliably detected.

1 is a graph showing the age distribution of sCOVID-19 patients and mCOVID-19 patients in Example 1. FIG. FIG. 1 shows the results of GWAS for Japanese comparing all COVID-19 patients and healthy subjects in Example 1. FIG. FIG. 2 shows the results of the international meta-GWAS comparing all COVID-19 patients and healthy subjects in Example 2. FIG. FIG. 4 shows the results of the integrated analysis of Japanese GWAS and international meta-GWAS comparing all COVID-19 patients and healthy subjects in Example 2. FIG. 4 is a LocusZoom plot of 400 kb around major SNPs in three gene regions including FOXP4-AS1, ABO, and IFNAR2 in Example 1. FIG. FIG. 2 is a diagram showing the results of GWAS of Japanese, comparing severe COVID-19 (sCOVID-19) patients and healthy subjects in Example 1. FIG. FIG. 2 shows the results of the international meta-GWAS comparing COVID-19 (sCOVID-19) patients and healthy subjects in Example 2. FIG. FIG. 10 is a diagram showing the results of an integrated analysis of Japanese GWAS and international meta-GWAS comparing COVID-19 (sCOVID-19) patients and healthy subjects in Example 2. FIG. 2 is a LocusZoom plot of 400 kb around major SNPs in two gene regions containing FOXP4-AS1 and IFNAR2 in Example 2. FIG. FIG. 1 shows the results of GWAS for Japanese comparing COVID-19 (sCOVID-19) patients and mild COVID-19 (mCOVID-19) patients in Example 1. FIG. FIG. 2 shows the results of an international meta-GWAS comparing COVID-19 (sCOVID-19) and mild COVID-19 (mCOVID-19) patients in Example 2. FIG. FIG. 2 shows the results of a pooled analysis of Japanese GWAS and international meta-GWAS comparing COVID-19 (sCOVID-19) patients and mild COVID-19 (mCOVID-19) patients in Example 2. Fig. 3 is a LocusZoom plot of 400 kb around major SNPs in the gene region containing ILF17A and ILF17F in Example 2; eQTL data for three GWAS-identified SNPs comparing severe and mild COVID-19 patients.

Method and kit for detecting genetic factors of COVID-19 exacerbation according to one embodiment of the present invention (hereinafter sometimes abbreviated as "method of this embodiment" and "kit of this embodiment") details are described below.

<COVID-19>
As used herein, the novel coronavirus infection (COVID-19) is an abbreviation for Coronavirus disease 2019, and is an infectious disease caused by SARS coronavirus-2 (SARS-CoV-2) infection. SARS coronavirus-2 belongs to the β-coronavirus genus and is believed to be genetically closely related to coronaviruses isolated from the horseshoe bat genus.

<Method for detecting genetic factors of aggravation of COVID-19>
The method of the present embodiment is a method of detecting genetic factors for the severity of COVID-19,
Detecting one or more single nucleotide polymorphisms (SNPs) present in the region from the interleukin-17A (ILF17A) locus to the interleukin-17F (ILF17F) locus in a DNA-containing sample from the subject.

As shown in the examples below, the inventors used Illumina in 503 COVID-19 patients registered in the Biobank of the National Center for Global Health and Medicine and 1,276 Japanese healthy controls. Genome-wide SNP typing was performed using the company's Japanese Screening Array (JSA), and a genome-wide association analysis (GWAS) was performed to compare a group of patients with severe and mild SARS-Cov-2 infection. As a result, SNPs present in the region from the ILF17A locus to the ILF17F locus were identified as SNPs associated with aggravation of COVID-19. Examples of SNPs present in the region from the ILF17A locus to the ILF17F locus include accession numbers rs11962054, rs13192563, rs13192246, rs9474169, etc. in the NCBI SNP Database. Each of these SNPs may be used as a detection target alone, or two or more of them may be used as a detection target in combination.

Furthermore, as shown in the examples below, the inventors integrated the statistics of the international meta-GWAS published by COVID-19hg and the statistics of the Japanese GWAS, and found that from the ILF17A locus to the ILF17F locus The inventors found that the above SNP present in the region satisfies the genome-wide significance level, thereby completing the present invention.

According to the method of the present embodiment, genetic factors of aggravation of COVID-19 in a subject can be easily and reliably detected.

The subject-derived DNA-containing sample is not particularly limited as long as it is collected from the subject's body. Fluid, tears, sputum, mucus, lymph, ascites, pleural fluid, amniotic fluid, bladder lavage, bronchoalveolar lavage, hair, stool, cells or tissues directly collected from a living body, and the like, but are not limited thereto. It is preferable to extract DNA from these samples and use it as a sample to be used for detection, which will be described later.

The method for extracting DNA is not particularly limited, and any known method can be used for extraction. For example, the phenol/chloroform method, the cetyltrimethylammonium bromide (CTAB) method, and the like can be mentioned. A commercially available kit may be used for DNA extraction. Examples of such kits include Wizard Genomic DNA Purification Kit (manufactured by Promega) and the like.

Alternatively, the DNA may be cDNA synthesized by extracting mRNA and using the mRNA as a template. The method for extracting mRNA is not particularly limited, and it can be extracted using a known method. Examples include the guanidine isothiocyanate method. A commercially available kit may be used for the extraction of mRNA. Examples of such kits include NucleoTrap (registered trademark) mRNA Kit (manufactured by Clontech) and the like. The method for synthesizing cDNA is also not particularly limited, and it can be synthesized using a known method. For example, cDNA can be synthesized from RNA by reverse transcriptase-polymerase chain reaction (RT-PCR) using random primers or polyT primers.

Detection of the above SNPs present in the region from the ILF17A locus to the ILF17F locus can be detected using a known SNP detection method. For example, direct sequencing method, polymerase chain reaction (PCR) method, restriction fragment length polymorphism (RFLP) method, hybridization method, TaqMan (registered trademark) PCR method (hereinafter, the description of "registered trademark" is omitted) ), a method using mass spectrometry, and the like.

In the direct sequencing method, the region containing the SNP present in the region from the ILF17A locus to the ILF17F locus to be detected in the subject-derived DNA-containing sample is cloned into a vector or amplified by PCR, This is done by determining the base sequence of the region. As a cloning method, cloning can be performed by screening a cDNA library using an appropriate probe. Alternatively, it can be cloned by amplifying by PCR reaction using appropriate primers and ligating into an appropriate vector. Furthermore, it can be subcloned into another vector, but is not limited to these. Examples of vectors include pBlue-Script SK (+) (manufactured by Stratagene), pGEM-T (manufactured by Promega), pAmp (manufactured by Gibco-BRL), p-Direct (manufactured by Clontech), pCR2.1-TOPO (manufactured by Invitrogene ) and other commercially available plasmid vectors, virus vectors, artificial chromosome vectors, and cosmid vectors can be used. A known method can be used for determining the base sequence. Examples include, but are not limited to, manual sequencing using a radioactive marker nucleotide and automatic sequencing using a dye terminator. Based on the nucleotide sequence thus obtained, it is determined whether or not the specimen has the above SNP present in the region from the ILF17A locus to the ILF17F locus.

In the PCR method, an oligonucleotide primer that hybridizes only to the sequence having the above SNPs present in the region from the ILF17A locus to the ILF17F locus (hereinafter referred to as "primer for detecting SNPs in the region from the ILF17A locus to the ILF17F locus"). (sometimes referred to as ). As described above, since there are multiple SNPs in the region from the ILF17A locus to the ILF17F locus, the SNP detection primers for the region from the ILF17A locus to the ILF17F locus are primers that can detect all SNPs. A single primer may be used, or two or more primers capable of detecting each SNP may be used in combination. The primers are used to amplify the sample's DNA. If the primers for detecting SNPs in the region from the ILF17A locus to the ILF17F locus generate a PCR product, the specimen will have one or more of the SNPs present in the region from the ILF17A locus to the ILF17F locus. . If no PCR product is generated, it indicates that the sample does not have the SNP present in the region from the ILF17A locus to the ILF17F locus.

In the RFLP method, first, a region containing one or more of the SNPs present in the detection target region from the ILF17A locus to the ILF17F locus is amplified by PCR. This PCR product is then cut with a restriction enzyme appropriate to the region containing one or more of the above SNPs present in the region from the ILF17A locus to the ILF17F locus. The restriction enzyme-digested PCR products are separated by gel electrophoresis and visualized by ethidium bromide staining. Presence of one or more SNPs in the region from the ILF17A locus to the ILF17F locus in the sample by comparing the length of the fragment with the molecular weight marker and the PCR product not treated with restriction enzymes as a control. can be detected.

The hybridization method is based on the property that the sample-derived DNA hybridizes with a DNA molecule complementary thereto (for example, an oligonucleotide probe). is a method for determining the presence or absence of the above SNP. Various techniques for hybridization and detection, such as colony hybridization, plaque hybridization, Southern blotting, and other known hybridizations, can be used for this hybridization method. For detailed procedures of the hybridization method, see "Molecular Cloning, A Laboratory Manual 3rd ed." (Cold Spring Harbor Press (2001); especially Sections 6-7), "Current Protocols in Molecular Biology" (John Wiley, 1987; ); especially Section 6.3-6.4), "DNA Cloning 1: Core Techniques, A Practical Approach 2nd ed." (Oxford University (1995); especially Section 2.10 for hybridization conditions) can. Furthermore, hybridization can also be detected using a DNA chip. In this method, an oligonucleotide probe specific to the above SNP present in the region from the ILF17A locus to the ILF17F locus is designed and attached to a solid support. Then, a DNA sample derived from the specimen is brought into contact with the DNA chip to detect hybridization.

The TaqMan PCR method uses TaqMan probes and Taq polymerase specific to the above SNPs present in the region from the ILF17A locus to the ILF17F locus, and is a method in which SNP detection and SNP-containing region amplification are performed in parallel. be. A TaqMan probe is an oligonucleotide of about 20 bases labeled with a fluorescent substance at the 5' end and a quencher at the 3' end, and is designed to hybridize to the SNP site of interest. Taq polymerase has 5' to 3' nuclease activity. When a region containing the SNP site of interest is amplified using these TaqMan probes and PCR primers designed to amplify the region containing the SNP site of interest in the presence of Taq polymerase, the TaqMan probe is converted to template DNA in parallel with the amplification. hybridizes to the SNP site of interest. When the extension reaction from the forward primer side reaches the TaqMan probe hybridized to the template, the 5' to 3' nuclease activity of Taq polymerase cleaves the fluorescent substance bound to the 5' end of the TaqMan probe. . As a result, the liberated fluorescent substance is no longer affected by the quencher and emits fluorescence. Measurement of fluorescence intensity enables SNP detection.

As a method using mass spectrometry, for example, a SNP typing method applying MALDI-TOF/MS method may be combined with a primer extension method. This method enables high-throughput analysis, and by the steps of 1) PCR, 2) purification of PCR products, 3) primer extension reaction, 4) purification of extension products, 5) mass spectrometry, and 6) genotyping. To analyze. First, a region containing the SNP site of interest is amplified from genomic DNA by PCR. PCR primers are designed so as not to overlap with the SNP site bases. It is then purified by enzymatic removal using exonuclease and shrimp alkaline phosphatase or by ethanol precipitation. A primer extension reaction is then performed using a genotyping primer designed so that the 3' end immediately flanks the SNP site. The PCR products are denatured at elevated temperature and excess genotyping primers are added and allowed to anneal. When ddNTP and DNA polymerase are added to the reaction system and subjected to thermal cycle reaction, an oligomer one base longer than the genotyping primer is produced. Oligomers one base longer generated in this extension reaction differ according to alleles due to the above design of genotyping primers. The purified elongation reaction product is subjected to mass spectrometry and analyzed from the mass spectrum.

Other detection methods include SNP typing methods capable of high throughput, methods applying single-molecule fluorescence spectrometry, and the like. For example, MF20/10S (manufactured by Olympus) is a system that employs this method. Specifically, using a confocal laser optical system and a highly sensitive photodetector, complementary and non-complementary primers are used in an ultra-small area of about 1 femtoliter (1/1000 trillion liter). This is to measure and analyze the single-molecule-level translational diffusion time of fluorescent labeled primers amplified by the PCR method.

A method using a DNA chip is also one type of typing that allows high throughput. A DNA chip has many types of DNA probes arrayed and immobilized on a substrate, and a labeled DNA sample is hybridized on the chip to detect fluorescent signals from the probes.

An example of the SNP typing method using a gene amplification method other than the PCR method is the Snipper method. This method is a SNP typing method that applies the RCA (rolling circle amplification) method, which is a DNA amplification method in which complementary strand DNA is synthesized while DNA polymerase moves over circular single-stranded DNA as a template. The probe is an oligo DNA with a length of 80 bases or more and 90 bases or less, and contains sequences of 10 base lengths and 20 base lengths or less complementary to the vicinity of the 5' end and 3' end of the target SNP site at both ends, It is designed to anneal to the target DNA and become circular. Also, the 3' end of the probe is designed to have a sequence complementary to the target SNP site. If the 3' end of the probe is perfectly complementary to the target SNP site, the probe will be circularized, but if the 3' end of the probe is mismatched, the probe will not be circularized. The probe also has a backbone sequence of 40 to 50 nucleotides in length and contains sequences complementary to two types of RCA amplification primers.

Other examples of SNP typing methods using gene amplification methods other than the PCR method include, for example, typing methods using the UCAN method and the LAMP method.

The UCAN method is a method that applies the ICAN method, which is an isothermal gene amplification method developed by Takara Bio. The UCAN method uses DNA-RNA-DNA chimeric oligonucleotides (DRD) as primer precursors. This DRD primer precursor is designed such that the DNA at the 3' end is modified so that replication of the template DNA by DNA polymerase does not occur, and the RNA portion binds to the SNP site. When this DRD primer precursor is incubated with the template, the coexisting RNase H cleaves the RNA portion of the paired DRD primer only when the DRD primer and template are perfectly matched. As a result, the modified DNA is removed from the 3' end of the primer and a new one is formed, so that the elongation reaction by the DNA polymerase proceeds and the template DNA is amplified. On the other hand, if the DRD primer and template DNA do not match, RNase H will not cleave the DRD primer and no DNA amplification will occur. The amplification reaction after the perfectly matched DRD primer precursor is cleaved by RNase H proceeds by the ICAN reaction mechanism.

The LAMP method is a gene isothermal amplification method developed by Eiken Chemical, which defines six regions of the target gene (F3c, F2c, F1c from the 3' end, B3, B2, B1 from the 5' end). , using four types of primers (FIP primer, F3 primer, BIP primer, B3 primer) for the six regions. For the purpose of typing, only the target SNP site (1 base) is sufficient between F1 and B1, and the FIP primer and BIP primer are designed so that the 1 base of the SNP is at the 5' end. In the absence of SNPs, a DNA synthesis reaction occurs from the dumbbell structure, which is the origin structure of the LAMP method, and the amplification reaction proceeds continuously. When SNP is present, DNA synthesis reaction from dumbbell structure does not occur, and amplification reaction does not proceed.

The Invader method is a method that uses two types of non-fluorescent labeled probes (allele probe, invader probe), one type of fluorescent labeled probe (FRET probe), and Cleavase, which is an endonuclease, without using a nucleic acid amplification method. . Allele probes have a sequence complementary to the template DNA on the 3'-end side from the SNP site, and a sequence unrelated to the template DNA called a flap on the 5'-end side of the probe. The invader probe has a complementary sequence on the 5' end side from the SNP site of the template DNA, and the portion corresponding to the SNP site has any base. The FRET probe has a sequence complementary to the flap sequence on the 3' end side. One 5' end is labeled with a fluorescent dye and a quencher, but the FRET probe is designed to form a double strand intramolecularly and is usually quenched. When these are reacted with the template DNA, the 3' end (arbitrary base portion) of the invader probe penetrates into the SNP site when the allele probe forms a double strand with the template DNA. Cleavase recognizes the structure invaded by the base and cleaves the flap portion of the allele probe. When this released flap then binds to the complementary sequence of the FRET probe, the 3' end of the flap penetrates the intramolecular double-stranded portion of the FRET probe. Cleavase recognizes the structure in which the base of the flap penetrates into the FRET probe, and cleaves the fluorescent dye of the FRET probe, as in the case of the allele probe and the invader probe. As the fluorochrome moves away from the quencher, fluorescence is generated. If the allele probe does not match the template DNA, the specific structure recognized by Cleavase is not formed, and the flap is not cleaved.

When primers are used for SNP detection, they are designed to be suitable for the region to be amplified and the typing method. For example, it is preferable to be able to fully amplify the region, and sequences can be designed based on the sequences near the ends of the region. Techniques for designing primers are well known in the art, and primers that can be used in the method of the present embodiment satisfy conditions that allow specific annealing, such as length and base composition that allow specific annealing ( melting temperature). The length of the region to be amplified is not limited as long as it does not interfere with typing, and may be increased or decreased as appropriate depending on the detection method. Moreover, although a part of the region to be amplified contains the SNP site, there is no restriction on the position of the site in the region to be amplified, and it may be arranged at an appropriate position according to the detection method (typing method). Therefore, when designing primers, the positional relationship between the primer and the SNP site can be freely designed according to the detection method, and the region containing the SNP to be detected (for example, a continuous base length of 50 bases or more and 500 bases or less). As long as you do so, you can design primers while taking into account the characteristics of your typing method. The length that exhibits the function as a primer is preferably 10 to 100 bases, more preferably 15 to 50 bases, and even more preferably 15 to 30 bases. In designing, it is preferable to confirm the melting temperature (Tm) of the primer, which is the temperature at which 50% of any nucleic acid strand forms a hybrid with its complementary strand. In order for the template DNA and the primer to form double strands and anneal, the annealing temperature must be optimized. It is not desirable because it will cause Known primer design software can be used to confirm the Tm.

When probes are used to detect SNPs, they are designed to recognize SNP sites. In designing a probe, the SNP site may be recognized anywhere in the probe according to the typing method, and may be recognized at the end of the probe depending on the typing method. When the SNP detection polynucleotide is used as a probe, the length of the base sequence complementary to the genomic DNA is usually 15 to 200 bases, preferably 15 to 100 bases, and 15 to 50 bases. Although more preferred, it may be longer or shorter depending on the typing method.

In the method of this embodiment, when one or more of the above SNPs present in the region from the ILF17A locus to the ILF17F locus are detected in the specimen, COVID-19 due to SARS-CoV-2 infection may become severe. indicates that there is

The method of the present embodiment is directly linked to improving the accuracy of predicting the risk of aggravation of COVID-19. It becomes possible.

The presence of the above SNPs present in the region from the ILF17A locus to the ILF17F locus may be either homozygous or heterozygous. It is considered that the risk may be higher.

In the method of the present embodiment, in addition to the detection of the above SNPs present in the region from the ILF17A locus to the ILF17F locus, other SNPs and haplotypes related to the possibility of aggravation of COVID-19 are detected. may By detecting these SNPs and haplotypes in combination with the detection of the above SNPs present in the region from the ILF17A locus to the ILF17F locus, it is possible to determine the possibility of aggravation of COVID-19 with higher accuracy. and increase the reliability of diagnosis.

Other SNPs associated with potential severity of COVID-19 include, for example:
SNP in the LZTFL1 (leucine zipper transcription factor like 1) locus: accession number rs35081325 in the NCBI SNP Database;
SNP in the CCHCR1 (Coiled-coil alpha-herical rod protein 1) locus: accession number rs111837807 in the NCBI SNP Database;
SNP in FOXP4-AS1 (Forkhead box protein 4-antisense RNA 1) locus: accession number rs1853837 in NCBI SNP Database;
SNP in VSTM2A (V-set and transmembrane domain containing 2A) locus: accession number rs71525842 in NCBI SNP Database;
SNPs in OAS (2′,5′-oligoadenylate synthase) family loci: accession number rs2269899 in the NCBI SNP Database;
SNP in the TAC4 (tachykinin 4) locus: accession number rs77534576 in the NCBI SNP Database;
SNP in the DPP9 (Dipeptidyl peptidase 9) locus: accession number rs2109069 in the NCBI SNP Database;
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These SNPs may be detected singly or in combination of two or more. When these SNPs are detected in a sample, it can be determined that the sample may have aggravation of COVID-19.
Among them, as shown in the examples below, in addition to the international meta-GWAS that compared COVID-19 severe patients and healthy subjects, the relevance was also reproduced in the GWAS in Japanese, so the FOXP4-AS1 locus and SNPs at one or more loci selected from the group consisting of IFNAR2 loci are preferred.

In addition to the SNPs related to the presence or absence of the possibility of aggravation of COVID-19 described above, SNPs related to the presence or absence of the possibility of developing COVID-19 (or susceptibility to SARS-CoV-2) are detected. may

Examples of SNPs associated with the presence or absence of the possibility of developing COVID-19 (or susceptibility to SARS-CoV-2) include the following.
SNP at the LZTFL1 locus: accession number rs35081325 in the NCBI SNP Database;
SNP at the CCHCR1 locus: accession number rs111837807 in the NCBI SNP Database;
SNPs at the FOXP4-AS1 locus: accession numbers rs2894439, rs9367106, rs12660421, rs1853837, rs55889968, rs12175265, rs1886814, rs4714474, rs9381074 in the NCBI SNP Database;
SNP at the ABO locus: accession number rs8176719 in the NCBI SNP Database;
SNP in the TMEM65 (transmembrane protein 65) locus: accession number rs72711165 in the NCBI SNP Database;
SNPs in the OAS family locus: accession number rs10774671 in the NCBI SNP Database;
SNP in KANSL1 (KAT8 regulatory NSL complex subunit 1) locus: accession number rs1819040 in NCBI SNP Database;
SNP at the DPP9 locus: accession number rs2109069 in the NCBI SNP Database;
SNPs at the IFNAR2 locus: accession numbers rs2248420, rs17860142, rs3153, rs12482014, rs12482193, rs12482060, rs17860165 in the NCBI SNP Database.

These SNPs may be detected singly or in combination of two or more. When these SNPs are detected in a sample, it can be determined that the sample has the possibility of developing COVID-19 (or has susceptibility to SARS-CoV-2).
Among them, as shown in the examples below, in addition to the international meta-GWAS that compared all COVID-19 patients and healthy subjects, the relevance was also reproduced in the GWAS in Japanese, so the FOXP4-AS1 locus , ABO loci and IFNAR2 loci are preferred.

For example, SNPs related to the presence or absence of the possibility of aggravation of COVID-19 described above in the specimen and the possibility of onset of COVID-19 described above (or susceptibility to SARS-CoV-2) SNPs related to the presence or absence of If detected, determine that the specimen may have developed COVID-19 (or have susceptibility to SARS-CoV-2) and may have aggravated COVID-19 can be done.

Further, for example, an SNP related to the presence or absence of the possibility of aggravation of COVID-19 described above is detected in the specimen, and the possibility of developing COVID-19 described above (or susceptibility to SARS-CoV-2) If the SNP associated with the presence or absence of is not detected, the specimen has a low or no likelihood of developing COVID-19 (or has little or no susceptibility to SARS-CoV-2), It can be determined that there is a possibility of becoming severe in case of infection with SARS-CoV-2.

Also, for example, SNPs related to the presence or absence of the possibility of aggravation of COVID-19 described above are not detected in the specimen, and the possibility of developing COVID-19 described above (or susceptibility to SARS-CoV-2 ) is detected, the specimen may develop COVID-19 (or have susceptibility to SARS-CoV-2), but the possibility of aggravation of COVID-19 can be determined to be low or absent.

Also, for example, SNPs related to the presence or absence of the possibility of aggravation of COVID-19 described above in the specimen and the possibility of the onset of COVID-19 described above (or, related to the presence or absence of susceptibility to SARS-CoV-2) If none of the SNPs are detected, the specimen has a low or no likelihood of developing COVID-19 (or has little or no susceptibility to SARS-CoV-2), and COVID- It can be determined that the possibility of aggravation of 19 is low or not.

<Kit for detecting genetic factors of aggravation of COVID-19>
The kit of this embodiment contains one or more nucleic acid probes or primers that detect one or more SNPs present in the region from the IL17A locus to the IL17F locus. The kit of the present embodiment is useful as a test kit for the possibility of aggravation of COVID-19. The one or more SNPs present in the region from the IL17A locus to the IL17F locus include the same as those exemplified in the above "Method for detecting genetic factors of aggravation of COVID-19". In addition, with regard to nucleic acid probes or primers, a person skilled in the art would know that one or more of It can be produced as appropriate using the method described in the SNP detection method.

In addition to the nucleic acid probes or primers described above, the kit of this embodiment can further include reagents, positive controls, solvents and solutes commonly used for SNP typing. Examples of such reagents include deoxynucleotide triphosphates (dNTPs) and DNA polymerase. Solvents and solutes include, for example, distilled water, pH buffer reagents, salts, proteins, surfactants, and the like.

The nucleic acid probe or primer may contain a sequence unrelated to one or more SNPs present in the region from the IL17A locus to the IL17F locus. Also, the nucleic acid probe or primer may be a chimera of DNA and RNA. In addition, the nucleic acid probe or primer may be labeled with a fluorescent substance, a binding affinity substance such as biotin or digoxin, an enzyme, a radioisotope, a luminescent substance, or the like. Examples of fluorescent substances include fluorescamine and fluorescein isothiocyanate. Examples of enzymes include peroxidase, alkaline phosphatase, malate dehydratase, α-glucosidase, α-galactosidase and the like. Examples of radioactive isotopes include ¹²⁵ I, ¹³¹ I, ³ H, ¹⁴ C and the like. Luminescent substances include, for example, luciferin, lucigenin, luminol, and luminol derivatives.

In addition to the nucleic acid probes or primers described above, the kit of the present embodiment may also contain a reference sample, a detector, and the like for use as a comparison standard or for creating a standard curve. The detector includes, for example, spectrometers, radiation detectors, light scattering detectors, and the like, which can detect the label of the above nucleic acid probe or primer.

In addition to nucleic acid probes or primers that detect one or more SNPs present in the region from the IL17A locus to the IL17F locus, the kit of the present embodiment is related to the possibility of other COVID-19 exacerbation. It can further comprise nucleic acid probes or primers that detect SNPs or haplotypes. Examples of the SNPs and haplotypes include those exemplified in the above "Method for detecting genetic factors of aggravation of COVID-19". In addition, with regard to nucleic acid probes or primers for detecting these SNPs and haplotypes, those skilled in the art will be familiar with the above "Method for detecting genetic factors of aggravation of COVID-19" from the IL17A locus to the IL17F locus. It can be prepared as appropriate using the method described in the method for detecting one or more SNPs present in the region.

In addition to nucleic acid probes or primers that detect one or more SNPs present in the region from the IL17A locus to the IL17F locus, the kit of this embodiment includes the possibility of COVID-19 onset (or SARS-CoV-2 can further comprise nucleic acid probes or primers that detect SNPs associated with the presence or absence of susceptibility to Examples of the SNP include those exemplified in the above "method for detecting genetic factors of exacerbation of COVID-19". In addition, with regard to nucleic acid probes or primers for detecting these SNPs, those skilled in the art will understand that the region from the IL17A locus to the IL17F locus can be prepared as appropriate using the method described in the method for detecting one or more SNPs present in .

EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

<Material and measurement method>
[COVID-19 patients and clinical data]
Using data from a total of 503 adult COVID-19 patients admitted to the National Center for Global Health and Medicine or treated at home or in accommodation from January 30, 2020 to January 11, 2021, COVID-19 Presence or absence of underlying disease in -19 patients was obtained from questionnaire-based clinical information. In this example, COVID-19 patients were treated according to the latest (December 25, 2020) clinical guidelines of the Ministry of Health, Labor and Welfare, severe COVID-19 patients (sCOVID-19) and mild COVID-19 patients (mCOVID-19 ) were divided into two groups. Patients defined as sCOVID-19 presented with clinical signs of pneumonia (fever, cough, dyspnea, and rapid breathing) with any of the following symptoms; Peripheral oxygen saturation (SpO2) ≤93 at room temperature %, need for oxygen administration or ventilation, and need for support with an extracorporeal membrane oxygenation (ECMO) device. Patients defined as mCOVID-19 have various signs and symptoms of COVID-19 (e.g., fever, cough, sore throat, malaise, headache, muscle pain, nausea, vomiting, diarrhea, loss of taste and smell). but had no shortness of breath, dyspnea, or abnormal chest imaging. These standards are mostly consistent with international standards established by the NIH (https://www.covid19treatmentguidelines.nih.gov/). Permission for this analysis was approved by the ethics department of the National Center for Global Health and Medicine and written informed consent was obtained from all COVID-19 patients (NCGM-G-003472).

[Data from Japanese healthy subjects]
Of 1,273 healthy Japanese adults, 419 (Tokyo healthy controls, THC) who lived in the Tokyo area before the outbreak of COVID-19 were collected and used extensively for genomic analysis. Informed consent was obtained from all 419 individuals. The remaining 854 were purchased from the Japan Health Science Foundation (JHSF) as Pharma SNP Consortium (PSC) samples. Human immortalized B cell lines were generated from blood samples of approximately 1,000 Japanese volunteers and deposited with the Japan Research Bioresource Collection (JCRB)/JHSF, Health Science Research Resource Bank (HSRRB).

[Genome-wide genotyped and filtered samples]
All 1,776 genomic DNA samples from 503 Japanese COVID-19 patients and 1,273 Japanese healthy subjects were genetically screened using the Illumina Infinium Japanese Screening Array (JSA) as per the protocol. determined the type. Of the 1,776 samples, 1,759 samples had a genotype call rate of over 97% for all SNPs, meeting the criteria for accuracy evaluation of heterozygosity. Twenty-nine samples, including 9 COVID-19 patients and 20 PSC healthy individuals, were defined as close relatives (PI≧0.1) as a result of determining homogeneity and were excluded from further analysis. 75 samples, including 32 COVID-19 patients, 19 THC healthy subjects, and 24 PSC healthy subjects, were detected as outliers (IQR=1.5) by principal component analysis and excluded from further analysis. was done. Ultimately, 1,655 samples, including 462 COVID-19 patients and 1,193 healthy subjects (consisting of 400 THC individuals and 793 PSC individuals), used the first component and the second component. Identical clusters were formed by principal component analysis. The average call rate for all SNPs in 1,655 samples was 99.49% (97.25-99.73%).

[Genotype estimation (genotype imputation) and statistical analysis]
Genotype imputation was performed on filtered SNP array data using BEAGLE 5.1. VCF-formatted genotype data were processed to match the reference panel using the conform-gt program, then genotype imputation was performed using BEAGLE 5.1 with default settings. The reference panel used for genotype imputation was self-generated. This panel consisted of 2,493 individuals from the International 1000 Genomes, 820 individuals from the Human Genome Diversity Project, 278 individuals from the Simons Genome Diversity Project, 90 individuals from the Korean Personal Genome Diversity Project, and 90 individuals from the BioJapan Project. It consists of 9,338 haplotypes in 4,669 individuals from a diverse population including 026 individuals. Data from Biobank Japan are restricted access data approved from NBDC Human Data (JGAS000114), other data were downloaded from public databases. Variants (SNPs and indels) of low quality (DR2<0.5) were excluded and variants with a genotypic probability of less than 0.9, the reference value for genotypic accuracy evaluation, were considered missing values. Statistical analysis of COVID-19 onset, severity and variants used logistic regression. Variants with a minor allele frequency of less than 1% or a low genotypic call rate (<95%) were excluded from the analysis. Statistical analyzes were performed assuming additive genetic effects. Genome-wide association studies were performed using plink1.9.

[Integrated statistical analysis integrating Japanese GWAS and international meta-GWAS]
To combine the Japanese GWAS P-values with the international meta-GWAS P-values, the Stouffer's Z-score method was used in this analysis. Briefly stated, first, the P-value of the one-tailed right-sided test was based on the direction of association detected in the Japanese GWAS, the amount of the international meta-GWAS was calculated from the P-value of the test, then the standard normal Z-scores were calculated from the one-tailed right-tailed P-values using the cumulative distribution function. In addition, the Japanese GWAS and international meta-GWAS Z-scores were combined using the formula shown below.

A two-sided P value for each SNP was calculated from Z _combined obtained by the above formula. The beta coefficient (β) for each SNP was obtained as an inverse variance-weighted estimator from the formula below, and the standard deviation (SE) of β was obtained as the square root of the variance from the formula below.

[Example 1]
(Detection of genetic factors for exacerbation of COVID-19 in Japanese)
1. Clinical Characteristics of 503 Japanese COVID-19 Patients Of the 503 Japanese COVID-19 patients in this analysis, 19 patients recovered from COVID-19, although there was no information on its severity. rice field. In addition to age and sex, the presence or absence of six underlying diseases (hypertension, dyslipidemia, type II diabetes mellitus (TIIDM), bronchial asthma, hyperuricemia, and obesity) in 484 patients with severity information was shown. summarized in 1.

Of the 109 severe COVID-19 (sCOVID-19) patients, 86 were male and 23 were female, with an average age of 56.1 years, with the youngest at 27 and the oldest at 88. rice field. Meanwhile, of the 375 mild COVID-19 (mCOVID-19) patients, 178 were male and 197 were female, with an average age of 44.8 years, with the youngest being 20 and the oldest being 89. was. Of the 6 underlying diseases, 5 were more common in sCOVID-19 patients than in mCOVID-19 patients. Hypertension accounted for 41.3% (45 of 109) of sCOVID-19 patients and 14.4% (54 of 375) of mCOVID-19 patients. Dyslipidemia accounted for 23.9% (26/109) of sCOVID-19 patients and 12.0% (45/375) of mCOVID-19 patients. TIIDM accounted for 22% (24/109) of sCOVID-19 patients and 5.6% (21/375) of mCOVID-19 patients. Hyperuricemia accounted for 19.3% (21/109) of sCOVID-19 patients and 5.3% (20/375) of mCOVID-19 patients. Obesity accounted for 15.6% (17/109) of sCOVID-19 patients and 3.7% (14/375) of mCOVID-19 patients. An opposite trend was observed for bronchial asthma; accounting for 3.7% (4/109) of sCOVID-19 patients and 6.1% (23/375) of mCOVID-19 patients.
In addition to age and gender, we compared the frequency of six underlying diseases between the two groups of sCOVID-19 and mCOVID-19 patients, and seven items, excluding bronchial asthma, were statistically significant in univariate analysis. (See Table 2 below, where statistically significant P-values are in bold). In multivariate analysis, in addition to age and sex, hyperuricemia and obesity showed a statistically significant association with P<0.05 (see Table 2 below).

The age distribution of sCOVID-19 and mCOVID-19 patients is shown in FIG.
These results suggested that older men with underlying medical conditions are at increased risk of developing sCOVID-19.

2. GWAS using Japanese COVID-19 patients and healthy subjects
A genome-wide association study (GWAS) using putative genotypes from 462 Japanese COVID-19 patients and 1,193 healthy controls was performed in the following three comparisons.
i) all COVID-19 patients and healthy individuals;
ii) sCOVID-19 patients and healthy subjects;
iii) sCOVID-19 and mCOVID-19 patients.

Regression analysis used the sex deduced from the genotypes of chromosomes X and Y for the comparisons in i) and ii) above, and the deduced sex, age, and six underlying diseases for the comparisons in iii) above. Applied with or without. The analysis results of each GWAS of i) to iii) above are shown in FIGS. 2A, 3A, and 4A.

The SNP did not reach the genome-wide significance level (p=5e-08). However, as a top hit SNP, from the comparison in i) above, rs796171020 present on chromosome 6 (the closest gene is DDX39BP2) was identified with a P value of 1.73e-07 and an odds ratio (OR) of 2.22. (see FIG. 2A).
The comparison in ii) above identified rs76954434 (closest gene is LINC00355) located on chromosome 13 with a P-value of 7.81e-08 and an odds ratio (OR) of 10.4 (see Figure 3A).
The comparison in iii) above identified rs376628389 (closest gene is IL17A) located on chromosome 6 with a P value of 5.72e-07 and an odds ratio (OR) of 2.59 (see Figure 4A).

[Example 2]
(Integrated analysis integrating Japanese GWAS and international meta-GWAS)
International Meta-GWAS summary statistics are the latest COVID-19 GWAS results from public download sites supported by the NHLBI IntramuralResearchprogram and the NIHBiowulf High Performance Computing Cluster (https://grasp.nhlbi.nih.gov/COVID19GWASResults.aspx) downloaded as 7,885 hospitalized COVID-19 patients and 961,804 healthy controls performed at COVID-19 hg to conduct a pooled analysis of Japanese GWAS comparing all COVID-19 patients with healthy controls We downloaded the international meta-GWAS statistics (corresponding to the comparison in i) above) comparing The results are shown in Figure 2B.
For a pooled analysis comparing sCOVID-19 patients with healthy controls, also performed on COVID-19 hg, 4,336 very severe respiratory confirmed COVID-19 patients and 623, An international meta-GWAS statistic (corresponding to the comparison in ii) above) comparing 902 healthy subjects was downloaded. The results are shown in Figure 3B.
Also, for a pooled analysis comparing sCOVID-19 and mCOVID-19 patients, conducted on COVID-19hg, 269 very severe respiratory confirmed COVID-19 patients and 688 International meta-GWAS statistics (corresponding to comparisons in iii) above) comparing non-hospitalized COVID-19 patients were also downloaded. The results are shown in Figure 3B.

Since the number of international meta-GWAS samples is overwhelmingly larger than the number of Japanese GWAS samples, the P-value was integrated using the Stouffer Z-score method (see "Materials and measurement methods" above for details). (see "Integrated Statistical Analysis Integrating Japanese GWAS and International Meta-GWAS"). Odds ratios (ORs) cannot be estimated accurately because it is not a meta-analysis of individual studies, but whether the associations of SNPs detected in the Japanese GWAS or the international meta-GWAS are replicated, or , to see if there are any new SNPs that meet the genome-wide significance level. The analysis results of each GWAS of i) to iii), which integrate the Japanese GWAS and the international meta-GWAS, are shown in FIGS. 2C, 3C, and 4C.

As shown in FIG. 2C, three gene regions including FOXP4-AS1, ABO, and IFNAR2 showed significant association in pooled analysis. A LocusZoom plot of 400 kb around the major SNPs in these three gene regions is shown in FIG. 2D. Summary statistics showing significant associations in the pooled GWAS are shown in Table 3 below.

These three gene regions were originally detected in the international meta-GWAS, and it was confirmed that the association of these three gene regions was replicated in the Japanese GWAS. However, IFNAR2 was not significant in Japanese GWAS due to the small number of samples.

As shown in FIG. 3C, significant associations were detected from two gene regions including FOXP4-AS1 and IFNAR2. These were the same as those detected by comparison of all COVID-19 and healthy subjects. A LocusZoom plot of 400 kb around the major SNPs of the two gene regions is shown in Figure 3D. Summary statistics showing significant associations in the pooled GWAS are shown in Tables 4-1 and 4-2 below.

FOXP4-AS1 was not significant in the original international meta-GWAS, but met the genome-wide significance level by pooled analysis with the Japanese GWAS. SNP rs1853837, which is significant in the FOXP4-AS1 gene, was not among the SNPs detected in all COVID-19 patients compared to healthy controls, but was a high frequency risk allele in sCOVID-19 patients. The same trend was observed. Although the IFNAR2 gene was originally significant in the international meta-GWAS, the association was duplicated in the Japanese GWAS.

There were no SNPs meeting the genome-wide significance level in either the international meta-GWAS or the Japanese GWAS. However, pooled analysis showed that SNPs present in the gene region containing IL17A and IL17F met the genome-wide significance level (rs13192246, P=1.42e-08), as shown in FIG. 4C. All four SNPs that met the genome-wide significance level in the pooled analysis were shown to have high frequency of risk alleles in severe COVID-19 patients. A LocusZoom plot of 400 kb around the major SNP in the gene region is shown in FIG. 4D. Summary statistics showing important associations in the combined GWAS are shown in Table 5 below.

[Discussion]
The IL17A/IL17F gene region, which showed the lowest P-value in the GWAS comparing Japanese severe and mild COVID-19 patients, met the genome-wide significance level as a result of the integrated analysis with the international meta-GWAS. These results suggest that the association between COVID-19 severity and IL17A/IL17F genes is not unique to Japanese, but represents a new disease susceptibility gene detected by integrated analysis with the international meta-GWAS. was suggested.

FIG. 5 is eQTL data for three SNPs identified in GWAS comparing severe and mild COVID-19 patients.
As shown in Figure 5, risk associated with COVID-19 severity from eQTL data using the genotype-tissue expression (GTEx) database of three SNPs that met genome-wide significance levels in the IL17A/IL17F gene region. Carrying the allele was shown to significantly reduce IL17F mRNA expression levels.

However, the eQTL data corresponded only to the expression level in testis. Although direct comparison of IL17F mRNA expression levels in severe and mild COVID-19 patients is needed in the future, IL17F expression levels reported to be associated with the prevention of mucosal epithelial infection are higher than those in severe COVID-19 patients. So it is very interesting that the probability is significantly lower.

IL17 has been reported to be calculated from γδT cells and innate lymphocytes in addition to the helper T cell subset Th17, and is involved in various diseases such as multiple sclerosis, inflammatory bowel disease, and psoriasis.

There are reports of analyzes using knockout mice regarding the functional differences between IL17A and IL17F. IL176A, but not IL17F, plays a major role in the development of autoimmune diseases such as arthritis and allergic inflammatory reactions, and IL17F is equivalent to IL17Ato or protects against mucosal epithelial infections with Staphylococcus aureus and Citrobacter rondentium. , IL17F has been shown to play an important role.

Of the 9 genes found in the international meta-GWAS comparing all COVID-19 patients and healthy controls, 3 genes FOXP4-AS1, ABO and IFNAR2 were duplicated in the Japanese GWAS. SNPs present in these three gene regions met the genome-wide significance level in pooled analysis, but none of the SNPs in the IFNAR2 gene region were significant in Japanese GWAS (P>0.05). However, since the odds ratio (OR) of each SNP was in the same direction as the international meta-GWAS, the number of cases was small, and it was considered that the Japanese GWAS did not have sufficient power.

In the ABO gene region, only one SNP: rs8176719 was at the genome-wide significance level (see Fig. 2D), but this SNP is a well-known deletion that determines type O blood, and Japan with type O blood. It has been shown that even humans are less likely to develop COVID-19.

Six gene associations, including the LZTFL1 gene with the lowest P-value in the international meta-GWAS, were not replicated in the Japanese GWAS. Also, the SNPs genotyped in the international meta-GWAS were not included in the Japanese GWAS. There were no SNPs with P<0.01, although the ORs could not be directly compared. The SNP near the DDX39BP2 gene, rs796171020, which had the lowest P-value in the Japanese GWAS comparing all COVID-19 patients and healthy subjects, did not meet the genome-wide significance level even in the pooled analysis.

From the above results, it was shown that 3 of the 9 genes found in the international meta-GWAS also play an important role in the research and development of COVID-19 in Japanese.

FOXP4-AS1 and IFNAR2 genes also met the genome-wide significance level in pooled analyzes comparing severe COVID-19 patients and healthy controls. In particular, FOXP4-AS1 was not shown to be associated with severe COVID-19 in the international meta-GWAS, but an integrated analysis with the Japanese GWAS revealed an association with severe COVID-19. rice field.

We could not directly compare the international meta-GWAS SNPs and the Japanese GWAS SNPs by OR, but there were no SNPs with P<0.01.

These results suggest that three genetic factors, including the IL17F gene newly identified in this analysis, are involved in the development of severe COVID-19 in Japanese.

The analysis also revealed that the newly identified IL17F gene associated with COVID-19 severity was a common genetic factor across populations. All study samples were collected from Japanese COVID-19 patients at hospital discharge, so it was not possible to determine whether IL18F could be used as a marker of severity. It is expected that if a decrease in serum IL17F level is confirmed, it will become an effective and important serum marker for predicting the severity of COVID-19 patients. In the future, it is hoped that the efficacy of IL17F as a diagnostic marker will be examined by collecting samples from COVID-19 patients at hospitalization.

According to the method and kit of the present embodiment, genetic factors of COVID-19 exacerbation in subjects can be detected simply and reliably.

Claims (6)

A method for detecting genetic factors of COVID-19 severity, comprising:
A method comprising detecting one or more single nucleotide polymorphisms present in a region from the interleukin-17A locus to the interleukin-17F locus in a DNA-containing sample derived from a subject. The method according to claim 1, wherein the single nucleotide polymorphism is one or more selected from the group consisting of accession numbers rs11962054, rs13192563, rs13192246, and rs9474169 in the NCBI SNP Database. Detecting a single nucleotide polymorphism at one or more loci selected from the group consisting of forkhead box protein 4-antisense RNA 1 locus and Interferon alpha and beta receptor subunit 2 loci in the subject-derived DNA-containing sample. 3. The method of claim 1 or 2, further comprising: A kit for detecting genetic factors of COVID-19 severity, comprising:
A kit comprising one or more nucleic acid probes or primers that detect one or more single nucleotide polymorphisms present in the region from the interleukin-17A locus to the interleukin-17F locus. 5. The kit according to claim 4, wherein the single nucleotide polymorphism is one or more selected from the group consisting of accession numbers rs11962054, rs13192563, rs13192246, and rs9474169 in the NCBI SNP Database. It further comprises one or more nucleic acid probes or primers that detect single nucleotide polymorphisms at one or more loci selected from the group consisting of forkhead box protein 4-antisense RNA 1 loci and interferon alpha and beta receptor subunit 2 loci. , a kit according to claim 4 or 5.

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