Method for examining autoimmune disease

Abstract

Provided is a technique for examining an autoimmune disease. This method for examining an autoimmune disease involves (1) a step for examining the presence or absence of endogenous human herpesvirus 6B in genomic DNA of a biological sample sampled from a subject.

Description

How to test for autoimmune diseases

[0001] The present invention relates to a method for testing an autoimmune disease.

[0002] Many autoimmune diseases, such as systemic lupus erythematosus, pulmonary alveolar proteinosis, and multiple sclerosis, are difficult to diagnose using a single biomarker, and diagnosis is only possible by combining multiple hematological findings, histological findings, and clinical symptoms. However, there are many challenges to be overcome in diagnosis, such as the high invasiveness of histological testing and the high level of diagnostic expertise required, and the discovery of new biomarkers is desired.

[0003] Human herpesvirus 6 (HHSV-6) is known to be the causative agent of exanthema subitum. In some humans, HHSV-6 exists integrated into the host's genomic DNA (endogenous HHSV-6) (Non-Patent Document 1). However, the relationship between endogenous HHSV-6 and disease remains unclear.

[0004] Liu, X. et al. Endogenization and excision of human herpesvirus 6 in human genomes. PLoS Genet 16, e1008915 (2020).

[0005] An objective of the present invention is to provide a testing technique for autoimmune diseases.

[0006] In view of the above-mentioned problems, the present inventors have conducted extensive research and have found that the above-mentioned problems can be solved by (1) a method for testing for autoimmune diseases, which includes a step of examining the presence or absence of endogenous human herpesvirus 6B in genomic DNA of a biological sample collected from a subject. Based on this finding, the present inventors have conducted further research and have completed the present invention. Specifically, the present invention encompasses the following aspects.

[0007] Item 1. (1) A method for testing for an autoimmune disease, comprising the step of examining the presence or absence of endogenous human herpesvirus 6B in genomic DNA of a biological sample collected from a subject.

[0008] Item 2. The method according to Item 1, further comprising: (2a) determining that the subject is suffering from the autoimmune disease, has a high risk of developing the autoimmune disease, or has a high risk of the autoimmune disease becoming severe, if endogenous human herpesvirus 6B is detected in the step (1); and / or (2b) determining that the subject is not suffering from the autoimmune disease, has a low risk of developing the autoimmune disease, or has a low risk of the autoimmune disease becoming severe, if endogenous human herpesvirus 6B is not detected in the step (1).

[0009] Item 3. The method according to Item 1 or 2, wherein the autoimmune disease is at least one selected from the group consisting of systemic lupus erythematosus, pulmonary alveolar proteinosis, and multiple sclerosis.

[0010] Item 4. The method according to any one of Items 1 to 3, wherein the autoimmune disease is at least one selected from the group consisting of systemic lupus erythematosus and pulmonary alveolar proteinosis.

[0011] Item 5. The method according to Item 2, wherein the autoimmune disease used in assessing the risk of progression to severe disease is systemic lupus erythematosus.

[0012] Item 6. The method according to any one of Items 1 to 5, wherein the biological sample is at least one selected from the group consisting of body fluid, skin, and mucosa.

[0013] Item 7. The method according to any one of Items 1 to 6, which is applied to a Japanese population.

[0014] Item 8. A test agent for use in the method of any one of Items 1 to 7, comprising an agent for detecting endogenous human herpesvirus 6B.

[0015] Item 8A: Use of an agent for detecting endogenous human herpesvirus 6B for producing a test agent for use in the method according to any one of Items 1 to 7.

[0016] Item 8B: A detection agent for endogenous human herpesvirus 6B for use as a test agent in the method according to any one of Items 1 to 7.

[0017] Item 8C: Use of an agent for detecting endogenous human herpesvirus 6B as a test agent for use in the method according to any one of Items 1 to 7.

[0018] The present invention can provide a method for testing for autoimmune diseases, a test agent for autoimmune diseases, etc. The test according to the present invention uses the presence or absence of endogenous human herpesvirus 6B in the genomic DNA of a subject as an indicator, and therefore cells from any tissue or body fluid in a living body can be used as a test sample, and it is possible to determine the risk of developing a disease at an earlier stage.

[0019] The prevalence of eHHV-6A and eHHV-6B is shown. A scatterplot of the prevalence of eHHV-6A and eHHV-6B in subjects with autoimmune diseases and healthy controls is shown, with the marker size indicating the sample size for each disease. The red line indicates a prevalence of eHHV-6 = 1%. A significant positive correlation between eHHV-6B and SLEDAI scores is shown. The graph shows a boxplot of SLEDAI scores in SLE patients with and without eHHV-6B. The SLEDAI scores of subjects with eHHV-6B were significantly higher than those without eHHV-6B (mean SLEDAI scores for eHHV-6B-positive and -negative SLE patients = 30.5 and 6.0, respectively; Wald test P = 1.3 × 10). －8 The histogram shows the distribution of the number of SLE patients with and without eHHV-6B (top panel). The horizontal histogram shows the distribution of SLEDAI scores for SLE patients with and without eHHV-6B, using different colors (right panel).

[0020] In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0021] 1. Testing Method In one aspect, the present invention relates to (1) a method for testing for autoimmune diseases (sometimes referred to herein as the "testing method of the present invention"), which comprises the step of examining the presence or absence of endogenous human herpesvirus 6B in genomic DNA of a biological sample collected from a subject. This method is described below.

[0022] The type of autoimmune disease to be tested is not particularly limited. Autoimmune diseases of all classes, grades, and stages according to various classification standards for autoimmune diseases can be tested. Furthermore, the site of the lesion is also not particularly limited. From the viewpoint of test accuracy and the like, particularly preferred examples of autoimmune diseases include systemic lupus erythematosus, pulmonary alveolar proteinosis, and multiple sclerosis. In one embodiment of the present invention, the autoimmune disease is at least one selected from the group consisting of systemic lupus erythematosus and pulmonary alveolar proteinosis. Furthermore, from the viewpoint of the accuracy of determining the risk of aggravation, which will be described later, the autoimmune disease is particularly preferably systemic lupus erythematosus.

[0023] The subject is not particularly limited as long as it is a mammal that can serve as a host for human herpesvirus 6 B. The subject is preferably a human.

[0024] The condition of the subject is not particularly limited. Examples of the subject include a subject whose condition is unknown whether it has an autoimmune disease, a subject whose condition has already been determined by another method to have an autoimmune disease, a subject whose condition has already been determined by another method to not have an autoimmune disease, and a subject undergoing treatment for an autoimmune disease. The testing method of the present invention can, for example, determine the presence, risk of onset, and risk of aggravation of an autoimmune disease in a subject whose condition is unknown; the risk of aggravation in a subject who has an autoimmune disease; and the risk of onset and risk of aggravation in a subject who does not have an autoimmune disease. In this specification, "risk of onset" means the possibility of onset, and "risk of aggravation" means the possibility of aggravation.

[0025] The testing method of the present invention can be applied to a variety of races. In one embodiment, the testing method of the present invention can be applied to the Japanese population. The Japanese population is literally defined and is not particularly limited, and can be determined, for example, from the base sequence of mitochondrial genome DNA, the base sequence of the Y chromosome, etc. For example, whether or not a person is a Japanese population can be determined from whether or not they have a specific base sequence or the proportion of a specific haplogroup. As a more specific example, a population in which 20% or more (preferably 30% or more) of male subjects have the Y chromosome haplogroup D1a2a and 5% or more (preferably 10% or more) of male subjects have the Y chromosome haplogroup O1b2 can be defined as a Japanese population.

[0026] The number of subjects in a population to which the testing method of the present invention is applied is not particularly limited, but is, for example, 10 or more, preferably 50 or more, more preferably 100 or more, and even more preferably 1000 or more.

[0027] The biological sample is not particularly limited as long as it contains chromosomal genomic DNA contained in the nuclei of cells of a subject. Examples of biological samples include body fluids, skin, mucous membranes, and body tissues. Among these, body fluids, skin, and mucous membranes are preferred, and body fluids are more preferred, from the viewpoints of ease of collection and minimal invasiveness.

[0028] Examples of body fluids include blood, follicular fluid, menstrual blood, saliva, cerebrospinal fluid, synovial fluid, urine, tissue fluid, sweat, tears, etc. Examples of mucous membranes include oral mucosa, nasal mucosa, etc.

[0029] The biological sample may be one directly collected from a living organism, or may be a sample obtained by concentrating and purifying genomic DNA.

[0030] The biological sample may be used alone or in combination of two or more types.

[0031] Biological samples can be collected from subjects by methods known to those skilled in the art. For example, whole blood can be collected by drawing blood using a syringe or the like. It is desirable that blood be collected by a medical professional such as a doctor or nurse. Serum is a portion of blood from which blood cells and specific blood coagulation factors have been removed, and can be obtained, for example, as the supernatant after blood clotting. Plasma is a portion of blood from which blood cells have been removed, and can be obtained, for example, as the supernatant after centrifugation under conditions that do not cause blood clotting.

[0032] In step (1), the presence or absence of endogenous human herpesvirus 6B in the genomic DNA of a subject is examined. The present invention has found that the presence of endogenous human herpesvirus 6B enables a determination that the subject is suffering from an autoimmune disease, has a high risk of developing an autoimmune disease, or has a high risk of an autoimmune disease becoming severe, and that the absence of endogenous human herpesvirus 6B enables a determination that the subject is not suffering from an autoimmune disease, has a low risk of developing an autoimmune disease, or has a low risk of an autoimmune disease becoming severe. Based on this, a technology for testing for autoimmune disease by determining the presence or absence of endogenous human herpesvirus 6B has been developed.

[0033] Endogenous human herpesvirus 6B is the genomic DNA of human herpesvirus 6B inserted into the chromosomal genomic DNA of a subject, and is not particularly limited insofar as it is so.

[0034] The nucleotide sequence of the genomic DNA of human herpesvirus 6B is known (NCBI Reference Sequence: NC_000898.1) and is shown in SEQ ID NO:1.

[0035] The method for detecting the presence or absence of endogenous human herpesvirus 6B is not particularly limited as long as it is a method capable of specifically detecting DNA of a specific base sequence, and various known methods or methods based on such methods can be used, such as PCR (e.g., real-time PCR), DNA microarray, Southern hybridization, and DNA sequencing.

[0036] In the above method, DNA-binding molecules (e.g., primers, probes, etc.) can be used. The primer pair and probe can be synthesized based on the base sequence of endogenous human herpesvirus 6B. The base lengths of the primers and probes are not particularly limited. The base length of the primer can be, for example, 10 to 50 nucleotides, preferably 15 to 30 nucleotides. The base length of the probe can be, for example, 10 to 5,000 nucleotides, preferably 10 to 1,000 nucleotides, and more preferably 20 to 150 nucleotides.

[0037] The primer pair and probe may be made of natural nucleic acids such as RNA and DNA, or may be made of a combination of natural nucleic acids with chemically modified nucleic acids or pseudo nucleic acids, if necessary. Examples of chemically modified nucleic acids and pseudo nucleic acids include PNA (Peptide Nucleic Acid), LNA (Locked Nucleic Acid; registered trademark), methylphosphonate DNA, phosphorothioate DNA, and 2'-O-methyl RNA. Furthermore, the primer and probe may contain a fluorescent substance and / or a quencher substance, or a radioisotope (e.g., 32 P, 33 P, 35 The antibody may be labeled or modified with a labeling substance such as ATP (S), or a modifying substance such as biotin, (strept)avidin, or magnetic beads.

[0038] The labeling substance is not limited, and commercially available substances can be used. For example, fluorescent substances such as FITC, Texas, Cy3, Cy5, Cy7, Cyanine3, Cyanine5, Cyanine7, FAM, HEX, VIC, fluorescamine and its derivatives, and rhodamine and its derivatives can be used. Quencher substances such as AMRA, DABCYL, BHQ-1, BHQ-2, and BHQ-3 can be used. The labeling position of the labeling substance in the primer and probe can be determined appropriately depending on the properties of the modifying substance and the intended use. Generally, the 5' or 3' end is often modified. Furthermore, one primer and probe molecule may be labeled with one or more types of labeling substance. The design of the nucleotide sequences of primers and probes and the selection of labeling substances are well known and are disclosed in molecular biology experimental protocol books such as Molecular Cloning: A Laboratory Manual by Sambrook, J and Russell, DW (3rd ed., Cold Spring Harbor Laboratory Press, 2001).

[0039] The endogenous human herpesvirus 6B to be examined for its presence or absence in step (1) does not need to be full-length, and its length is not particularly limited as long as it allows the presence of human herpesvirus 6B genomic DNA to be determined with a certain probability. The length is, for example, 50 to 300 bases, preferably 80 to 200 bases.

[0040] Furthermore, the endogenous human herpesvirus 6B whose presence or absence is examined in step (1) may be a single contiguous region in the genomic DNA of human herpesvirus 6B, or may be two or more non-overlapping or partially overlapping regions in the genomic DNA of human herpesvirus 6B. The number of such regions may be, for example, 2 to 20 or 3 to 10.

[0041] Human herpesvirus 6A has a nucleotide sequence highly identical to that of human herpesvirus 6B. In step (1), as long as endogenous human herpesvirus 6B can be detected, the method may also be capable of simultaneously detecting endogenous human herpesvirus 6A. However, from the viewpoint of test accuracy, a method capable of specifically detecting endogenous human herpesvirus 6B (i.e., endogenous human herpesvirus 6A is difficult to detect or is not detected at all) is preferred. When endogenous human herpesvirus 6B is specifically detected, for example, the genomic DNA sequence of human herpesvirus 6B can be compared with the genomic DNA sequence of human herpesvirus 6A (NCBI Reference Sequence: NC_001664.4), and a sequence that is present in the genomic DNA sequence of human herpesvirus 6B but is absent or only partially present in the genomic DNA sequence of human herpesvirus 6A can be used as the detection target region.

[0042] The testing method of the present invention, which includes step (1), can provide information on the presence or absence of endogenous human herpesvirus 6B, which is a testing indicator for autoimmune diseases, and can thereby assist in assessing the risk of onset / aggravation of autoimmune diseases, diagnosing autoimmune diseases, and the like.

[0043] The test results obtained by the test method of the present invention including step (1) can be used to elucidate the pathology of autoimmune diseases, predict the prognosis of autoimmune diseases, stratify patients, select treatment methods (personalized medicine, treatment responsiveness), etc.

[0044] In one embodiment, the testing method of the present invention preferably comprises: (2a) a step of determining that the subject is suffering from the autoimmune disease, has a high risk of developing the autoimmune disease, or has a high risk of the autoimmune disease becoming severe, if the endogenous human herpesvirus 6B is detected in the step (1); and / or (2b) a step of determining that the subject is not suffering from the autoimmune disease, has a low risk of developing the autoimmune disease, or has a low risk of the autoimmune disease becoming severe, if the endogenous human herpesvirus 6B is not detected in the step (1).

[0045] 2. Diagnosis of Autoimmune Disease with Higher Accuracy When a subject is determined to have an autoimmune disease by the test method of the present invention including step (2), the autoimmune disease can be diagnosed with higher accuracy by combining the test method of the present invention with a step of having a doctor diagnose the autoimmune disease. Furthermore, since the test method of the present invention can detect autoimmune diseases more accurately, combining the test method of the present invention with the above step can more efficiently and accurately diagnose "the subject having an autoimmune disease."

[0046] 3. Prevention and Treatment of Autoimmune Diseases If a subject is determined to have an autoimmune disease using the test method of the present invention including step (2), the test method of the present invention can be further combined with the test method of the present invention. Alternatively, if a subject is diagnosed with an autoimmune disease as described above in "2. Diagnosis of Autoimmune Diseases with Higher Accuracy," the test method of the present invention can be combined with a step of having a doctor's diagnosis. (3) A step of treating the disease in the subject determined or diagnosed to have the autoimmune disease can be further combined with the test method of the present invention and a step of having a doctor's diagnosis. Furthermore, because the test method of the present invention can detect autoimmune diseases more accurately, combining the test method of the present invention with step (3) or the combination of the test method of the present invention with a doctor's diagnosis can more efficiently and reliably treat subjects suffering from an autoimmune disease. Furthermore, if a subject is determined to have a high risk of developing an autoimmune disease using the test method of the present invention including step (2), appropriate preventive measures can be taken.

[0047] The method for treating autoimmune diseases is not particularly limited, but a representative example is medication. The pharmaceuticals used in medication are not particularly limited, but examples include therapeutic agents for systemic lupus erythematosus such as nonsteroidal anti-inflammatory drugs and steroids, and therapeutic agents for multiple sclerosis such as interferon preparations, immunosuppressants such as fingolimod, α4 integrin antibodies, and CD20 antibodies. One, two, or three or more types of pharmaceuticals can be used in combination. Furthermore, whole lung lavage can be performed for pulmonary alveolar proteinosis.

[0048] Methods for preventing autoimmune diseases are not particularly limited, and examples include avoidance and / or suppression of onset triggering factors and / or exacerbating factors (specifically, for example, suppression of sun exposure, application of sunscreen, suppression of viral infections, suppression of stress factors, and avoidance / suppression of drugs such as hormones). These can be used singly, two or three or more types in combination.

[0049] 4. Test Agent In one aspect, the present invention relates to a test agent (sometimes referred to herein as the "test agent of the present invention") for use in the testing method of the present invention, which comprises an agent for detecting endogenous human herpesvirus 6B. This will be described below.

[0050] The detecting agent of the present invention is not particularly limited as long as it can detect endogenous human herpesvirus 6 B. Examples of the detecting agent include primers and probes for endogenous human herpesvirus 6 B.

[0051] The detection agent of the present invention may be modified, as long as its function is not significantly impaired. Modifications include the addition of labels such as fluorescent dyes, enzymes, proteins, radioisotopes, chemiluminescent substances, biotin, etc.

[0052] Fluorescent dyes suitable for use in the present invention include those typically used to label nucleotides for the detection and quantification of nucleic acids. Examples include, but are not limited to, HEX (4,7,2',4',5',7'-hexachloro-6-carboxylfluorescein, a green fluorescent dye), fluorescein, NED (trade name, manufactured by Applied Biosystems, a yellow fluorescent dye), 6-FAM (trade name, manufactured by Applied Biosystems, a yellow-green fluorescent dye), and rhodamine or its derivatives (e.g., tetramethylrhodamine (TMR)). Nucleotides can be labeled with fluorescent dyes using any suitable known labeling method (see Nature Biotechnology, 14, 303-308 (1996)). Alternatively, commercially available fluorescent labeling kits (e.g., Oligonucleotide ECL 3'-Oligolabeling System, manufactured by Amersham-Pharmacia) can be used.

[0053] The detection agent of the present invention can also be used by immobilizing it on any solid phase, and therefore the test agent of the present invention can be provided in the form of a substrate on which the detection agent is immobilized (for example, a microarray chip on which a probe is immobilized).

[0054] The solid phase used for immobilization is not particularly limited as long as it can immobilize polynucleotides, etc., and examples thereof include glass plates, nylon membranes, microbeads, silicon chips, capillaries, and other substrates. The immobilization of the detection agent to the solid phase is not particularly limited. For example, in the case of a microarray, a commercially available spotter (e.g., manufactured by Amersham) can be used. Immobilization methods are well known in the art depending on the type of immobilized probe (e.g., photolithographic technology (Affymetrix) or in situ synthesis of oligonucleotides using inkjet technology (Rosetta Inpharmatics)).

[0055] The primers, probes, etc. are not particularly limited as long as they selectively (specifically) recognize endogenous human herpesvirus 6B. Here, "selectively (specifically) recognize" means, for example, that endogenous human herpesvirus 6B is specifically amplified in PCR, but is not limited thereto, and may be any primers, probes, etc. that allow a person skilled in the art to determine that the detected or amplified product is derived from endogenous human herpesvirus 6B.

[0056] Specific examples of primers and probes include at least one selected from the group consisting of the polynucleotides described in (a) below and the polynucleotides described in (b) below: (a) a polynucleotide having at least 15 consecutive bases in the base sequence of endogenous human herpesvirus 6B and / or a polynucleotide complementary to said polynucleotide, and (b) a polynucleotide having at least 15 bases that hybridizes under stringent conditions to the base sequence of endogenous human herpesvirus 6B or a base sequence complementary thereto.

[0057] A complementary polynucleotide or complementary base sequence (complementary strand, reverse strand) refers to a polynucleotide or base sequence that is complementary to the full-length polynucleotide sequence of endogenous human herpesvirus 6B, or a partial sequence thereof having at least 15 consecutive bases in length (hereinafter, for convenience, these are also referred to as the "positive strand"). However, such a complementary strand need not necessarily be perfectly complementary to the base sequence of the target positive strand; it may also be complementary enough to hybridize with the target positive strand under stringent conditions. Here, stringent conditions can be determined based on the melting temperature (Tm) of the nucleic acid to which the complex or probe binds, as taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego, CA). For example, typical post-hybridization washing conditions include approximately 1x SSC, 0.1% SDS, and 37°C. It is preferable that the complementary strand maintains its hybridization state with the target positive strand even when washed under these conditions. While not particularly limited, more stringent hybridization conditions include approximately 0.5x SSC, 0.1% SDS, and 42°C, and even more stringent hybridization conditions include approximately 0.1x SSC, 0.1% SDS, and 65°C. Specifically, examples of such complementary strands include a strand consisting of a nucleotide sequence that is completely complementary to the nucleotide sequence of the target positive strand, and a strand consisting of a nucleotide sequence that shares at least 90%, preferably 95%, more preferably 98% or more, and even more preferably 99% or more identity with the target positive strand.

[0058] Primers, probes, etc. can be designed, for example, using various design programs based on the nucleotide sequence of endogenous human herpesvirus 6B. Specifically, candidate sequences for primers or probes obtained by applying the nucleotide sequence of endogenous human herpesvirus 6B to a design program, or sequences containing at least a portion of such sequences, can be used as primers or probes.

[0059] The base length of a primer, probe, or the like is not particularly limited as long as it has a length of at least 15 consecutive bases as described above, and can be appropriately set depending on the application. For example, when used as a primer, the base length can be, for example, 15 to 35 bases, and when used as a probe, the base length can be, for example, 15 to 35 bases.

[0060] The test agent of the present invention may contain a detection agent other than the detection agent of the present invention (e.g., a probe for detecting nucleic acids such as mRNA and miRNA, or an antibody). In this case, the test agent of the present invention may be a test agent that can test for other diseases or conditions in addition to autoimmune diseases. In this case, the detection agent of the present invention is included as a detection agent for testing for autoimmune diseases. From this perspective, in one aspect, the test agent of the present invention is a test agent for autoimmune diseases that contains a detection agent for testing for a target disease that consists of the detection agent of the present invention.

[0061] The test agent of the present invention may be in the form of a composition. The composition may contain other components as needed. Examples of other components include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, chelating agents, etc.

[0062] The test agent of the present invention may be in the form of a kit. In addition to the detection agent or the composition containing the same, the kit may contain other materials that can be used to detect endogenous human herpesvirus 6B in the body fluids of a subject. Specific examples of such materials include various reagents (e.g., buffer solutions), instruments (e.g., instruments for collecting, purifying, and isolating biological samples), and the like.

[0063] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0064] Example 1. Analysis of the Association Between Autoimmune Disease and Endogenous Human Herpesvirus 6. 1-1. Overview. A dataset of 3,422 samples from Japanese individuals was analyzed. Details of the sample characteristics, sample collection, and WGS (Whole Genome Sequencing) platform are described below. The WGS dataset consisted of 251 patients with systemic lupus erythematosus (SLE), 216 patients with pulmonary alveolar proteinosis (PAP), 36 patients with multiple sclerosis (MS), and 2,919 healthy controls (HC). Two bioinformatics pipelines were used to examine the presence or absence of endogenous human herpesvirus 6 (eHHV-6: the genomic DNA of human herpesvirus 6 inserted into the host's chromosomal genomic DNA). Briefly, WGS reads that did not map to the human reference genome were remapped to the eHHV-6 reference genome. Adjusted odds ratios (ORs) for the risk of each disease associated with eHHV-6 infection status were calculated using logistic regression and Fisher's exact function. For each disease, linear or logistic regression and Fisher's test were used to estimate the relationship between clinical indicators of each disease and eHHV-6 infection status.

[0065] <1-2. Subjects> Group A consisted of 867 Japanese individuals, consisting of 251 with SLE, 216 with PAP, 36 with MS, and 364 with HC. Group B consisted of genomic DNA from 429 unrelated Japanese HCs obtained from Epstein-Barr virus-infected B lymphoblast cell lines. Group C consisted of CRAM files obtained from WGS of 2,126 HCs. Principal component analysis was performed on the HapMap3 data. To confirm the East Asian genetic background, principal component analysis was also performed using SNP-array data from the same individuals.

[0066] <1-3. WGS Data Preprocessing> Genomic DNA was extracted from whole blood samples from individuals in population A. DNA samples from population A and population B were sequenced at Macrogen Japan Co., Ltd. (Japan). Sequencing libraries were prepared using reagents from the TruSeq DNA PCR-free Library Prep kit (Illumina Inc., USA) according to the manufacturer's protocol. 2 × 150 bp paired-end whole-genome sequencing was performed using HiSeqX or NovaSeq (Illumina Inc., USA). Sequenced reads were aligned to the human reference build hs37d5 using the BWA-MEM algorithm (version 0.7.13 or 0.7.8). Reads from the raw alignment were flagged for duplicates using Picard MarkDuplicates (version 2.10.10 or 2.17.2). The average number of aligned reads was 386 million per sample, corresponding to an average genome coverage of 18×. WGS CRAM files for Population C contained 370 million 2×150 bp paired-end reads per sample, aligned to hs37d5 with BWA-MEM (version 0.7.13) and followed with Picard MarkDuplicates (version 2.10.10), resulting in an average genome coverage of 17×. A total of 3,422 BAM or CRAM files were prepared for downstream analysis.

[0067] 1-4. Detection of eHHV-6A / B from WGS: 3,422 WGS datasets were analyzed for eHHV-6 detection using a previously developed bioinformatics pipeline for eHHV-6 detection and reconstruction (PLoS Genet 17, e1009324 (2021)). Briefly, reads not mapping to the human reference genome were extracted, and these reads were mapped to reference HHV-6A (NCBI accession NC_001664.4) and HHV-6B (NC_000898.1). Subsequently, duplicate flagging and PCR duplication removal were performed. The mapping depth and coverage of each virus were then calculated. A sample was considered to reflect eHHV-6 carriage if 5% of the HHV-6 genome was covered with a read depth of 2× or greater. The workflow described here has been compiled as a Python pipeline and is available in the following GitHub repository: https: / / github.com / shohei-kojima / integrated_HHV6_recon. The "main.py" in this repository contains the entire workflow described here.

[0068] 1-5. Statistical Analysis: Using a logistic regression model, we calculated the adjusted case-control odds ratio (OR) with 95% confidence interval (CI) for the risk of each disease according to eHHV-6 infection status. Next, we performed a multivariate logistic regression model with age and sex as independent variables. We also performed a two-tailed Fisher's exact test, with a Wald test P value of <0.05 and a Fisher test intermediate P value of <0.05 considered significant for each disease and healthy controls. All analyses were performed in R (version 4.0.5) using the R-packages MASS53 (version 7.3.58) and exact2x254 (version 1.6.6). For each disease, we used a linear or logistic regression model with age and sex as independent variables to estimate the relationship between clinical indicators of each disease and eHHV-6 infection status. When clinical indicators were binary, we also used Fisher's test. In the regression analysis, listwise deletion was applied for cases with missing values.

[0069] 1-6. Results The prevalence of eHHV-6 and its association with each disease are shown in Table 1 and Figure 1. No difference in the prevalence of eHHV-6A was observed in patients with any disease compared to HCs. However, the prevalence of eHHV-6B was significantly higher in patients with SLE, PAP, and MS compared to HCs (1.6%, 1.4%, and 2.8% vs. 0.21%; Fisher's test mid-P = 0.0060, 0.022, and 0.085, respectively). eHHV-6B was associated with SLE, PAP, and MS, with adjusted ORs of 6.9 (95% CI = 1.61-27.2, Wald test P = 0.0061) for SLE, 7.0 (95% CI = 1.43-27.7, P = 0.0075) for PAP, and 10.9 (95% CI = 0.55-71.6, P = 0.034) for MS. These results suggest that eHHV-6B carriage contributes to the risk of these diseases with a larger effect size than previously known genetic and environmental factors.

[0070] The association between eHHV-6B and clinical indicators of SLE is shown in Table 2 and Figure 2. eHHV-6B was significantly positively correlated with a high SLE Disease Activity Index (SLEDAI) score (mean SLEDAI = 30.5 and 6.0 in eHHV-6B-positive and -negative SLE patients; P = 1.3 × 10 －8 Three of the four eHHV-6B-positive SLE patients had very high SLEDAI scores (≥18), suggesting that eHHV-6B has a significant clinical impact on disease activity (Figure 2). Three of the four eHHV-6B-positive SLE patients were anti-RNP antibody positive, and the final patient was anti-Scl-70 antibody positive. Two eHHV-6B-positive SLE patients had other autoimmune diseases: one anti-RNP antibody-positive patient had scleroderma and Sjögren's syndrome, and the other anti-Scl-70 antibody-positive patient had scleroderma and antiphospholipid syndrome.

[0071]

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[0073] Study Example 2: Analysis of the Association Between Autoimmune Disease and Endogenous Human Herpesvirus 62 We conducted an independent, large-scale replication study of the association between SLE and eHHV-6 using WGS data from the NIH All of Us program. Because cohort participants were genetically diverse and SLE risk varies considerably by family, we used logistic regression models to perform association tests across the entire multifamily dataset of 121,494 individuals of European ancestry and 243,247 individuals. Specifically, we performed the following:

[0074] We developed a rapid screening method to identify potential eHHV-6 carriers from biobank-scale WGS datasets. This screening method detects HHV-6 reads by aligning unmapped reads with the HHV-6 reference genome. First, we use the "samtools view" command to extract unmapped reads stored at the end of BAM or CRAM files and convert the reads to fastq format. The number of unmapped reads varies between individuals and is affected by the mapping algorithm. To minimize computational burden, our screening approach does not use more than 1 million unmapped reads by default. To avoid mismapping of telomere repeats in the telomere repeat and HHV-6 reference genome, we filter reads containing at least one telomere repeat (TAACCC). After filtering potential telomeric reads, the sequences are aligned to the HHV-6A and HHV-6B reference genomes (NCBI accessions NC_001664.4 and NC_000898.1, respectively) using hisat2 with a relaxed mismatch penalty (--mp 2,1 --no-spliced-alignment). To reduce the computational burden, paired-end reads are treated as single-end reads when mapping to HHV-6. Then, reads that successfully map to HHV-6 are counted. To avoid counting non-HHV-6 reads that are partially aligned with HHV-6, reads without clipping or indels are counted. Finally, the number of screened reads and reads mapped to HHV-6 are output. This method is available as the "quick_check.py" script on GitHub: https: / / github.com / shohei-kojima / integrated_HHV6_recon. Because WGS reads are mapped to the human reference genome by DRAGEN, most CRAM files contain more than 1 million unmapped reads (typically about 15 million unmapped reads).To reduce the computational load, 1 million reads stored at the end of the CRAM file were used for screening. A total of 245,472 datasets were screened for the All of Us Controlled Tier Dataset v7. For all datasets in which the screening method detected six or more HHV-6 reads, the HHV-6 reconstruction method "main.py" in the integrated_HHV6_recon package was applied to detect eHV-6 carriers.

[0075] SLE was defined using diagnoses coded by ICD9 (710.0) and ICD10 (M32). To increase the reliability of the phenotype, subjects with two or more diagnoses on unique dates separated by at least one year were defined as cases. Regarding PAP, another autoimmune disease with significant eHHV-6B risk in the Japanese population, a replication study was not performed given its ultra-rare nature (<10 cases per million population worldwide). We included all subjects in the association test except for those with two or more copies of full-length eHHV-6, presumed to be "solo-DR." We used the following logistic regression model: SLE ~ eHHV-6 + age + age^2 + sex + top 10 genetic PCs. To avoid high type I error, score tests were performed using the saddle-point approximation implemented in the R package SPAtest (https: / / cran.r-project.org / web / packages / SPAtest / index.html; v3.1.2).

[0076] The results are shown in Table 3. Consistent with the Japanese discovery cohort, no association was observed with the prevalence of eHHV-6A in either population (P > 0.15). In contrast, eHHV-6B was associated with a higher risk of SLE, with adjusted ORs of 2.7 (95% CI = 1.54-4.76; P = 0.0020) in those of European ancestry and 2.4 (95% CI = 1.47-3.82; P = 0.0012) in those of mixed ancestry. These results indicate that eHHV-6B is associated with SLE in people of diverse ancestry.

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Claims

1. (1) A method for testing for autoimmune diseases, comprising a step of examining the presence or absence of endogenous human herpesvirus 6B in the genomic DNA of a biological sample collected from a subject.

2. moreover, (2a) determining that the subject is suffering from the autoimmune disease, has a high risk of developing the autoimmune disease, or has a high risk of the autoimmune disease becoming severe, when the endogenous human herpesvirus 6B is detected in the step (1); and / or (2b) determining that the subject is not suffering from the autoimmune disease, has a low risk of developing the autoimmune disease, or has a low risk of the autoimmune disease becoming severe, when the endogenous human herpesvirus 6B is not detected in the step (1). The method of claim 1 , comprising:

3. 2. The method according to claim 1, wherein the autoimmune disease is at least one selected from the group consisting of systemic lupus erythematosus, pulmonary alveolar proteinosis, and multiple sclerosis.

4. The method according to claim 1, wherein the autoimmune disease is at least one selected from the group consisting of systemic lupus erythematosus and pulmonary alveolar proteinosis.

5. The method according to claim 2, wherein the autoimmune disease in the assessment of the risk of progression is systemic lupus erythematosus.

6. The method according to any one of claims 1 to 5, wherein the biological sample is at least one selected from the group consisting of body fluid, skin, and mucosa.

7. The method according to any one of claims 1 to 5, which is applied to a Japanese population.

8. A test agent for use in the method according to any one of claims 1 to 5, comprising an agent for detecting endogenous human herpesvirus 6B.