Methods for predicting depression and depression testing kits

Abstract

The present invention aims to provide a method for predicting the risk of developing SITH-1 related disorders such as depression. [Solution] A method for acquiring data to predict the risk of developing SITH-1-related disease, comprising detecting gene polymorphisms in the region related to the expression of the human herpesvirus 6 (HHV-6) SITH-1 gene in a sample taken from a subject, A method for predicting a high risk of developing SITH-1-related disease when the aforementioned gene polymorphism is detected.

Description

Technical Field

[0001] The present invention relates to a method for predicting the risk of developing a disease in which Small protein encoded by the Intermediate stage Transcript of HHV-6-1 (SITH-1) of human herpesvirus 6 (HHV-6), such as depression, is a risk factor (hereinafter, also referred to as a SITH-1-related disease), and a test kit for SITH-1-related diseases.

Background Art

[0002] It is known that depression has a heritability of 30 to 50%, but no effective causative gene has been identified on the chromosome, and it is called "missing heritability". Stress-related diseases such as depression are caused by stress. Therefore, it is important to control stress to an appropriate amount, such as arranging a work environment with less stress, in order to prevent the onset of such diseases. However, since stress resistance (stress resilience) and vulnerability vary greatly among individuals, it is useful to know the stress resilience of each individual for disease prevention. If an individual can know their stress resilience, it is considered possible to prevent diseases caused by stress by adjusting the type and amount of work to keep the stress load at an appropriate level.

[0003] The present inventors have previously discovered SITH-1, which is a causative gene of depression, in the gene of human herpesvirus 6 (HHV-6), a virus that coexists with humans, and have found that the expression of SITH-1 protein causes depression (Non-Patent Document 1). The SITH-1 protein is a protein that enhances brain stress and brain inflammation. However, since HHV-6 is latent-infected in almost 100% of people, it is not possible to distinguish between people who are likely to develop depression in the future and those who are not based only on the presence or absence of SITH-1, which is a causative gene of depression, and it is not possible to predict the risk of developing depression.

Prior Art Documents

[0004] [Non-Patent Document 1] iScience 2020 Jun 26;23(6):101187 [Overview of the project] [Problems that the invention aims to solve]

[0005] The present invention aims to provide a method for predicting the risk of developing SITH-1 related disorders such as depression, and a testing kit for SITH-1 related disorders. [Means for solving the problem]

[0006] The inventors, through diligent research to solve the above problems, discovered that among HHV-6 cells that express SITH-1, a factor that enhances stress and brain inflammation, there are HHV-6 cells that are more likely to express SITH-1 and HHV-6 cells that are not. Furthermore, they found that the number of R1A sequences in the R1 region, a repeating element region, is related to the gene polymorphism of HHV-6 cells involved in SITH-1 expression. The present invention was completed by discovering for the first time that the risk of developing depression, inflammatory bowel disease (e.g., ulcerative colitis, Crohn's disease), Alzheimer's disease, and post-COVID-19 symptoms (sequelae) can be predicted before the onset of these diseases, with SITH-1 being a risk factor.

[0007] In other words, the present invention is as follows: [1] The development of SITH-1-related disease, including the detection of genetic polymorphisms in the region related to the expression of the human herpesvirus 6 (HHV-6) SITH-1 gene in samples taken from subjects. A method for acquiring data to predict risk, A method for predicting a high risk of developing SITH-1-related disease when the aforementioned gene polymorphism is detected. [2] The method according to [1], wherein the HHV-6 is HHV-6B. [3] The method according to [1] or [2], wherein the gene polymorphism is a repeat sequence polymorphism in a repeat element region. [4] The method according to [3], wherein the repeating element region is the R1 region. [5] The repeating sequence polymorphism in the R1 region repeats the R1A sequence represented by sequence number 8. The method described in [4], which is a polymorph. [6] The method according to [5], wherein the number of repeats of the R1A sequence is 17 or less. [7] The method according to any one of [1] to [6], wherein the sample is a saliva sample. [8] The method according to any one of [1] to [7], wherein the SITH-1 related disease is selected from the group consisting of depression, inflammatory bowel disease, Alzheimer's disease and post-COVID-19 symptoms. [9] A test kit for assessing the risk of developing SITH-1 related disease, A kit containing reagents for detecting genetic polymorphisms in the region related to the expression of the SITH-1 gene in human herpesvirus 6 (HHV-6).

[10] The kit according to [9], wherein the reagent is for detecting gene polymorphisms in the region related to the expression of the SITH-1 gene of HHV-6B.

[11] The kit according to [9] or

[10] , wherein the reagent is a reagent for detecting repeat sequence polymorphisms in a region related to the expression of the SITH-1 gene.

[12] The kit according to any one of [9] to

[11] , wherein the reagent is for detecting repeat sequence polymorphisms in the R1 region of the region related to the expression of the SITH-1 gene.

[13] The kit according to

[12] , wherein the reagent comprises a primer for amplifying the R1 region and a primer for sequencing the amplified R1 region.

[14] A kit for testing saliva samples, as described in any of [9] to

[13] .

[15] A kit according to any one of [9] to

[14] , wherein the SITH-1 related disease is selected from the group consisting of depression, inflammatory bowel disease, Alzheimer's disease, and post-COVID-19 symptoms. [Effects of the Invention]

[0008] Until now, the diagnosis of SITH-1-related diseases has been based on measuring the presence of antibodies against SITH-1 in the patient's blood. While this SITH-1 test can determine the expression level of SITH-1 at the time of blood collection, it has been difficult to predict future levels. The present invention allows for the examination of genetic polymorphisms in the region related to the expression of the SITH-1 gene of HHV-6, which remains latent in patients throughout their lives, thereby enabling the diagnosis of SITH-1-related diseases. This method makes it possible to predict the risk of developing SITH-1 related diseases, such as cerebrospinal fluid, from childhood. Furthermore, because saliva samples can be used as specimens, subjects can collect their own samples non-invasively. [Brief explanation of the drawing]

[0009] [Figure 1] Figure 1 shows the results of the analysis of repeat sequences present in the HHV-6B SITH-1 enhancer region. A shows the HHV-6B latent transcript and repeat element regions. The top shows the location and arrangement of the major repeat element regions, R1, R2, and R3, and the structure of the direct repeat (DR) terminals. The middle shows the HHV-6 latent transcript (H6LT). The bottom shows the locations of the R1, R2, and R3 regions in the SITH-1 mRNA. All mRNA figures are shown in orientation relative to the HHV-6 genome. Thin lines represent introns, and thick arrows represent exons. All exons and introns are drawn to scale. B shows the positional relationship between the PCR primer and the R1 region sequence. C shows the nucleotide sequence and structure of the repeat sequence in the R1 region of the HHV-6B HST strain. Sequences shorter than 12 nucleotides are underlined. D shows a representative example of a repeat sequence present in the R1 region. [Figure 2]Figure 2 shows the association between repeat sequences in the R1 region and patients with major depressive disorder (MDD). A compares the total number of repeat sequences in the R1 region between normal controls (NC) and MDD patients. B compares the number of R1A repeats between NC and MDD patients. C compares the number of R1B repeats between NC and MDD patients. D compares the anti-SITH-1 antibody titers between NC and MDD patients. E is a correlation diagram between the total number of repeat sequences in the R1 region and anti-SITH-1 antibody titers in NC and MDD patients. F is a correlation diagram between the number of R1A repeats and anti-SITH-1 antibody titers in NC and MDD patients. G is a correlation diagram between the number of R1B repeats and anti-SITH-1 antibody titers in NC and MDD patients. H shows the results of ROC analysis regarding the number of R1A repeats and the development of MDD. A, B, C, D: Mann-Whitney U test (bars indicate median). E, F, G: Spearman's rank correlation coefficients. [Figure 3] Figure 3 shows the relationship between the number of R1A repeats and the symptoms of MDD patients. A shows that the difference in anti-SITH-1 antibody titers in MDD patients differs depending on whether the number of R1A repeats is 17 or less or greater than 17. The bar represents the median. B shows that the score of item 3 of the HAMD (HAMD3) in MDD patients differs depending on whether the number of R1A repeats is 17 or less or greater than 17. C shows that the score of item 8 of the HAMD (HAMD8) in MDD patients differs depending on whether the number of R1A repeats is 17 or less or greater than 17. D shows that the score of item 12 of the HAMD (HAMD12) in MDD patients differs depending on whether the number of R1A repeats is 17 or less or greater than 17. A, B, C, D: Mann-Whitney U test. [Figure 4]Figure 4 shows the effect on gene expression of the repetitive R1A sequence related to MDD in the astrocytoma cell line U373. A shows the structure of the SEAP expression reporter plasmid, specifically indicating the positions of the NF-κB binding site, the TATA-like promoter (PTAL), the insertion site of the R1 region, the SEAP gene, and the SEAP Kozak sequence. B shows the comparison results of SEAP production when using a reporter plasmid inserted with the R1A sequence with 17 repeats, a reporter plasmid inserted with the R1B sequence with 17 repeats, and a reporter plasmid without the R1 region inserted. C shows the comparison results of SEAP production of plasmids inserted with the R1A sequence with 7, 12, or 17 repeats. D shows the comparison results of SEAP production of plasmids inserted with the R1B sequence with 7, 12, or 17 repeats. B: Tukey's multiple comparison test after one-way ANOVA. C, D: Dunnett's multiple comparison test after one-way ANOVA. [Figure 5] It is a figure comparing the anti-SITH-1 antibody levels in healthy subjects and Alzheimer's disease patients. [Figure 6] It is a figure comparing the anti-SITH-1 antibody levels in healthy subjects and ulcerative colitis patients. [Figure 7] It is a figure comparing the anti-SITH-1 antibody levels in healthy subjects and Crohn's disease patients. [Figure 8] It is a figure comparing the anti-SITH-1 antibody levels in healthy subjects and patients with long-term effects of COVID-19.

Mode for Carrying Out the Invention

[0010] <Method for Predicting the Risk of Developing SITH-1-Related Diseases> One embodiment of the present invention is a data acquisition method for predicting the risk of developing SITH-1-related diseases, which includes detecting gene polymorphisms in a region related to the expression of the SITH-1 gene of human herpesvirus 6 (HHV-6) in a sample collected from a subject, and it is a method in which when the gene polymorphism is detected, it is predicted that the risk of developing SITH-1-related diseases is high. The data obtained by this method can be used to assist doctors and others in predicting the risk of developing SITH-1-related diseases.

[0011] The present invention relates to a method for predicting the risk of developing SITH-1-related diseases by detecting gene polymorphisms in regions related to the expression of the SITH-1 gene of HHV-6. HHV-6 is classified into HHV-6A and HHV-6B, and either virus may be targeted in the present invention. Since HHV-6B infects almost all humans for the first time during infancy and then persists in a latent infection throughout life, it can be preferably used in the present invention. Since HHV-6B infects almost all humans for the first time during infancy and then persists in a latent infection throughout life, it can be preferably used in the present invention. It can be preferably used.

[0012] The gene polymorphisms in the regions related to the expression of the SITH-1 gene of HHV-6 detected by the present invention are not particularly limited as long as they can predict the risk of developing SITH-1-related diseases. Examples of gene polymorphisms include tandem repeat polymorphisms in a region containing a plurality of specific types of sequences repeated multiple times (also referred to as a repeat element region) in the latent gene coding region of HHV-6B. A tandem repeat polymorphism means a gene polymorphism caused by a difference in the number of repetitions of a base sequence. The inclusion of multiple repetitions means that the same sequence may be continuously repeated and included multiple times, or other sequences may be included between consecutive identical sequences. Also, a plurality of specific types of sequences may be randomly included. Typically, the length per unit of the repeat sequence is 12 bases, but one or more bases may be increased or decreased. Examples of the repeat element region in the latent gene coding region of HHV-6B include the R1, R2, and R3 regions. The gene polymorphisms detected by the present invention may be gene polymorphisms contained in any of the R1, R2, and R3 regions, but are preferably gene polymorphisms contained in the R1 region, which is upstream of exon 1 of the mRNA of the SITH-1 protein, a risk factor for depression, and partially overlaps with exon 1.

[0013] Examples of the repeat element region in the latent gene coding region of HHV-6B include the R1, R2, and R3 regions. The gene polymorphisms detected by the present invention may be gene polymorphisms contained in any of the R1, R2, and R3 regions, but are preferably gene polymorphisms contained in the R1 region, which is upstream of exon 1 of the mRNA of the SITH-1 protein, a risk factor for depression, and partially overlaps with exon 1.

[0014] The repeating sequence polymorphisms in the R1 region are not particularly limited, but examples include polymorphisms that repeat one or more sequences selected from the R1A to R1U sequences represented by sequence numbers 8 to 28, respectively. For example, a polymorphism that repeats the R1A sequence represented by sequence number 8. It is preferable to do so. For example, a high risk of developing SITH-1-related disease can be predicted by using as an indicator that the number of R1A repeats is 17 or less. In this case, the lower limit of the number of R1A repeats is not particularly limited, but may be any of 2 or more to 16 or more. For example The number of repetitions in the R1A sequence is 2 or more and 17 or less, 5 or more and 17 or less, 10 or more and 17 or less, 13 When the number is above and below 17, above and below 16 and below 17, or 17, a high risk of developing SITH-1-related disease can be predicted. Also, the number of repeats in the R1A sequence and the repeats of the R1B-R1U sequences, etc. The risk of developing SITH-1-related diseases can be predicted by combining the number of factors.

[0015] SITH-1-related diseases are not particularly limited, but examples include diseases selected from the group consisting of depression, inflammatory bowel disease (e.g., ulcerative colitis, Crohn's disease), Alzheimer's disease, and post-COVID-19 symptoms. Depression is preferred.

[0016] The subject is a human being.

[0017] The sample is not limited to any sample that can detect the region related to the expression of the HHV-6 SITH-1 gene. Although not definitively established, saliva samples are preferred because they can be collected non-invasively.

[0018] The method for detecting genetic polymorphisms can be carried out by methods known to those skilled in the art. The DNA in the sample is As a template, a gene polymorphism can be detected by amplifying a predetermined region by the PCR method and then performing sequencing using the DNA of the amplified predetermined region. PCR method For DNA amplification by the PCR method, standard methods can be used, but nested PCR may be used to enhance the specificity and yield of the target DNA.

[0019] For example, when detecting a gene polymorphism contained in the R1 region, using the DNA in the sample as a template, perform primary PCR using the primers of SEQ ID NOs: 1 to 2, and perform secondary PCR using the primers of SEQ ID NOs: 3 to 4 to amplify the R1 region. Then, perform sequencing using the sequencing primers of SEQ ID NOs: 5 to 6 on the amplified DNA of the R1 region, whereby the gene polymorphism can be detected. At this time, the reagent conditions and thermal profiles used for specific PCR and sequencing are not particularly limited and can be appropriately changed as desired.

[0020] <Kit for detecting the risk of developing SITH-1 related diseases> Another embodiment of the present invention is a kit for detecting the risk of developing SITH-1 related diseases, which contains reagents for detecting gene polymorphisms in a region related to the expression of the SITH-1 gene of human herpesvirus 6 (HHV-6). For details, reference can be made to the description in the section of <Method for predicting the risk of developing SITH-1 related diseases>.

[0021] As described in the above section of <Method for predicting the risk of developing SITH-1 related diseases>, the SITH-1 of HHV-6 The genetic polymorphisms detected in the region related to gene expression are preferably those in the region related to the expression of the SITH-1 gene in HHV-6B. Furthermore, it is preferable to detect repeat sequence polymorphisms in the region related to the expression of the SITH-1 gene, and more preferably to detect repeat sequence polymorphisms in the R1 region of the region related to the expression of the SITH-1 gene. For this reason, the kit of this embodiment preferably includes reagents for detecting these genetic polymorphisms.

[0022] For example, when detecting repeat sequence polymorphisms in the R1 region of the SITH-1 gene expression region, it is preferable that the detection reagent includes a primer for amplifying the R1 region and a primer for sequencing the amplified R1 region. Although not particularly limited, the primer for amplifying the R1 region is used when performing nested PCR, in the primary PCR. Using primers SEQ ID NOs. 1-2, and then primers SEQ ID NOs. 3-4 can be used in secondary PCR, allowing for more efficient amplification of the target DNA. It is possible to do so. Also, primers for sequencing the amplified R1 region DNA are Primers of sequence numbers 5-6 can be used.

[0023] Based on DNA sequencing results in the R1 region, for example, a high risk of developing SITH-1-related disease can be predicted if the number of repeats in the R1A sequence is 17 or less. In this case, the lower limit of the number of repeats in the R1A sequence is not particularly limited, but it may be any of the following: 2 or more to 16 or more. For example, if the number of repeats in the R1A sequence is 2 or more and 17 or less, 5 or more and 17 or less, or 10 or less... When the number of repeats in the R1A sequence and the number of repeats in the R1B-R1U sequence are above 17, above 13 and below 17, above 16 and below 17, or 17, the risk of developing SITH-1 related disease can be predicted to be high. The risk of developing SITH-1-related diseases can be predicted by combining this with the number of repetitions in sequences and other factors.

[0024] SITH-1-related diseases are not particularly limited, but examples include diseases selected from the group consisting of depression, inflammatory bowel disease (e.g., ulcerative colitis, Crohn's disease), Alzheimer's disease, and post-COVID-19 symptoms. Depression is preferred.

[0025] The sample to be tested should be one that can detect the region related to the expression of the HHV-6 SITH-1 gene. While not particularly limited, saliva samples are preferred because they can be collected non-invasively.

[0026] The kit of this embodiment may include any reagents or equipment other than the primers described above. While not particularly limited, examples include a positive control, PCR reagents (e.g., DNA polymerase, buffer, dNTPs, water, etc.), sequencing reagents (e.g., DNA polymerase, Examples include buffers, dNTPs, water, ddNTPs, fluorescent dyes, etc., and 96-well plates. Furthermore, it includes reagents and preservation solutions for extracting DNA from the sample to be tested, as well as sample collection equipment, etc. That's good too. [Examples]

[0027] The examples are provided for disclosure purposes only and are not intended to limit the scope of the invention. .

[0028] <cell> U373MG (human astrocytoma cell line, ATCC) and HEK293A (human fetal kidney cell line, Thermo Fisher) (Scientific) Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) The cells were cultured at 37°C and 5% CO2.

[0029] <Subject> In this study, 77 patients with mental disorders were enrolled. They were diagnosed by psychiatrists using the DSM-IV criteria. Among the 77 patients, 48 had depression, 18 had bipolar disorder, 7 had anxiety disorder, 1 had schizophrenia, 1 had somatic symptom disorder, 1 had epilepsy, and 1 was a healthy subject without a diagnosable disease. 77 people were used as NC. On the same day as blood and saliva collection, the depressive symptoms of all participants were evaluated using the Japanese version of the Beck Depression Inventory (BDI, range 0 - 63), which is a subjective measure of the depressive state. In addition, the objective depressive symptoms of MDD were evaluated by psychiatrists using the Japanese version of the Montgomery-Asberg Depression Rating Scale (MADRS, range 0 - 60). Subjects aged 65 or older were excluded, and NC with a BDI score of 17 or higher (the recommended cut-off value for screening by BDI) was also excluded.

[0030] <Purification of Viral DNA> Saliva samples for the analysis of HHV-6 DNA were collected using test tubes (Salivette; Sarstedt AG & Co.). After centrifugation, the flow-through was stored at -80°C until analysis. Viral DNA was extracted from 400 μL of saliva samples by automated separation using a BioRobot EZ1 workstation and an EZ1 Virus Mini Kit v2.0 (QIAGEN, Inc.) according to the manufacturer's protocol . The DNA was eluted with 90 μL of elution buffer.

[0031] <Measurement of HHV-6 Copy Number> Using the purified saliva DNA as a template, first-round PCR was performed under the following conditions. The amplification was carried out in a total volume of 50 μL containing 1 μL of KOD FX Neo DNA polymerase (TOYOBO), 25 μL of 2×PCR buffer for KOD FX Neo, 10 μL of 2 mM dNTP, 0.3 μL of 50 μM PCR forward primer, 0.3 μL of 50 μM PCR reverse primer, 4 μL of the extracted viral DNA solution, and 9.4 μL of PCR-grade water. The primers used ​The following are the names: H6R1_F2:5'-CAGCTTCTGTACTAATTGAGGTGTCATG(SEQ ID NO: 1)-3';H6R1_R2:5'-CGGAGTCATTCCAATCCTAGCTC(SEQ ID NO: 2)-3'. The thermal profile was performed for 20 cycles of 2 minutes at 94°C, followed by 10 seconds at 98°C and 60 seconds at 68°C. The PCR product was obtained using QIAquick PCR. The cells were purified using a kit (Qiagen) and eluted with 30 μL of Tris-EDTA buffer. Secondary PCR was then performed under the following conditions. The amplification reagent conditions were the same as for primary PCR. The primers used were as follows: H6R1_F1:5'-CACAAGTTTGAGGGGAAGGTATGTTGGAC(sequence) Number 3)-3';H6R1_R1:5'-CAAATCCAACAGTGGCAACAGCACCATG(Sequence ID 4)-3'. The thermal profile was performed for 45 cycles of 2 minutes at 94°C, followed by 10 seconds at 98°C and 30 seconds at 68°C. Secondary PCR The PCR products were subsequently subjected to electrophoresis. For those in which a band was observed by electrophoresis, the PCR products were purified using the QIAquick PCR purification kit and eluted with 30 μL of Tris-EDTA buffer. The purified PCR products were sequenced using the BigDye 3.1 kit (Thermo Fisher Scientific Inc.) and the Applied Biosystems 3730xl DNA Analyzer (Thermo Fisher Scientific Inc.) according to the manufacturer's protocol. The primers used were as follows: H6R_Seq1: 5'-GCACTTCTAACGGTGATGAATGTAC (SEQ ID NO: 5)-3'; H6R_Seq2: 5'-CCAAACTAATGGCGAAAACTCTG (SEQ ID NO: 6)-3'.

[0032] <Measurement of anti-SITH-1 antibody titer> The anti-SITH-1 antibody titer was measured by a known method. The fusion protein N-SITH-CAML-C of SITH-1 and CAML was used as an antigen. HEK293T cells were cultured on a Lab-Tek chamber slide (Nunc), and pCMV-N-SITH-CAML-C was transfected using Lipofectamine LTX Reagent with Plus Reagent (Thermo Fisher Scientific). Two days after transfection, HEK293T cells expressing SITH1-CAML were fixed with an acetone-methanol solution. As a control, cells transfected with pCMV-CAML were prepared according to the same procedure. Plasma samples were diluted 1:80 with PBS (containing 2% BSA and 0.05% Tween 20) and stained by indirect immunofluorescence test. The samples were reacted on the slide at 37 °C for 1 hour. After washing with PBS (containing 0.05% Tween 20), the slide was reacted with Alexa Fluor 488 goat anti-human antibody diluted 200-fold with PBS (containing 2% BSA and 0.05% Tween 20) at 37 °C for 30 minutes. After washing with PBS (containing 0.05% Tween 20), a coverslip was placed on the slide. All samples were observed under the same conditions with an Olympus BX51 microscope equipped with a DP-73 CCD camera. The fluorescence intensity of some stained cells in each well was analyzed using ImageJ (NIH), and the average was calculated. The antibody titer was shown as the ratio of [the value obtained by subtracting the AFI of untransfected HEK293T cells from the average fluorescence intensity (AFI) of HEK293T cells transfected with pCMV-N-SITH-CAML-C] to [the value obtained by subtracting the AFI of untransfected cells from the AFI of HEK293T cells transfected with pCMV-CAML]. <Preparation of HHV-6 R1 SEAP plasmid>

[0033]

[0034] pNFκB-SEAP vector (Takara Bio Inc.) for herpes simplex virus thymidinequina Downstream from the HSV-TK minimal promoter, there are two TATA-like promoter (pTAL) regions. Restriction sites XhoI and Sse8387I were inserted using standard methods. Sequences with 7, 12, or 17 repeats of either the R1A sequence: GAGGCCCTGCTG (SEQ ID NO: 8) or the R1B sequence: GAGGCTCTGCTG (SEQ ID NO: 9), each having an XhoI site at 5' and a Sse8387I site at 3', were artificially synthesized using a commercial DNA synthesis service. These sequences were inserted into the pNFκB-SEAP vector using XhoI and Sse8387I. .

[0034] <SEAPアッセイ> In assays using U373MG cells, the Nucleofector (Lonza, Basel, Switzerland) and Cell Line Nucleofector Kit V (Program T-20) (Lonza) were used, with a result of 1 × 10⁶ 6 Each cell was given 2 μg of SEAP reporter plasmid and 0.5 μg of pMetLuc-Control vector as nucleofects. The cells were incubated for 48 hours. HEK293A cells. For this, following the manufacturer's instructions, use Lipofectamine LTX Reagent with PLUS Reagent (Thermo Fisher Scientific Inc.) to combine 0.5 μg of SEAP reporter plasmid and 50 ng of pMetLuc-Control plasmid in 1 × 10⁶ units. 5 The drug was co-introduced into individual cells. The introduced cells were kept for 48 hours. Incubate and use secreted luciferase expressed in the pMetLuc-Control vector, The delivery efficiency was standardized. SEAP activity was measured using the Great EscAPe SEAP Chemiluminescence Kit 2.0 (Takara Bio Inc.). Secretory luciferase activity was measured using the Secretory Luciferase Reporter System (Takara Bio Inc.). SEAP and secretory luciferase activity were measured for 1 second using the TriStar LB941 luminometer (Berthold Technologies).

[0035] <Quantification and Statistical Analysis> To test the normality of the data, we used the Shapiro-Wilk W test. (Parametric) For the comparison between the two groups, an independent t-test was used. For the non-parametric comparison between the two groups, The Mann-Whitney U test was used. For comparisons by sex, the chi-squared test was used. For comparisons between three groups, one-way analysis of variance (ANOVA) was used. Furthermore, if a significant difference was found, further analysis was performed as needed. For post-hoc testing, Tukey's multiple comparison test or Dunnett's multiple comparison test was used. Relationships between variables were evaluated using Spearman's rank correlation coefficient. A p < 0.05 value was considered statistically significant. Statistical analysis was performed using SPSS Statistics 21 (IBM Corporation) for Windows and Prism 9 (GraphPad Software) for macOS.

[0036] <Example 1> Analysis of repeating sequences present in the HHV-6B SITH-1 enhancer region HHV-6B has repeating element regions R1, R2, and R3 in its latent gene coding region, which can cause genetic polymorphisms. The R1 region is related to the SITH-1 protein, a risk factor for depression. It is located upstream of exon 1 of the mRNA and partially overlaps with exon 1 (Figure 1A). Therefore, we analyzed the structure of the R1 region of HHV-6B during the incubation period in MDD patients and normal controls (NC). HHV-6 is frequently reactivated and released into saliva during the incubation period, so double nesting HHV-6B DNA in saliva was amplified using an imager (Figure 1B). The subjects were 77 psychiatric outpatients (46 of whom had MDD) and 77 non-cancerous individuals (NCs). PCR amplification was performed on 33 MDD patients and 62 NCs. This can be done, and direct sequencing of the amplified products can be performed on 28 MDD patients and 35 NC patients. It's here. Gel electrophoresis of the PCR products revealed a single band in 30 of the 33 MDD patients and in 61 of the 62 NC patients. As a control, other diseases (such as bipolar disorder and schizophrenia) were used. HHV-6B DNA was amplified in psychiatric outpatients with these conditions, but no amplification products specific to these diseases were detected. There were no significant differences in age, sex ratio, BMI, or salivary HHV-6B DNA levels between MDD patients and NC (non-cancerous neuropathy) in direct sequencing, but antibody titers against SITH-1 were significantly higher in MDD patients. Ta. We found that the R1 region (SEQ ID NO: 7) of the HST strain, a representative HHV-6 virus strain, contains more than 21 types of repeat sequences formed by 12 nucleotides (Figure 1C). These identified sequences were named R1A to R1U (SEQ ID NOs: 8 to 28) respectively (Figure 1D). In the R1 region sequencing of 28 MDD patients and 35 NC patients, which were directly sequenced, the sequences named R1A and R1B were identified. It was shown that the number of repetitions in this array is far greater than that of other arrays.

[0037] <Example 2> Relationship between repeating sequences in the R1 region and depression To identify repeat sequences and gene polymorphisms in the R1 region associated with MDD, The relationship between structure and MDD was investigated. There was no significant difference in the total number of repeat sequences in the R1 region between NC and MDD patients (Figure 2A), but the number of R1A repeats was significantly lower in MDD patients (Figure 2B). However, there was no significant difference in the number of R1B repeats between NC and MDD patients (Figure 2C), and there were no significant differences in the 19 types of R1C-R1U sequences or other minor elements. Antibody titers for SITH-1 were significantly higher in MDD patients than in NC patients (Figure 2D), which was previously observed. This is consistent with the reported association between anti-SITH-1 antibodies and MDD (Non-Patent Literature 1). Analysis of the correlation between the total number of repeat sequences and the anti-SITH-1 antibody titer revealed no correlation between them (Figure 2E), however, there was a negative correlation between the number of R1A repeats and the anti-SITH-1 antibody titer. A correlation was observed (Figure 2F). There was a correlation between the number of R1B sequence repeats and the anti-SITH-1 antibody titer. It could not be seen (Figure 2G). The results above indicate that a low number of R1A repeats is associated with the development of MDD. In this regard, receiver operating characteristic (ROC) analysis showed that 67.9% of MDD patients and 28.6% of non-cancerous cells (NCs) had 17 or fewer R1A repeats, with an odds ratio of 5.28 (Figure 2H).

[0038] <Example 3> Relationship between the number of R1A repeats and symptoms in patients with depression The number of R1A repeats in MDD patients is lower than in NC patients, and ROC analysis showed that the number of repeats is 17 or less. A difference was shown between the case where it is below and the case where it exceeds 17. (R1A sequence repeat count) Furthermore, a negative correlation was shown between the anti-SITH-1 antibody titer, which reflects the SITH-1 expression level (Figure 2F). In fact, when the number of repeats of the R1A sequence is 17 or less, the anti-SITH-1 antibody titer is negatively correlated with the R1A sequence The rate was significantly higher compared to cases where the number of repetitions exceeded 17 (Figure 3A). When evaluating the symptoms of MDD patients according to the Hamilton Depression Rating Scale (HAMD-17), the score of HAMD item 3 (HAMD3), an indicator of suicidal tendency or ideation, and the score of HAMD item 8 (HAMD8), an indicator of psychomotor inhibition, are when the number of R1A repetitions is 17 or less. The scores were significantly higher (Figures 3B-3C). Furthermore, the score for item 12 of the HAMD (HAMD12), an indicator of gastrointestinal physical symptoms, tended to be higher when the number of R1A repetitions was 17 or less. (Figure 3D). No significant differences were found in other HAMD items. Model mice expressing SITH-1 in the olfactory bulb, the latent site of HHV-6B, exhibit depressive symptoms. Studies on the pathogenesis of these model mice have shown that SITH-1 causes depressive symptoms by enhancing the hypothalamic-pituitary-adrenal (HPA) axis response, and this enhanced HPA axis activity is known to be associated with suicidal tendencies, psychomotor depression, and gastrointestinal somatic symptoms. Based on these findings, the repetition of the R1A sequence transmitted to offspring through familial transmission is being investigated. The number of gene polymorphisms is thought to increase HPA axis activity through increased SITH-1 expression. As a result, in MDD patients with 17 or fewer R1A repeats, the number of R1A repeats is 17. It is believed that suicidal tendencies, psychomotor retardation, and gastrointestinal symptoms were more pronounced in patients exceeding a certain threshold.

[0039] <Example 4> Association between the number of R1A repeats and family history of depression in patients To investigate the impact of R1A sequence polymorphisms on the heritability of MDD, the association between the number of R1A sequence repeats and family history was examined in MDD patients. The results showed that 47.4% of MDD patients with 17 or fewer R1A sequence repeats had a family history of MDD, whereas MDD patients with more than 17 repeats... It was found that none of the participants had a family history of MDD. Fisher's exact test yielded a p-value of 0.026 (Table 1). There were no significant differences between the two groups in terms of age or sex. Since family history is associated with the heritability of MDD, it was shown that it is important to consider the effect of the number of R1A repeats when studying the inheritance of MDD. [Table 1]

[0040] <Example 5> Effect of R1A sequence on gene expression in astrocytoma cell line U373 When the relationship between the R1A sequence and MDD was investigated, it was found that the R1A sequence suppresses the production of SITH-1 protein. It was shown that it has the function of doing so. Because a typical promoter sequence was not found upstream of the SITH-1 mRNA transcription start site. A reporter plasmid was constructed containing a highly versatile NF-κB cis-element and a secreted alkaline phosphatase (SEAP) gene downstream of a TATA-like promoter. To reproduce the situation where the R1A sequence partially overlaps with the upstream region of exon 1 of SITH-1 mRNA, the R1A sequence was inserted immediately before the translation initiation signal of the Kozak consensus sequence of the SEAP gene (Figure 4A). The astrocytoma cell line U373 was used for the reporter assay to analyze the function of the R1A sequence. Astrocytes are the site where HHV-6B expresses the SITH-1 protein. The branching point for the number of R1A repeats between MDD patients and NC is 17, therefore the number of R1A repeats We investigated SEAP production of a reporter plasmid into which a sequence with R1A was inserted. As a result, R1A It was shown that inserting a sequence with 17 repeats in the column significantly suppressed SEAP production. Furthermore, reporter-plus sequencing with a sequence containing 17 repeats in the R1B sequence was also performed. It was shown that using Mido resulted in a weaker inhibitory effect on SEAP production compared to using R1A. (Figure 4B). To investigate the relationship between the number of R1A repeats and the suppression of SEAP production, the reporter plus The effects of Mido on SEAP production were compared. There was no significant difference in the reporter plasmid's ability to suppress SEAP production when a sequence with 7 repeats in the R1A sequence was inserted compared to when a sequence with 12 repeats in the R1A sequence was inserted. On the other hand, when a sequence with 17 repeats in the R1A sequence was inserted, the reporter plasmid's ability to suppress SEAP production was significantly suppressed (Figure 4C). A similar analysis was performed on the R1B sequence, and it was found that when a sequence with 7 repeats in the R1B sequence was inserted... When a column is inserted, and when an array with 17 repeats in the R1B sequence is inserted, the reporter —While it was shown that the plasmid's ability to suppress SEAP production was significantly inhibited, the strength of the inhibitory effect was not necessarily correlated with the number of R1B sequence repeats (Figure 4D). Furthermore, whether the suppression by the R1A sequence obtained in the astrocytoma cell line U373 is a common effect is being investigated. When a similar reporter assay was performed using the 293A epithelial cell line, the same results were obtained as when using the U373 cell line. Based on these results, the R1A sequence has the effect of suppressing the production of downstream encoded proteins. Furthermore, it was shown that this effect becomes stronger as the number of R1A repeats increases. This suggests that in the case of HHV-6B, the production of SITH-1 protein is suppressed because there is DNA with a large number of R1A sequences in the upstream sequence of SITH-1 mRNA.

[0041] <Example 6> Relationship between SITH-1 and various diseases To investigate whether SITH-1 expression is a risk factor for various diseases, antibody titers for SITH-1 were compared between patients with various diseases and NCs (non-clinical neuropathy). First, when antibody titers for SITH-1 were examined between Alzheimer's disease (AD) patients and non-corporeal candidiasis (NCs), the positive rate of anti-SITH-1 antibodies in AD patients was 84.8%, while the positive rate of anti-SITH-1 antibodies in NCs was 30.0%. The odds ratio was shown to be 8.36. Anti-SITH-1 antibody titers were significantly higher in AD patients than in non-clinical patients (p<0.0001), indicating that SITH-1 expression is a risk factor for AD (Figure 5). Next, antibody titers for SITH-1 were examined between ulcerative colitis (UC) patients and non-contractile disorders (NC). The positivity rate for anti-SITH-1 antibodies was 40.2% in UC patients, and 3.6% in NC patients, with an odds ratio of 17.9. Anti-SITH-1 antibody titers were significantly higher in UC patients than in NC patients (p<0.01), indicating that SITH-1 expression is a risk factor for UC (Figure 6). Furthermore, when antibody titers for SITH-1 were examined between Crohn's disease (CD) patients and non-cancer (NC), the anti-SITH-1 antibody positivity rate was 41.1% in CD patients and 3.6% in NC patients, with an odds ratio of 18.6. This showed that anti-SITH-1 antibody titers were significantly higher in CD patients than in NC patients (p<0.01), indicating that SITH-1 expression is a risk factor for CD (Figure 7). Furthermore, antibody titers related to SITH-1 were investigated between patients with post-COVID-19 syndrome (PCC) and NC. At that time, the anti-SITH-1 antibody positivity rate in PCC patients was 62.4%, while the anti-SITH-1 antibody positivity rate in NC patients was 8.3%. Yes, and the odds ratio was shown to be 28.2. Anti-SITH-1 antibody titers were higher in PCC patients than in NC patients. The results also showed a significantly higher level (p<0.0001), indicating that SITH-1 expression is a risk factor for PCC. (Figure 8).

Claims

1. A method for acquiring data to predict the risk of developing SITH-1-related disease, comprising detecting gene polymorphisms in the region related to the expression of the human herpesvirus 6 (HHV-6) SITH-1 gene in a sample taken from a subject, A method for predicting a high risk of developing SITH-1-related disease when the aforementioned gene polymorphism is detected.

2. The method according to claim 1, wherein the HHV-6 is HHV-6B.

3. The method according to claim 1, wherein the gene polymorphism is a repeat sequence polymorphism in a repeat element region.

4. The method according to claim 3, wherein the repeating element region is the R1 region.

5. The repeating sequence polymorphism in the R1 region is a polymorphism that repeats the R1A sequence represented by sequence number 8. The method according to claim 4, which is a type.

6. The method according to claim 5, wherein the number of repeats of the R1A sequence is 17 or less.

7. The method according to claim 1, wherein the sample is a saliva sample.

8. The method according to any one of claims 1 to 7, wherein the SITH-1 related disease is selected from the group consisting of depression, inflammatory bowel disease, Alzheimer's disease, and post-COVID-19 symptoms.

9. This is a test kit for assessing the risk of developing SITH-1 related diseases. A kit containing reagents for detecting genetic polymorphisms in the region related to the expression of the SITH-1 gene in human herpesvirus 6 (HHV-6).

10. The kit according to claim 9, wherein the reagent is a reagent for detecting gene polymorphisms in a region related to the expression of the SITH-1 gene of HHV-6B.

11. The kit according to claim 9, wherein the reagent is a reagent for detecting repeat sequence polymorphisms in a region related to the expression of the SITH-1 gene.

12. The kit according to claim 9, wherein the reagent is a reagent for detecting repeat sequence polymorphisms in the R1 region of a region related to the expression of the SITH-1 gene.

13. The kit according to claim 12, wherein the reagent comprises a primer for amplifying the R1 region and a primer for sequencing the amplified R1 region.

14. A kit according to claim 9 for testing saliva samples.

15. The kit according to any one of claims 9 to 14, wherein the SITH-1 related disease is selected from the group consisting of depression, inflammatory bowel disease, Alzheimer's disease, and post-COVID-19 symptoms.

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