Method for detecting worsening of skin itching due to atopic dermatitis

Abstract

To provide a method for detecting a deterioration in the severity of pruritus due to AD by using a marker for detecting a deterioration in the severity of pruritus due to AD; and the marker for detecting a deterioration in the severity of pruritus due to AD.SOLUTION: A method for detecting a deterioration in the severity of pruritus due to atopic dermatitis in a subject includes a step for measuring the expression level of a CBLB gene or an expression product thereof in a biological sample collected from the subject.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for detecting the exacerbation of skin itching due to atopic dermatitis using a detection marker for the exacerbation of skin itching due to atopic dermatitis.

Background Art

[0002] Atopic dermatitis (hereinafter also referred to as "AD") is an eczematous skin disease that mainly occurs in individuals with an atopic predisposition. Typical symptoms of AD include chronic and recurrent itching, rashes, erythema, etc. that occur bilaterally, as well as parakeratosis, reduced barrier function, dry skin, etc. Most cases of AD occur in infants and tend to improve with growth, but in recent years, adult-type and refractory AD have also been increasing. In AD, various etiological factors are involved complexly, resulting in the formation of diversity in symptoms and phenotypes, and it is known that exacerbation and remission repeat (Non-Patent Document 1). For example, it has been reported that when moisturizing is not continued after remission is induced by topical medications, approximately 40% of AD patients relapse within 14 days, and approximately 60% of AD patients relapse within 28 days (Non-Patent Document 2). Therefore, in treating AD, it is necessary to accurately grasp the severity of AD, including the diversity of symptoms and phenotypes, and its course. Among them, skin itching is considered one of the most important factors in AD, and appropriate management and treatment based on its severity are important because scratching behavior causes further deterioration of dermatitis and sleep disorders.

[0003] Traditional methods for evaluating the severity of Alzheimer's disease (AD) involve assessment based on the physician's visual observation. These observations include various features such as dryness, erythema, scaling, papules, scratch marks, edema, crusting, vesicles, erosions, and pruritic nodules. Scoring indices for these features include the Eczema Area and Severity Index (EASI) and the Severity Scoring of Atopic Dermatitis (SCORAD). In AD, the severity of skin itching is mostly assessed by the patient themselves, based on visual observation and tactile sensation. Scoring indices for this assessment include the Patient-Oriented Eczema Measure (POEM), Patient-Oriented SCORAD (PO-SCORAD), Visual Analog Scaling (VAS), and Numerical Ratio. An ng Scale (NRS) exists.

[0004] Furthermore, for an objective understanding of the pathogenesis of the disease, genes or their expression products contained in skin biopsies, blood, stratum corneum, etc., or the presence of specific cell types (these are sometimes collectively referred to as biomarkers) are often used. Conventionally, biomarkers proposed for evaluating the presence or absence and severity of AD include peripheral blood eosinophil count, serum total IgE levels, lactate dehydrogenase (LDH) levels, and serum thymus and activation-regulated chemokine (TARC) and squamous cell carcinoma antigen 2 (SCCA2) (Non-patent documents 3, 4).

[0005] In treating Alzheimer's disease (AD), it is considered important to control the amount and type of medication under an appropriate treatment plan by a physician based on the evaluation indicators mentioned above, in order to maintain remission for as long as possible and to prevent sudden exacerbations. For example, even if symptoms do not appear to be present externally or subjectively, there are cases where latent inflammation continues in the tissues and cells inside the skin, and treatment methods that maintain remission by continuing to apply topical medications and moisturizers even during periods when symptoms are not present are called proactive therapy (Non-Patent Literature 5). However, patients may discontinue treatment on their own during the remission period when no symptoms are present, so it is considered important to provide sufficient explanation and guidance to AD patients.

[0006] However, the conventional methods for evaluating the severity of various types of Alzheimer's disease (AD) described above only assess the presence or absence and severity of AD at the time of evaluation or biomarker collection, and do not predict the subsequent progression of symptom exacerbation or improvement. Therefore, objectively understanding whether AD symptoms will worsen over the next 14 to 28 days is useful information for physicians in selecting treatment methods and for patients in adhering to treatment.

[0007] In recent years, technologies have been developed to investigate the current and future physiological state of the human body by analyzing nucleic acids such as DNA and RNA in biological samples. Nucleic acids derived from living organisms can be extracted from bodily fluids such as blood, secretions, and tissues. More recently, it has been reported that RNA contained in skin surface lipids (SSL) can be used as a sample for biological analysis (Patent Document 1). It has also been reported that AD marker genes can be detected from SSL (Patent Document 2). [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] International Public Gazette No. 2018 / 008319 [Patent Document 2] Japanese Patent Publication No. 2020-074769 [Non-patent literature]

[0009] [Non-Patent Document 1] Kato et al., Journal of the Japanese Dermatological Association, 2018, 128:2431-2502. [Non-Patent Document 2] Lin et al., Adv Ther. 2017, 34:2601-2611. [Non-Patent Document 3] Sugawara et al., Allergy. 2002, 57:180-181. [Non-Patent Document 4] Ohta et al., Ann Clin Biochem. 2012, 49:277-284. [Non-Patent Document 5] Schmitt et al., Br J Dermatol. 2011, 164:415-28. [Overview of the Initiative] [Problems that the invention aims to solve]

[0010] This invention relates to a detection marker for the worsening of skin itching symptoms due to Alzheimer's disease (AD), and to the detection of skin itching due to AD. This invention provides a method for detecting the worsening of symptoms of AD, and a marker for detecting the worsening of skin itching caused by AD. Regarding the act of doing something. [Means for solving the problem]

[0011] The inventors collected SSL from patients with mild and moderate AD, and the R contained in SSL Comprehensive analysis of NA expression status as sequencing information revealed that the expression level of specific genes However, in patients, the severity of skin itching worsens at a certain time after SSL sampling, and in patients, it does not. There were significant differences among patients, and this gene was used as an indicator to predict the worsening of skin itching symptoms due to AD. We discovered that it is possible to measure it.

[0012] That is, the present invention relates to the following 1) to 3). 1) A step of measuring the expression level of the CBLB gene or its expression product in a biological sample collected from a subject, which includes detecting the exacerbation of skin itching due to atopic dermatitis in the subject. Detection method. 2) An oligonucleotide that specifically hybridizes with the gene or a nucleic acid derived therefrom, or an antibody that recognizes the expression product of the gene, and is used in the method of 1). A test kit for detecting the exacerbation of skin itching due to atopic dermatitis. 3) A detection marker for the exacerbation of skin itching due to atopic dermatitis, which consists of the CBLB gene or its expression product.

Effects of the Invention

[0013] According to the present invention, in AD patients who often repeat exacerbation and remission of symptoms, it is possible to easily predict whether the severity of skin itching will deteriorate in the near future, and each patient can receive optimal treatment and information provided based on the assumption of future exacerbation of skin itching symptoms.

Modes for Carrying Out the Invention

[0014] All patent documents, non-patent documents, and other publications cited in this specification are hereby incorporated by reference in their entirety.

[0015] In the present invention, the terms "nucleic acid" or "polynucleotide" mean DNA or RNA. DNA includes cDNA, genomic DNA, and synthetic DNA, and "RNA" includes total RNA, mRNA, rRNA, tRNA, non-coding RNA, and synthetic RNA.

[0016] In this invention, "gene" means a double-stranded DNA including human genomic DNA, as well as single-stranded DNA (positive strand) including cDNA, single-stranded DNA having a sequence complementary to the positive strand (complementary strand), and fragments thereof, in which the sequence information of the bases constituting the DNA contains some kind of biological information. Furthermore, the term "gene" in this invention includes not only "genes" represented by a specific base sequence, but also their homologs (i.e., orthologs), variants such as gene polymorphisms, and derivatives.

[0017] In this invention, the term "expression product" of a gene is a concept that encompasses both the transcript and the translation product of a gene. A "transcript" is RNA produced by transcription from a gene (DNA), and a "translation product" is a protein encoded by a gene that is synthesized through translation based on RNA.

[0018] In this invention, "atopic dermatitis (AD)" refers to a disease characterized by itchy eczema that repeatedly worsens and remits, and most patients with this disease are considered to have an atopic predisposition. Atopic predispositions include i) a family history or past medical history (one or more of the following diseases: bronchial asthma, allergic rhinitis / conjunctivitis, or atopic dermatitis), or ii) a predisposition to easily produce IgE antibodies.

[0019] In the present invention, "severity of pruritus due to AD" refers to the level of symptoms of pruritus due to AD, and includes not only broad classifications such as mild, moderate, and severe, but also classifications based on more minor differences. "Severity of pruritus due to AD" can be determined, for example, based on various known evaluation scores for assessing the degree of pruritus due to AD. In the present invention, such evaluation scores are referred to as "scores related to the severity of pruritus due to AD." Examples of scores related to the severity of pruritus due to AD include itching intensity evaluation scores using visual analogue scales (VAS), numerical rating scales (NRS), Patient Oriented Eczema Measure (POEM), and Patient Oriented SCORAD (PO-SCORAD). A VAS score is preferred, in which the patient visually marks the intensity of itching on a 100 mm line segment (a score from 0 to 100 is obtained) across the entire body. The score itself related to the severity of skin itching in AD may be used as the level of symptoms of skin itching in AD, as indicated by the "severity of skin itching due to AD."

[0020] In this invention, the term "detection" of worsening of the severity of itching due to Alzheimer's disease (AD) can be rephrased as terms such as examination, measurement, judgment, or evaluation support. However, the terms "detection," "examination," "measurement," "judgment," or "evaluation" of worsening of the severity of itching due to AD in this invention do not include a physician's diagnosis of worsening of the severity of itching due to AD.

[0021] As shown in the examples described later, patients with mild to moderate AD were used as subjects, and a 42-day (6-week) observation period was established. SSL samples were collected at predetermined times (day 0 (week 0) and day 28 (week 4)), and RNA expression analysis of the RNA contained in the SSL was performed according to the following procedures 1) to 4). When comparing the RNA expression levels of patients whose skin itching worsened (worsening group) and patients whose skin itching did not worsen (non-worsening group) 14 days (2 weeks) after SSL collection (day 14 (week 2) and day 42 (week 6)), it was found that the expression level of the CBLB gene was significantly elevated in the worsening group at both time points. 1) Obtain RNA expression data (read count value) extracted from SSL. 2) Convert the read count value to an RPM value corrected for differences in the total number of reads between samples. Add an integer 1 to this RPM+1 value and convert the resulting RPM+1 value to a base-2 logarithm (Log2(RPM+1) value) to use as an indicator of expression level. Based on this, select genes with different expression levels between the worsening group and the non-worsening group. Specifically, we first select genes for which the p-value in Welch's t-test is less than 0.05 between the two groups: the worsening group and the non-worsening group. Here, the "p-value" in a statistical test indicates the probability that a statistic more extreme than the statistic actually calculated from the data under the null hypothesis will be observed. Therefore, the smaller the "p-value," the more significant the difference between the comparison groups can be considered to be. 3) Next, select genes in which the mean expression level differs by more than 1 between the worsening group and the non-worsening group. Specifically, the mean Log2(RPM+1) value of the worsening group is calculated as {mean Log2(RPM+1) value of the non-worsening group}, and genes whose absolute value is greater than 1 are selected. 4) Genes for which the p-value is less than 0.05 between the worsening group and the non-worsening group, and for which the difference in expression level is greater than 1, are selected as differentially expressed genes between the worsening group and the non-worsening group.

[0022] Therefore, the CBLB gene or its expression product can serve as a marker for detecting the worsening of skin itching symptoms due to Alzheimer's disease (AD). Here, the gene name "CBLB" follows the Official Symbol listed in NCBI ([www.ncbi.nlm.nih.gov / ]), and its Gene ID is 868.

[0023] In this invention, "detection of worsening of skin itching due to AD" refers to detecting whether or not the subject's skin itching has worsened due to AD at a time period separated from the time of sample collection. In other words, detecting the worsening of skin itching due to AD means predicting whether or not the subject's skin itching has worsened at a time period separated from the time of sample collection. Here, the "separated period" is preferably 12 days or more, more preferably 13 days or more, and preferably 16 days or less, more preferably 15 days or less, and even more preferably 14 days.

[0024] In this invention, "worsening of the severity of skin itching due to AD" refers to a worsening of the severity of skin itching due to AD compared to the current level (at the time of sample collection). As mentioned above, the severity of skin itching due to AD can be determined based on various known evaluation scores for evaluating skin itching symptoms due to AD. Specifically, this refers to a worsening of the evaluation of skin itching due to AD using VAS scores, etc. VAS scoring is a method of scoring the intensity of itching by drawing a 100mm line on a piece of paper, with 0 (no itching at all) at the left end and 100 (the strongest itching imaginable) at the right end, and having the patient indicate where their score falls between 0 and 100. A worsening of the VAS score includes all cases in which the VAS score increases compared to the current VAS score (at the time of sample collection).

[0025] Furthermore, the genes that can serve as detection markers for the worsening of skin itching due to AD (hereinafter also referred to as "target genes") include genes that have substantially identical base sequences to the DNA that constitutes the target gene, insofar as they can serve as biomarkers for detecting the worsening of skin itching due to AD. Here, substantially identical base sequences mean, for example, that when searching using the homology calculation algorithm NCBI BLAST with the conditions expected value = 10; gap allowed; filtering = ON; match score = 1; mismatch score = -3, the base sequence of the target gene has 90% or more identity, preferably 95% or more, more preferably 98% or more identity, and even more preferably 99% or more identity.

[0026] The present invention provides a method for detecting the worsening of skin itching symptoms due to AD, which includes measuring the expression level of a target gene, in one embodiment, the CBLB gene or its expression product, in a biological sample taken from a subject.

[0027] In the present invention's method for detecting the worsening of the severity of itching due to AD, the gender, age, and race of the subjects are not particularly limited and may include infants to the elderly. Preferably, the subjects are people who need or desire the detection of the worsening of the severity of itching due to AD. For example, the subjects are people who have developed itching due to atopic dermatitis, people suspected of developing itching due to atopic dermatitis, or people who have a genetic predisposition to atopic dermatitis.

[0028] The biological samples used in the present invention may be any cells, tissues, and biomaterials in which the expression of the gene of the present invention changes in accordance with the worsening of the severity of skin itching due to AD. Specifically, examples include organs, skin, blood, urine, saliva, sweat, stratum corneum, surface lipids (SSL), tissue exudates and other bodily fluids, serum and plasma prepared from blood, and others such as feces and hair. Preferably, these are skin, stratum corneum, or surface lipids (SSL), and more preferably, surface lipids (SSL). The site of skin from which SSL is collected is not particularly limited and may be any part of the body such as the head, face, neck, trunk, hands and feet. Areas with high sebum secretion, such as the skin of the head or face, are preferred, and facial skin is more preferred. Furthermore, the site of skin from which SSL is collected may be either a lesion area where skin itching due to atopic dermatitis has occurred, or an unaffected area, but preferably, the lesion area or an unaffected area near the lesion area is preferred. Here, "near the lesion area" refers to an area within 10 cm adjacent to the lesion area.

[0029] Here, "superficial lipids (SSL)" refers to the lipid-soluble fraction present on the surface of the skin, and is sometimes called sebum. Generally, SSL mainly consists of secretions from exocrine glands such as sebaceous glands in the skin, and exists on the skin surface in the form of a thin layer covering the skin surface. SSL contains RNA expressed in skin cells. (See Patent Document 1 above). Furthermore, in the present invention, unless otherwise specified, "skin" is a general term for the region including the stratum corneum, epidermis, dermis, hair follicles, and tissues such as sweat glands, sebaceous glands, and other glands.

[0030] Any means used for the collection or removal of SSL from the skin can be employed to collect SSL from the subject's skin. Preferably, an SSL absorbent material, an SSL adhesive material, or an instrument for scraping off SSL from the skin, as described later, can be used. The SSL absorbent material or SSL adhesive material is not particularly limited as long as it is a material that has an affinity for SSL, and examples include polypropylene and pulp. More detailed examples of procedures for collecting SSL from the skin include methods of absorbing SSL onto a sheet material such as oil-blotting paper or oil-blotting film, methods of adhering SSL to a glass plate or tape, and methods of scraping off and collecting SSL with a spatula, scraper, etc. To improve the adsorption of SSL, an SSL absorbent material pre-containing a highly lipid-soluble solvent may be used. On the other hand, since the adsorption of SSL is inhibited if the SSL absorbent material contains highly water-soluble solvents or moisture, it is preferable that the content of highly water-soluble solvents or moisture is low. It is preferable to use the SSL absorbent material in a dry state.

[0031] RNA-containing SSLs collected from subjects may be stored for a certain period of time. To minimize the degradation of the contained RNA, it is preferable to store the collected SSLs under low-temperature conditions as quickly as possible after collection. The storage temperature conditions for the RNA-containing SSLs in this invention may be 0°C or lower, preferably -20±20°C to -80±20°C, more preferably -20±10°C to -80±10°C, even more preferably -20±20°C to -40±20°C, even more preferably -20±10°C to -40±10°C, even more preferably -20±10°C, and even more preferably -20±5°C. The storage period for the RNA-containing SSLs under these low-temperature conditions is not particularly limited, but is preferably 12 months or less, for example, 6 hours to 12 months, more preferably 6 months or less, for example, 1 day to 6 months, and even more preferably 3 months or less, for example, 3 days to 3 months.

[0032] In the present invention, the objects to be measured for the expression level of a target gene or its expression product include cDNA artificially synthesized from RNA, the DNA encoding that RNA, the protein encoded by that RNA, molecules that interact with that protein, molecules that interact with that RNA, or molecules that interact with that DNA. Here, molecules that interact with RNA, DNA, or proteins include DNA, RNA, proteins, polysaccharides, oligosaccharides, monosaccharides, lipids, fatty acids, and their phosphorylated, alkylated, and glycosidic compounds, as well as complexes of any of the above. Furthermore, the expression level comprehensively refers to the amount of expression or activity of the gene or expression product in question.

[0033] In the method of the present invention, in a preferred embodiment, SSL is used as the biological sample. In this case, the expression level of RNA contained in the SSL is analyzed. Specifically, the RNA is converted to cDNA by reverse transcription, and then the cDNA or its amplified product is measured. For RNA extraction from SSL, methods commonly used for RNA extraction or purification from biological samples can be employed, such as the phenol / chloroform method, the AGPC (acid guanidinium thiocyanate-phenol-chloroform extraction) method, or methods using columns such as TRIzol®, RNeasy®, or QIAzol®, or methods using special silica-coated magnetic particles, methods using Solid Phase Reversible Immobilization magnetic particles, or extraction using commercially available RNA extraction reagents such as ISOGEN.

[0034] For the reverse transcription, primers targeting a specific RNA to be analyzed may be used, but for more comprehensive nucleic acid preservation and analysis, random primers are preferable. A general reverse transcriptase or reverse transcription reagent kit can be used for the reverse transcription. Preferably, a reverse transcriptase or reverse transcription reagent kit with high accuracy and efficiency is used, such as M-MLV Reverse Transcriptase and its variants, or commercially available reverse transcriptase or reverse transcription reagent kits, such as the PrimeScript® Reverse Transcriptase series (Takara Bio Inc.) and the SuperScript® Reverse Transcriptase series (Thermo Scientific Inc.). SuperScript® III Reverse Transcriptase and SuperScript® VILO cDNA Synthesis kit (both from Thermo Scientific Inc.) are preferably used. In the reverse transcription extension reaction, it is preferable to adjust the temperature to preferably 42°C ± 1°C, more preferably 42°C ± 0.5°C, and even more preferably 42°C ± 0.25°C, while adjusting the reaction time to preferably 60 minutes or more, more preferably 80 to 120 minutes.

[0035] Methods for measuring expression levels can be selected from nucleic acid amplification methods such as PCR, real-time RT-PCR, multiplex PCR, SmartAmp, and LAMP, which use DNA that hybridizes to RNA, cDNA, or DNA as primers; hybridization methods (DNA chips, DNA microarrays, dot blot hybridization, slot blot hybridization, Northern blot hybridization, etc.) which use nucleic acids that hybridize to these as probes; methods for determining the base sequence (sequencing); or methods combining these.

[0036] In PCR, a primer pair targeting a specific DNA to be analyzed may be used to amplify only that specific DNA, or multiple primer pairs may be used to amplify multiple specific DNAs simultaneously. Preferably, the PCR is multiplex PCR. Multiplex PCR is a method of simultaneously amplifying multiple gene regions by using multiple primer pairs simultaneously in the PCR reaction system. Multiplex PCR can be performed using commercially available kits (for example, the Ion AmpliSeqTranscriptome Human Gene Expression Kit; Life Technologies Japan Co., Ltd., etc.). The temperature for the annealing and extension reactions in the PCR cannot be generalized as it depends on the primers used, but when using the above-mentioned multiplex PCR kit, it is preferably 62°C ± 1°C, more preferably 62°C ± 0.5°C, and even more preferably 62°C ± 0.25°C. Therefore, in the PCR, the annealing and extension reactions are preferably performed in one step. The duration of the annealing and extension reaction steps can be adjusted depending on the size of the DNA to be amplified, but is preferably 14 to 18 minutes. The conditions for the denaturation reaction in the PCR can be adjusted depending on the DNA to be amplified, but is preferably 95 to 99°C for 10 to 60 seconds. Reverse transcription and PCR at the above temperatures and times can be performed using a thermal cycler commonly used for PCR.

[0037] The purification of the reaction product obtained by the PCR is preferably carried out by size separation of the reaction product. Size separation allows the target PCR reaction product to be separated from primers and other impurities contained in the PCR reaction mixture. DNA size separation can be carried out, for example, by a size separation column, a size separation chip, or magnetic beads that can be used for size separation. Preferred examples of magnetic beads that can be used for size separation include Solid Phase Reversible Immobilization (SPRI) magnetic beads such as Ampure XP.

[0038] The purified PCR reaction product may be subjected to further processing necessary for subsequent quantitative analysis. For example, the purified PCR reaction product may be prepared into a suitable buffer solution for DNA sequencing, the PCR primer region in the PCR-amplified DNA may be cleaved, or an adapter sequence may be further added to the amplified DNA. For example, the purified PCR reaction product can be prepared into a buffer solution, the PCR primer sequence can be removed and the amplified DNA can be ligated with an adapter, and the resulting reaction product can be amplified as needed to prepare a library for quantitative analysis. These operations can be performed, for example, with the 5× included with the SuperScript® VILO cDNA Synthesis kit (Life Technologies Japan Co., Ltd.) This can be performed using the VILO RT Reaction Mix, the 5×Ion AmpliSeq HiFi Mix included with the Ion AmpliSeq Transcriptome Human Gene Expression Kit (Life Technologies Japan Co., Ltd.), and the Ion AmpliSeq Transcriptome Human Gene Expression Core Panel, following the protocol included with each kit.

[0039] When measuring the expression level of a target gene or nucleic acid derived therefrom using Northern blot hybridization, for example, a probe DNA is first labeled with a radioisotope, a fluorescent substance, etc. Then, the resulting labeled DNA is hybridized with RNA derived from a biological sample transferred to a nylon membrane, etc., according to a conventional method. Subsequently, the double helix formed between the labeled DNA and RNA is measured by detecting the signal originating from the label.

[0040] When measuring the expression level of a target gene or nucleic acid derived therefrom using RT-PCR, for example, cDNA is first prepared from RNA derived from a biological sample according to a conventional method, and a pair of primers (a positive strand that binds to the cDNA (- strand), and a reverse strand that binds to the + strand) prepared to amplify the target gene of the present invention are hybridized with this cDNA as a template. Then, PCR is performed according to a conventional method, and the resulting amplified double-stranded DNA is detected. For the detection of the amplified double-stranded DNA, a method can be used to detect labeled double-stranded DNA produced by performing the above PCR using primers that have been previously labeled with an RI, a fluorescent substance, etc.

[0041] When measuring the expression level of a target gene or nucleic acid derived therefrom using a DNA microarray, for example, an array in which at least one nucleic acid (cDNA or DNA) derived from the target gene of the present invention is immobilized on a support is used, labeled cDNA or cRNA prepared from mRNA is bound to the microarray, and the expression level of mRNA can be measured by detecting the label on the microarray. The nucleic acid immobilized on the array can be any nucleic acid that hybridizes specifically (i.e., substantially only to the target nucleic acid) under stringent conditions. For example, it may be a nucleic acid having the entire sequence of the target gene of the present invention, or it may be a nucleic acid consisting of a partial sequence. Here, "partial sequence" refers to a nucleic acid consisting of at least 15 to 25 bases. Here, stringent conditions are typically around "1×SSC, 0.1%SDS, 37°C". Washing conditions can be listed, and more stringent hybridization conditions include approximately "0.5×SSC, 0.1%SDS, 42°C," while even more stringent hybridization conditions include approximately "0.1×SSC, 0.1%SDS, 65°C." Hybridization conditions are described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press (2001), etc.

[0042] When measuring the expression level of a target gene or nucleic acid derived therefrom by sequencing, for example, analysis can be performed using a next-generation sequencer (e.g., the Ion S5 / XL system, Life Technologies Japan Co., Ltd.). RNA expression can be quantified based on the number of reads generated by sequencing (read count).

[0043] The probes or primers used in the above measurements, namely primers for specifically recognizing and amplifying the target gene of the present invention or nucleic acids derived therefrom, or probes for specifically detecting the RNA or nucleic acids derived therefrom, can be designed based on the base sequence constituting the target gene. Here, "specifically recognizing" means, for example, in the Northern blotting method, that substantially only the target gene of the present invention or nucleic acids derived therefrom can be detected, or for example, in the RT-PCR method, that substantially only the nucleic acids in question are amplified, so that the detected substance or product can be determined to be the gene or nucleic acids derived therefrom. Specifically, the present invention can utilize DNA consisting of the base sequence constituting the target gene, or oligonucleotides containing a certain number of nucleotides complementary to its complementary strand. Here, "complementary strand" refers to the other strand of a double-stranded DNA consisting of A:T (U in the case of RNA) and G:C base pairs. Furthermore, "complementary" is not limited to cases where the sequence is perfectly complementary in the given number of consecutive nucleotide regions, but is preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 98% or more of the base sequence identity. The base sequence identity can be determined by algorithms such as BLAST. When used as a primer, such oligonucleotides only need to be able to perform specific annealing and chain extension, and typically have a chain length of, for example, 10 bases or more, preferably 15 bases or more, more preferably 20 bases or more, and for example 100 bases or less, preferably 50 bases or less, and more preferably 35 bases or less. When used as a probe, it is sufficient that specific hybridization can be performed, and oligonucleotides that have at least a part or all of the sequence of DNA (or its complementary strand) consisting of the base sequence constituting the target gene of the present invention are used, and for example, oligonucleotides with a chain length of 10 bases or more, preferably 15 bases or more, and for example 100 bases or less, preferably 50 bases or less, and more preferably 25 bases or less are used. Here, "oligonucleotides" can be DNA or RNA, and may be synthetic or naturally occurring. Furthermore, the probes used for hybridization are usually labeled.

[0044] Furthermore, when measuring the translation product (protein) of the target gene of the present invention, molecules that interact with said protein, molecules that interact with RNA, or molecules that interact with DNA, methods such as protein chip analysis, immunoassay (e.g., ELISA), mass spectrometry (e.g., LC-MS / MS, MALDI-TOF / MS), 1-hybrid methods (PNAS 100, 12271-12276 (2003)), and 2-hybrid methods (Biol. Reprod. 58, 302-311 (1998)) can be used and can be appropriately selected depending on the target. For example, when a protein is used as the target of measurement, the method is carried out by contacting a biological sample with an antibody that specifically recognizes the expression product of the present invention, specifically an antibody that recognizes a structural characteristic site (epitope) that can distinguish the expression product protein from other proteins, detecting the polypeptide or protein in the sample bound to the antibody, and measuring its level. For example, using the Western blotting method, the above antibody is used as the primary antibody, and then the primary antibody is labeled with a radioisotope, fluorescent substance, or enzyme, and the signal derived from these labeling substances is measured with a radiation detector, fluorescence detector, etc. Furthermore, the antibodies against the above-mentioned translation products may be polyclonal or monoclonal antibodies. These antibodies can be manufactured according to known methods. Specifically, polyclonal antibodies can be obtained by using proteins expressed and purified in E. coli or other bacteria according to conventional methods, or by synthesizing partial polypeptides of such proteins according to conventional methods, immunizing non-human animals such as rabbits, and then obtaining them from the serum of the immunized animals according to conventional methods. On the other hand, monoclonal antibodies can be obtained from hybridoma cells prepared by immunizing non-human animals such as mice with proteins expressed and purified in E. coli or other bacteria according to conventional methods, or with partial polypeptides of said proteins, and then fusing the resulting spleen cells with myeloma cells. Monoclonal antibodies may also be produced using phage display (Griffiths, AD; Duncan, AR, Current Opinion in Biotechnology, Volume 9, Number 1, February 1998, pp. 102-108(7)).

[0045] Thus, the expression level of the target gene of the present invention or its expression product in a biological sample taken from a subject is measured, and the worsening of the severity of skin itching due to AD is detected based on the expression level. Specifically, the detection is performed by comparing the measured expression level of the target gene of the present invention or its expression product with a control level or a predetermined cutoff value (reference value). When analyzing the expression levels of multiple target genes by sequencing, it is preferable to use the following as indicators, as described above: the read count value, which is the expression level data; the RPM value obtained by correcting the difference in the total number of reads between samples from the read count value; the value obtained by converting the RPM value to a base-2 logarithm (Log2RPM value) or the base-2 logarithm obtained by adding an integer 1 (Log2(RPM+1) value); or the count value corrected using DESeq2 (Love MI et al. Genome Biol. 2014) (Normalized count value) or the base-2 logarithm obtained by adding an integer 1 (Log2(Normalized count+1) value). Alternatively, values ​​calculated using common RNA-seq quantitative values ​​such as fragments per kilobase of exon per million reads mapped (FPKM), reads per kilobase of exon per million reads mapped (RPKM), or transcripts per million (TPM) may also be used. Furthermore, signal values ​​obtained by microarray methods and their corrected values ​​may also be used. Furthermore, when analyzing only specific target genes using methods such as RT-PCR, it is preferable to either convert the expression level of the target gene to a relative expression level based on the expression level of housekeeping genes for analysis, or to quantify the absolute copy number (absolute quantification) using a plasmid containing the region of the target gene for analysis. The copy number obtained by digital PCR may also be used. Here, "control level" refers to the expression level of the target gene or its expression product in a patient population in which the severity of skin itching did not worsen over a certain period. This expression level may be determined by referring to statistical values ​​such as the mean and standard deviation of the expression level of the target gene or its expression product measured from the population. "Reference value" can be determined in advance based on the relationship between whether or not the severity of skin itching worsened over a certain period and the expression level of the target marker. For example, a population can be divided into a worsening group and a non-worsening group based on whether or not the severity of skin itching worsened over a certain period, and a value determined by referring to statistical values ​​such as the mean and standard deviation of the expression level of the target marker in each group can be determined as a reference value to determine whether or not a patient belongs to each group. It is preferable to determine the control level and reference value for each CBLB gene or its expression product.

[0046] Furthermore, by using measured values ​​of the expression levels of target genes or their expression products derived from AD patients whose skin pruritus worsens within a certain period, and the expression levels of target genes or their expression products derived from AD patients whose skin pruritus does not worsen within a certain period, a discriminant formula (predictive model) can be constructed to separate patients into groups whose skin pruritus worsens and those whose skin pruritus does not worsen. This discriminant formula can then be used to detect the worsening of skin pruritus due to AD. Specifically, using measured values ​​of the expression levels of target genes or their expression products derived from AD patients whose skin pruritus worsens within a certain period, and the expression levels of target genes or their expression products derived from AD patients whose skin pruritus does not worsen within a certain period, as training samples, a discriminant formula (predictive model) can be constructed to separate patients into groups whose skin pruritus worsens (worsening group) and those whose skin pruritus does not worsen (non-worsening group). Based on this discriminant formula, a cutoff value (reference value) is determined to distinguish each patient group with different skin pruritus worsening. Then, by similarly measuring the level of the target gene or its expression product from a biological sample taken from the subject, substituting the obtained measurement value into the discriminant formula, and comparing the result obtained from the discriminant formula with a reference value, it is possible to detect the worsening of skin itching symptoms due to AD in the subject.

[0047] For constructing discriminant formulas, publicly known algorithms such as those used in machine learning can be utilized. Examples of machine learning algorithms include Random Forest, Support Vector Machines with Linear Kernel (SVM linear), Support Vector Machines with rbf Kernel (SVM rbf), Neural Networks, Generalized Linear Models, Regularized Linear Discriminant Analysis, and Regularized Logistic Regression. By inputting validation data into the constructed prediction model and calculating predicted values, the model whose predicted values ​​best match the observed values, for example, the model with the highest accuracy, can be selected as the optimal prediction model. Additionally, the Recall, Precision, and their harmonic mean (F-score) can be calculated from the predicted and observed values, and the model with the highest F-score can be selected as the optimal prediction model.

[0048] The method for determining the cutoff value (reference value) is not particularly restricted and can be determined according to known methods. For example, it can be obtained from an ROC (Receiver Operating Characteristic Curve) curve created using a discriminant. In an ROC curve, the vertical axis plots the probability of a positive result in a positive patient (sensitivity), and the horizontal axis plots the value obtained by subtracting the probability of a negative result in a negative patient (specificity) from 1 (false positive rate). Regarding the "true positive (sensitivity)" and "false positive (1-specificity)" shown in the ROC curve, the value (Youden index) at which "true positive (sensitivity)" - "false positive (1-specificity)" is maximized can be used as the cutoff value (reference value).

[0049] The diagnostic kit for detecting the worsening of skin itching symptoms due to Alzheimer's disease (AD) according to the present invention contains diagnostic reagents for measuring the expression level of the target gene or its expression product in a biological sample isolated from a patient. Specifically, these include reagents for nucleic acid amplification and hybridization, including oligonucleotides (e.g., primers for PCR) that specifically bind (hybridize) to the target gene or nucleic acid derived therefrom, or reagents for immunological measurement, including antibodies that recognize the expression product (protein) of the target gene. The oligonucleotides, antibodies, etc., included in the kit can be obtained by known methods as described above. Furthermore, the test kit may include, in addition to the antibodies and nucleic acids mentioned above, labeling reagents, buffer solutions, chromogenic substrates, secondary antibodies, blocking agents, equipment necessary for the test, control reagents used as positive and negative controls, and tools for collecting biological samples (for example, oil-absorbing film for collecting SSL).

[0050] With regard to the embodiments described above, the present invention further discloses the following embodiments. <1> A method for detecting the worsening of skin itching due to atopic dermatitis in a subject, comprising the step of measuring the expression level of the CBLB gene or its expression product in a biological sample taken from the subject. <2> The process further includes comparing the measured expression level with a control level or cutoff value (reference value). <1> The method. <3> The process further includes determining the aforementioned control level or cutoff value (reference value), <2> The method. <4> The process further includes a step of detecting the worsening of skin itching due to atopic dermatitis by using a discriminant formula (predictive model) that separates patients into groups whose skin itching worsens and those whose skin itching does not worsen. <1> The method. <5> The process further includes constructing the aforementioned discriminant formula (predictive model), <4> The method. <6> The worsening of skin itching is the condition that occurs 12 to 16 days after the time of sample collection. <1> ~ <5> One of the following methods. <7> The severity of skin itching corresponds to the Visual Analog Scaling (VAS) score, which scores the intensity of itching, and represents the severity of skin itching caused by atopic dermatitis. <1> ~ <6> One of the following methods. <8> The expression level of a gene or its expression product is measured by the expression level of mRNA. <1> ~ <7> One of the following methods. <9> The gene or its expression product is RNA contained in the lipids on the skin surface of the subject. <1> ~ <8> One of the following methods. <10> The subject is a person who needs or desires the detection of worsening of the severity of skin itching due to atopic dermatitis. <1> ~ <9> One of the following methods. <11> The subject is a person who has developed itching due to atopic dermatitis, a person suspected of developing itching due to atopic dermatitis, or a person who has a genetic predisposition to atopic dermatitis. <1> ~ <9> One of the following methods. <12> Use of the CBLB gene or its expression product derived from biological samples taken from subjects as a detection marker for the worsening of skin itching due to atopic dermatitis. <13> The worsening of skin itching is the condition that occurs 12 to 16 days after the time of sample collection. <12> Use. <14> The severity of skin itching corresponds to the Visual Analog Scaling (VAS) score, which scores the intensity of itching, and represents the severity of skin itching caused by atopic dermatitis. <12> or <13> Use. <15> The gene or its expression product is mRNA contained in the surface lipids of the subject's skin. <12> ~ <14> Use of either of the following. <16> The system contains an oligonucleotide that specifically hybridizes with the gene or nucleic acid derived therefrom, or an antibody that recognizes the expression product of the gene. <1> ~ <11> A test kit for detecting the worsening of skin itching symptoms caused by atopic dermatitis, used in one of the following methods. <17> A marker for detecting the worsening of skin itching symptoms due to atopic dermatitis, consisting of the CBLB gene or its expression product. <18> The severity of skin itching corresponds to the Visual Analog Scaling (VAS) score, which scores the intensity of itching, and represents the severity of skin itching caused by atopic dermatitis. <17> The marker. [Examples]

[0051] The present invention will be described in more detail below based on examples, but the present invention is not limited thereto. Example 1: Exploration of differentially expressed genes in disease progression groups versus non-progression groups using SSL-derived RNA (Case 1) 1) Obtaining scores and SSL collection related to the severity of skin itching in AD patients The study included 25 adult males (ages 23-56) with Alzheimer's disease (AD). All subjects were diagnosed with mild to moderate AD by a dermatologist at the time of the initial measurement. At the initial measurement, subjects obtained a score for the severity of skin itching due to AD and had an STI sample taken. Fourteen days after the initial measurement, subjects obtained another score for the severity of AD. The AD severity score used was the subject's own whole-body skin itching VAS score (scoring the intensity of itching on a scale of 0 to 100). The itching VAS scores obtained at the initial measurement and 14 days later are referred to as the initial itching VAS score and the 14-day itching VAS score, respectively, and the STI sample taken at the initial measurement is referred to as the initial STI.

[0052] As mentioned above, SSL refers to surface skin lipids, which were collected from the entire face of each subject using an oil-absorbing film (5 x 8 cm, made of polypropylene, 3M). The oil-absorbing films were transferred to vials and stored at -80°C for approximately one month until they were ready for RNA extraction.

[0053] 2) RNA preparation and sequencing The oil-absorbing film described in 1) above was cut to an appropriate size, and RNA was transferred to the aqueous layer using QIAzol Lysis Reagent (Qiagen) according to the included protocol. RNA was extracted from the aqueous layer using a commercially available RNA extraction kit with an RNA extraction spin column, according to the included protocol. The extracted RNA was reverse transcribed using the SuperScript VILO cDNA Synthesis kit (Life Technologies Japan Co., Ltd.) at 42°C for 90 minutes to synthesize cDNA. Random primers included in the kit were used as primers for the reverse transcription reaction. From the obtained cDNA, a library containing DNA derived from the 20802 gene was prepared by multiplex PCR. Multiplex PCR was performed using the Ion AmpliSeqTranscriptome Human Gene Expression Kit (Life Technologies Japan Co., Ltd.) under the conditions [99°C, 2 min → (99°C, 15 sec → 62°C, 16 min) × 20 cycles → 4°C, Hold]. The obtained PCR products were purified using Ampure XP (Beckman Coulter, Inc.), followed by buffer reconstitution, primer sequence digestion, adapter ligation and purification, and amplification to prepare the library. The prepared library was loaded onto an Ion 540 Chip and sequenced using the Ion S5 / XL system (Life Technologies Japan, Inc.). The genes from which each read sequence originated were determined by gene mapping using the human genome reference sequence, hg19 AmpliSeq Transcriptome ERCC v1.

[0054] 3) Data used The initial itching VAS scores of 25 AD patients obtained in 1) above were compared with their itching VAS scores 14 days later (day 14) to calculate how much the itching VAS scores changed over 14 days. More specifically, the value obtained by subtracting the initial itching VAS score from the itching VAS score on day 14 for the same individual (Δ value) was calculated. A positive Δ value was defined as "worsening," and a negative or zero value was defined as "no worsening." Of the 25 AD patients, 7 subjects with a positive Δ value and a larger absolute value were selected as the worsening group, and 7 subjects with a negative Δ value and a larger absolute value were selected as the no-worsening group.

[0055] Of the read counts obtained from sequencing the initial SSL-derived RNA of the subjects as measured in 2) above, the read counts of the seven subjects selected as the deterioration group and the seven subjects selected as the non-deterioration group were used as data for the expression level of each RNA. Genes whose amplification region in sequencing spans at least two or more exons were selected as the target genes for analysis, and in order to correct for differences in total read counts between samples, the read counts of the target genes were converted to RPM (Reads per million mapped reads) values. Among these, 5881 genes for which a read count of 20 or more was obtained in more than 90% of the samples were used for the following analysis. Furthermore, in order to approximate the RPM values ​​to a normal distribution, they were converted to base 2 logarithms (Log2(RPM+1) values) by adding the integer 1. Following the above procedure, expression level data (Log2(RPM+1) values) for 5881 genes from 14 subjects were created.

[0056] 4) Data Analysis Based on the initial expression level data (Log2(RPM+1) values) of 5881 genes derived from SSL, created in step 3) above, we searched for genes with differing expression levels between the worsening group and the non-worsening group at the time of the initial measurement. First, we selected genes for which the p-value in Welch's t-test was less than 0.05 between the two groups, the worsening group and the non-worsening group. Next, we calculated {mean Log2(RPM+1) value of the worsening group} - {mean Log2(RPM+1) value of the non-worsening group} and selected genes for which the absolute value was greater than 1. When we extracted genes with a p-value of less than 0.05 and an absolute difference in expression level data (Log2(RPM+1) value) greater than 1 as differentially expressed genes between the worsening group and the non-worsening group, 199 genes were found.

[0057] Example 2: Exploration of differentially expressed genes in groups with worsening skin pruritus versus groups without worsening symptoms using SSL-derived RNA (Case 2) 1) Obtaining scores and SSL collection related to the severity of skin itching in AD patients A similar study was conducted using 23 subjects (23-56 years old, male) from the subjects of Example 1. 14 days after the initial assessment in Example 1 (28 days after the initial assessment in Example 1), the subjects underwent the acquisition of a score related to the severity of skin itching due to Alzheimer's disease (AD) and the collection of an STI (Skin Stimulation Sample) in the same manner as in Example 1. 14 days after the 28th day (42 days after the initial assessment in Example 1), the subjects again acquired a score related to the severity of skin itching due to AD in the same manner as in Example 1. The subject's own whole-body skin itching VAS score (scoring the intensity of itching on a scale of 0 to 100) was used as the score related to the severity of skin itching due to AD. The itching VAS scores acquired on the 28th and 42nd days are referred to as the 28th day itching VAS score and the 42nd day itching VAS score, respectively, and the STI collected on the 28th day is referred to as the 28th day STI.

[0058] SSL was recovered from the entire face of each subject using an oil-absorbing film (5 x 8 cm, made of polypropylene, 3M) in the same manner as in Example 1. The oil-absorbing film was transferred to a vial and stored at -80°C for approximately one month until use for RNA extraction.

[0059] 2) RNA preparation and sequencing RNA preparation and sequencing were performed from the oil-absorbing film collected in 1) above, using the same method as in Example 1.

[0060] 3) Data used The itching VAS scores of 23 AD patients obtained in 1) above were compared with their itching VAS scores 14 days later (day 42) to calculate how much the itching VAS score changed over 14 days. More specifically, the value obtained by subtracting the itching VAS score on day 28 from the itching VAS score on day 42 for the same individual (Δ value) was calculated. According to the definition in Example 1, out of 23 AD patients, 8 subjects with a positive Δ value and a larger absolute value were selected as the worsening group, and 8 subjects with a negative Δ value and a larger absolute value were selected as the non-worsening group.

[0061] Of the read counts obtained by sequencing SSL-derived RNA from subjects on day 28 as measured in 2) above, the read counts of the eight subjects selected as the deterioration group and the eight subjects selected as the non-deterioration group were used as data for the expression level of each RNA. Genes whose amplification region in sequencing spanned at least two or more exons were selected as the genes to be analyzed. To correct for differences in total read counts between samples, the read counts of the genes to be analyzed were converted to RPM (Reads per million mapped reads) values. Of these, 5586 genes for which a read count of 20 or more was obtained in more than 90% of the samples were used for the following analysis. Furthermore, in order to approximate the RPM values ​​to a normal distribution, they were converted to base 2 logarithms (Log2(RPM+1) values) by adding the integer 1. Following the above procedure, expression level data (Log2(RPM+1) values) for 5586 genes from 16 subjects were created.

[0062] 4) Data Analysis Based on the expression level data (Log2(RPM+1) values) of 5586 genes derived from SSL on day 28, as prepared in 3) above, the expression levels of the worsening group and the non-worsening group were measured on day 28. We searched for genes with differential expression between the two groups: the worsening group and the non-worsening group. First, we selected genes for which the p-value in Welch's t-test was less than 0.05. Next, we calculated {mean Log2(RPM+1) value of the worsening group} - {mean Log2(RPM+1) value of the non-worsening group} and selected genes for which the absolute value was greater than 1. When we extracted genes with a p-value of less than 0.05 and an absolute difference in expression level data (Log2(RPM+1) value) greater than 1 as differentially expressed genes between the worsening group and the non-worsening group, 18 genes were found to fit this description.

[0063] Example 3: Comparison of differentially expressed genes between Case 1 and Case 2 The 199 differentially expressed genes extracted in Example 1 (Case 1) were compared with the 18 differentially expressed genes extracted in Example 2 (Case 2). As shown in Table 1, the expression level of the CBLB gene was significantly higher in the worsening group compared to the non-worsening group in both Case 1 and Case 2 (p-value less than 0.05, and the difference in expression level data was greater than 1). Therefore, it was suggested that the increased expression level of SSL-derived CBLB may be an indicator of an increase in the pruritus VAS score 14 days after SSL collection, or a worsening of the severity of generalized skin pruritus corresponding to the pruritus VAS score.

[0064] [Table 1]

Claims

1. A method for detecting the worsening of skin itching due to atopic dermatitis in a subject, comprising the step of measuring the mRNA expression level of the CBLB gene in a lipid sample taken from the surface of the subject's skin, (1) If the measured expression level of the mRNA is greater than the reference value, the subject will experience a worsening of skin itching symptoms. (2) The process includes the step of predicting that if the measured value of the mRNA expression level is equal to or less than the reference value, the subject will not experience a worsening of skin itching symptoms. The reference value is a reference value predetermined based on the relationship between whether or not the severity of skin itching has worsened and the mRNA expression level, and is determined as a value that distinguishes between the worsening group and the non-worsening group based on whether or not the severity of skin itching has worsened. Detection method.

2. The method according to claim 1, wherein the worsening of the severity of skin itching is a condition that occurs at a time between 12 and 16 days after the time of sample collection.

3. The method according to claim 1 or 2, wherein the severity of skin itching corresponds to the severity of skin itching due to atopic dermatitis, which is represented by a Visual Analog Scaling (VAS) score that scores the intensity of itching.

4. A test kit for detecting the worsening of the severity of skin itching due to atopic dermatitis, used in the method according to any one of claims 1 to 3, comprising an oligonucleotide that specifically hybridizes with the aforementioned gene.

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