WO2022186296A1 - Method for detecting deterioration in severity of atopic dermatitis - Google Patents

Abstract

Provided are: a method for detecting a deterioration in the severity of AD by using a marker for detecting a deterioration in the severity of AD; and said marker for detecting a deterioration in the severity of AD.　This method for detecting a deterioration in the severity of atopic dermatitis in a subject comprises a step for measuring the expression level of at least one gene or an expression product thereof in a biological sample collected from the subject, said at least one gene being selected from among two kinds of genes including NCLN and ZNF429.

Description

Method for detecting exacerbation of atopic dermatitis

The present invention relates to a method for detecting aggravation of atopic dermatitis using a detection marker for aggravation of atopic dermatitis.

Atopic dermatitis (hereinafter also referred to as "AD") is an eczematous skin disease that mainly develops in people with atopic predisposition. Typical symptoms of AD include chronic and recurrent itching, eruptions, erythema, etc., occurring bilaterally, as well as hypokeratosis, impaired barrier function, dry skin, and the like. Most AD develops in infants and tends to improve with growth, but adult-type and refractory AD are increasing in recent years. It is known that in AD, a variety of symptoms and phenotypes are formed due to the combined involvement of various etiologies, and exacerbations and remissions are repeated (Non-Patent Document 1). For example, it has been reported that about 40% of AD patients relapse after 14 days, and about 60% of AD patients after 28 days, if moisturizing is not continued after induction of remission with topical drugs (non-patented Reference 2). Therefore, in treating AD, it is necessary to correctly understand the severity of AD, including the diversity of symptoms and phenotypes, and its course.

　Conventional methods for evaluating the severity of AD include evaluation based on a doctor's observations with the naked eye. There are various findings such as dry symptoms, erythema, scales, papules, scratch marks, edema, adhesion of crusts, vesicles, erosions, and pruritic nodules. There are Eczema Area and Severity Index (EASI) and Severity SCORing of Atopic Dermatitis (SCORAD) as indexes that score these. On the other hand, AD is also being evaluated by patients themselves based on observations with the naked eye and self-awareness through the sense of touch. SCORAD) and Visual Analog Scaling (VAS) exist.

In addition, for objective understanding of disease pathology, genes or their expression products contained in skin biopsies, blood, stratum corneum, etc., and the presence of specific cell types (these are also collectively referred to as biomarkers) ) is often used. Conventionally, biomarkers for evaluating the presence or absence and severity of AD include blood peripheral blood eosinophil count, serum total IgE level, lactate dehydrogenase (LDH) level, serum Thymus and Activation-Regulated Chemokine (TARC), squamous cell carcinoma antigen 2 (SCCA2), etc. have been proposed (Non-Patent Documents 3 and 4).

In the treatment of AD, based on the above-mentioned evaluation indicators, the amount and type of drugs should be controlled under the appropriate treatment policy by the doctor, the remission state should be maintained as long as possible, and sudden deterioration should not occur. It is considered important to strive to For example, there are cases where latent inflammation continues in the tissues and cells inside the skin, even if symptoms do not appear externally or subjectively, and topical drugs are applied even during periods when symptoms do not appear. A therapeutic method that maintains a remission state by continuing moisturizing is called proactive therapy (Non-Patent Document 5). However, in the remission period when symptoms do not appear, treatment may be discontinued at the discretion of the patient, and sufficient explanation and guidance to AD patients are considered important.

However, the above-described conventional methods for evaluating the severity of various AD are intended to evaluate the presence or absence and severity of AD at the time of evaluation or biomarker collection, and predict the course of subsequent exacerbation or remission of symptoms. The reality is that it is not something to do. Therefore, objectively grasping whether AD symptoms will progress over the next 14 to 28 days is useful information for physicians to select treatment methods and for patient adherence to treatment methods. I can say

In recent years, techniques 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. Biological nucleic acids can be extracted from body fluids such as blood, secretions, tissues, and the like. 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 an AD marker gene can be detected from SSL (Patent Document 2).

[Patent Document 1] International Publication No. 2018/008319 [Patent Document 2] JP-A-2020-074769 [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.

The present invention relates to the following 1) to 4).
1) Atopic dermatitis of a subject, which comprises the step of measuring the expression level of at least one gene selected from two genes of NCLN and ZNF429, or an expression product thereof, for a biological sample collected from the subject. A method for detecting exacerbation of symptoms.
2) Use of at least one gene selected from two types of genes, NCLN and ZNF429, derived from a biological sample collected from a subject, or an expression product thereof as a marker for detecting exacerbation of atopic dermatitis.
3) Detecting aggravation of atopic dermatitis used in the method of 1), which contains 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 test kit for
4) A marker for detecting exacerbation of atopic dermatitis, comprising at least one gene selected from two genes, NCLN and ZNF429, or an expression product thereof.

The present invention relates to a method for detecting aggravation of AD using a detection marker for aggravation of AD, and to providing a detection marker for aggravation of AD.

The present inventors collected SSL from mild and moderate AD patients, and comprehensively analyzed the expression state of RNA contained in SSL as sequence information. As a result, the expression level of a specific gene was After a certain period of time from , there was a significant difference between patients whose symptoms worsened and those who did not, and it was found that the worsening of AD symptoms can be predicted using the gene as an index.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to easily predict whether or not the degree of symptoms will worsen in the near future in AD patients who often repeat exacerbation and remission of symptoms, and to assume future symptom deterioration for each patient. It will be possible to receive the optimal treatment and information provided.

Detailed description of the invention

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

In the present invention, the term "nucleic acid" or "polynucleotide" means 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.

In the present invention, the term "gene" refers to double-stranded DNA containing human genomic DNA, single-stranded DNA (positive strand) containing cDNA, and single-stranded DNA (complementary strand) having a sequence complementary to the positive strand. , and fragments thereof, in which some biological information is contained in the sequence information of bases that constitute DNA.
In addition, the "gene" in the present invention includes not only "gene" represented by a specific nucleotide sequence, but also its homologues (i.e., homologs or orthologs), mutants such as genetic polymorphisms, and derivatives. be.

In the present invention, the "expression product" of a gene is a concept that includes transcription products and translation products of genes. A "transcription product" is RNA produced by transcription from a gene (DNA), and a "translation product" means a protein encoded by a gene that is translated and synthesized based on RNA.

In the present invention, "atopic dermatitis (AD)" refers to a disease whose main pathogen is itchy eczema with repeated exacerbations and remissions, and many of its patients are said to have atopic predisposition. . Examples of atopic predisposition include i) family history and medical history (one or more of bronchial asthma, allergic rhinitis/conjunctivitis, and atopic dermatitis), or ii) a predisposition to easily produce IgE antibodies. be done.

In the present invention, the "severity" of AD refers to the level of symptoms of AD, and includes not only rough classifications such as mild, moderate, and severe, but also classifications based on minor differences. The "severity" of AD can be determined, for example, based on various known evaluation scores for evaluating AD symptoms. In the present invention, the evaluation score is referred to as an "AD severity score". Examples of scores related to the severity of AD include EASI score and POEM score related to systemic rash due to AD, VAS score for itchy skin due to AD, VAS score for dry skin due to AD (atopic dermatitis clinical practice guideline, Japanese Dermatological Association, Japan Dermatological Society: 128(12), 2431-2502 (2018)), preferably EASI score and POEM score related to systemic rash due to AD, more preferably AD EASI score for whole body rash. As the “severity”, the score itself relating to the severity of AD may be used as the level of symptoms of AD.

In the present invention, "detection" of exacerbation of AD symptoms can also be rephrased with terms such as inspection, measurement, judgment, or evaluation support. The terms "detection", "examination", "measurement", "determination" or "evaluation" of exacerbation of AD in the present invention do not include diagnosis of exacerbation of AD by a doctor.

As shown in the examples described later, AD patients with mild and moderate severity were used as subjects, and an observation period of 42 days (6 weeks) was provided. On day 4 (week 4)), SSL was collected, and expression analysis of RNA contained in SSL was performed according to the following procedures 1) to 4). 14 days (2 weeks) after SSL sampling (14th day (2nd week) and 42nd day (6th week)) patient group (aggravated group) and patients who are not When the RNA expression levels of the groups (non-aggravated group) were compared, it was found that the expression levels of the two genes, NCLN and ZNF429, were significantly elevated in the aggravated group at any time.
1) Acquire expression level data (read count value) of RNA extracted from SSL.
2) Convert the read count value to an RPM value corrected for the difference in the total number of reads between samples, and add an integer 1 to the RPM+1 value converted to a base 2 logarithm value (Log ₂ (RPM+1) value) is used as an index of the expression level, and based on this, genes with different expression levels between the aggravated group and the non-aggravated group are selected.
Specifically, first, genes with a p-value of less than 0.05 in Welch's t-test between two groups, an exacerbated group and a non-exacerbated group, are selected. Here, "p value" indicates the probability of observing a statistic that is more extreme than the statistic actually calculated from the data under the null hypothesis in a statistical test. Therefore, the smaller the "p value", the more significant the difference between the comparison subjects.
3) Next, genes with a mean expression level difference of more than 1 between the exacerbated group and the non-exacerbated group are selected.
Specifically, {mean value of Log ₂ (RPM+1) value of exacerbated group}−{mean value of Log ₂ (RPM+1) value of non-exacerbated group} is calculated, and genes whose absolute value is greater than 1 are selected. .
4) Genes with a p-value of less than 0.05 between the exacerbated group and the non-exacerbated group and a difference in expression level of more than 1 are selected as expression-variable genes between the exacerbated group and the non-exacerbated group. do.

Therefore, a gene or its expression product selected from the two gene groups of NCLN and ZNF429 can serve as a detection marker for exacerbation of symptoms.
Here, the gene names "NCLN" and "ZNF429" follow the Official Symbol described in NCBI ([www.ncbi.nlm.nih.gov/]), and the Gene IDs are NCLN 56926 and ZNF429. is 353088.

In the present invention, "detection of exacerbation of symptoms" refers to detecting the presence or absence of exacerbation of AD symptoms of the subject at a time point after a period separated from the time point of sample collection. That is, the detection of exacerbation predicts the presence or absence of exacerbation at a time point separated from the sampling time point.
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 still more preferably 14 days.

In the present invention, "exacerbation of severity" refers to AD severity worse than the current level (at the time of sample collection). As described above, the "severity" of AD can be determined based on various known evaluation scores for evaluating AD symptoms. That is, EASI score and POEM score for systemic rash due to AD, VAS score for itchy skin due to AD, VAS score for dry skin due to AD (Atopic dermatitis clinical practice guideline, published by the Japanese Dermatological Association, Nisshinkai: 128 (12), 2431-2502 (2018)), etc. will deteriorate.
Of these, in the present invention, it is preferable to use the EASI score and the POEM score related to systemic eruptions due to AD, and it is more preferable to use the evaluation by the EASI score.
EASI evaluates the head and neck, trunk, upper limbs, and lower extremities, with four symptom-specific scores of erythema, edema/infiltration/papules, scratch marks, and lichenification at each evaluation site. It is a value between 0 and 72 calculated based on the percentage (%) of the area of the 4 symptoms in (Hanifin et al. Exp Dermatol, 10, 2001). When the EASI score worsens, for example, the existing severity classification using the EASI score (Chopra et al. Br J Dermatol.177, 2017) not limited to cases where the severity worsens from mild to moderate, All cases where the EASI score increases compared to the current EASI score (at the time of sampling) are included.

In addition, the gene that can be a detection marker for aggravation of AD (hereinafter also referred to as "target gene") includes DNA that constitutes the gene, as long as it can be a biomarker for detecting aggravation of AD. Genes having substantially the same nucleotide sequence as the nucleotide sequence of are also included. Here, the substantially identical base sequence is, for example, using the homology calculation algorithm NCBI BLAST, expected value = 10; gaps allowed; filtering = ON; match score = 1; mismatch score = -3 It means that it has 90% or more, preferably 95% or more, more preferably 98% or more, and still more preferably 99% or more identity with the base sequence of the DNA constituting the gene when searched with .

In the method for detecting exacerbation of AD symptoms of the present invention, a biological sample collected from a subject is subjected to a target gene, as one aspect, at least one gene selected from two types of genes, NCLN and ZNF429, or an expression product thereof. including the step of measuring expression levels.

In the method for detecting exacerbation of AD symptoms of the present invention, the subject's sex, age, race, etc. are not particularly limited, and may include infants to the elderly. Preferably, the subject is a human needing or desiring detection of exacerbation of AD. For example, the subject is a human developing atopic dermatitis, a human suspected of developing atopic dermatitis, or a human genetically predisposed to atopic dermatitis.

The biological samples used in the present invention may be cells, tissues, and biomaterials in which the expression of the gene of the present invention changes as AD worsens. Specific examples include organs, skin, blood, urine, saliva, sweat, stratum corneum, superficial skin lipids (SSL), body fluids such as tissue exudate, serum prepared from blood, plasma, feces, hair, and the like. preferably skin, stratum corneum, or superficial skin lipids (SSL), more preferably superficial skin lipids (SSL). The site of the skin from which the SSL is collected is not particularly limited, and includes any site of the body such as the head, face, neck, trunk, limbs, etc., and sites with high sebum secretion, such as the head or face. skin is preferred, and facial skin is more preferred. In addition, the site of the skin from which SSL is collected may be either an erupted area where atopic dermatitis develops or an erupted area where atopic dermatitis does not develop. A non-erupted area near the erupted area is preferred. Here, the vicinity of the rash refers to a range within 10 cm adjacent to the rash.

Here, "superficial skin lipid (SSL)" refers to the fat-soluble fraction present on the surface of the skin, and is sometimes called sebum. In general, SSL mainly contains secretions secreted 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. (Refer to said patent document 1). In the present invention, unless otherwise specified, "skin" is a general term for areas including stratum corneum, epidermis, dermis, hair follicles, and tissues such as sweat glands, sebaceous glands and other glands.

Any means used to collect or remove SSL from the skin can be used to collect SSL from the subject's skin. Preferably, an SSL absorbent material, an SSL adhesive material, or an instrument that scrapes the SSL off the skin, as described below, can be used. The SSL absorbent material or SSL adhesive material is not particularly limited as long as it has affinity for SSL, and examples thereof include polypropylene and pulp. More detailed examples of procedures for collecting SSL from the skin include a method of absorbing SSL into sheet-like materials such as blotting paper and blotting film, a method of adhering SSL to a glass plate, tape, etc., a spatula, a scraper, etc. and a method of scraping off and recovering the SSL. In order to improve the adsorptivity of SSL, an SSL absorbent material previously impregnated with a solvent having high fat solubility may be used. On the other hand, if the SSL absorptive material contains a highly water-soluble solvent or moisture, the adsorption of SSL is inhibited, so it is preferable that the content of the highly water-soluble solvent and moisture is small. The SSL absorbent material is preferably used dry.

　The RNA-containing SSL collected from the subject may be stored for a certain period of time. The collected SSL is preferably stored under low temperature conditions as soon as possible after collection in order to minimize degradation of the contained RNA. The temperature condition for storing the RNA-containing SSL in the present invention may be 0°C or lower, preferably -20±20°C to -80±20°C, more preferably -20±10°C to -80±10°C. , More preferably -20 ± 20°C to -40 ± 20°C, more preferably -20 ± 10°C to -40 ± 10°C, more preferably -20 ± 10°C, still more preferably -20 ± 5°C . The storage period of the RNA-containing SSL under the low-temperature conditions is not particularly limited, but is preferably 12 months or less, for example, 6 hours or more and 12 months or less, more preferably 6 months or less, for example, 1 day or more and 6 months or less, More preferably, it is 3 months or less, for example, 3 days or more and 3 months or less.

In the present invention, targets for measuring the expression level of the target gene or its expression product include cDNA artificially synthesized from RNA, DNA encoding the RNA, proteins encoded by the RNA, and interactions with the proteins. molecules that interact with the RNA, molecules that interact with the DNA, and the like. Here, molecules that interact with RNA, DNA or protein include DNA, RNA, protein, polysaccharides, oligosaccharides, monosaccharides, lipids, fatty acids, phosphorylated products thereof, alkylated products, sugar adducts, etc., and Any one of the above complexes may be mentioned. In addition, the expression level comprehensively means the expression level and activity of the gene or expression product.

In the method of the present invention, as a preferred embodiment, SSL is used as a biological sample. In this case, the expression level of RNA contained in SSL is analyzed, specifically after converting RNA into cDNA by reverse transcription. , the cDNA or its amplification product is measured.
For extraction of RNA from SSL, methods commonly used to extract or purify RNA from biological samples, such as the phenol/chloroform method, the AGPC (acid guanidinium thiocyanate-phenol-chloroform extraction) method, or TRIzol® ), a method using a column such as RNeasy (registered trademark), QIAzol (registered trademark), a method using special magnetic particles coated with silica, a method using Solid Phase Reversible Immobilization magnetic particles, a commercially available method such as ISOGEN Extraction with an RNA extraction reagent or the like can be used.

For the reverse transcription, primers targeting specific RNAs to be analyzed may be used, but random primers are preferably used for more comprehensive nucleic acid storage and analysis. A common reverse transcriptase or reverse transcription reagent kit can be used for the reverse transcription. Preferably, a highly accurate and efficient reverse transcriptase or reverse transcription reagent kit is used, examples of which include M-MLV Reverse Transcriptase and variants thereof, or commercially available reverse transcriptase or reverse transcription reagent kit, Examples include PrimeScript (registered trademark) Reverse Transcriptase series (Takara Bio Inc.) and SuperScript (registered trademark) Reverse Transcriptase series (Thermo Scientific). SuperScript (registered trademark) III Reverse Transcriptase, SuperScript (registered trademark) VILO cDNA Synthesis kit (both from Thermo Scientific) and the like are preferably used.
In the elongation reaction in the reverse transcription, the temperature is preferably adjusted to 42°C ± 1°C, more preferably 42°C ± 0.5°C, even more preferably 42°C ± 0.25°C, while the reaction time is preferably It is preferable to adjust the time to 60 minutes or more, more preferably 80 to 120 minutes.

Examples of methods for measuring expression levels include PCR, real-time RT-PCR, multiplex PCR, SmartAmp, LAMP, etc., using DNAs that hybridize to RNA, cDNA, or DNA as primers. nucleic acid amplification methods, hybridization methods using nucleic acids that hybridize to these as probes (DNA chips, DNA microarrays, dot blot hybridization, slot blot hybridization, Northern blot hybridization, etc.), methods for determining base sequences ( sequencing), or a combination thereof.

In PCR, a primer pair targeting a specific DNA to be analyzed may be used to amplify only one specific DNA, but multiple primer pairs may be used to amplify a plurality of specific DNAs at the same time. good too. Preferably, said PCR is multiplex PCR. Multiplex PCR is a method for simultaneously amplifying multiple gene regions by simultaneously using multiple primer pairs in a PCR reaction system. Multiplex PCR can be performed using a commercially available kit (eg, Ion AmpliSeq Transcriptome Human Gene Expression Kit; Life Technologies Japan Co., Ltd., etc.).
The temperature of the annealing and extension reaction in the PCR depends on the primers used and cannot be generalized. .5°C, more preferably 62°C ± 0.25°C. Therefore, in the PCR, annealing and extension reactions are preferably performed in one step. The time for the annealing and extension reaction steps can be adjusted depending on the size of the DNA to be amplified, etc., but is preferably 14 to 18 minutes. The denaturation reaction conditions in the PCR can be adjusted depending on the DNA to be amplified, but are preferably 95-99° C. for 10-60 seconds. Reverse transcription and PCR at temperatures and times as described above can be performed using a thermal cycler commonly used for PCR.

Purification of the reaction product obtained by the PCR is preferably carried out by size separation of the reaction product. Size separation allows separation of the desired PCR reaction product from primers and other impurities contained in the PCR reaction. Size separation of DNA can be performed by, for example, a size separation column, a size separation chip, magnetic beads that can be used for size separation, or the like. Preferred examples of magnetic beads that can be used for size separation include Solid Phase Reversible Immobilization (SPRI) magnetic beads such as Ampure XP.

Purified PCR reaction products may be subjected to further processing necessary for subsequent quantitative analysis. For example, for DNA sequencing, a purified PCR reaction product is prepared into an appropriate buffer solution, a PCR primer region contained in PCR amplified DNA is cleaved, an adapter sequence is added to the amplified DNA, and an adapter sequence is added to the amplified DNA. may be added. For example, a purified PCR reaction product is prepared in a buffer solution, PCR primer sequences are removed from the amplified DNA and adapter ligation is performed, and the resulting reaction product is amplified as necessary for quantitative analysis. of libraries can be prepared. These operations are performed, for example, using the 5x VILO RT Reaction Mix attached to the SuperScript (registered trademark) VILO cDNA Synthesis kit (Life Technologies Japan Co., Ltd.) and the Ion AmpliSeq Transcriptome Human Gene Expression Kit (Life Technologies Japan Co., Ltd.) 5×Ion AmpliSeq HiFi Mix and Ion AmpliSeq Transcriptome Human Gene Expression Core Panel attached to the kit can be used according to the protocol attached to each kit.

When measuring the expression level of a target gene or a nucleic acid derived therefrom using the Northern blot hybridization method, for example, the probe DNA is first labeled with a radioactive isotope, a fluorescent substance, or the like, and then the resulting labeled DNA is labeled. , and hybridize with biological sample-derived RNA transferred to a nylon membrane or the like according to a conventional method. After that, there is a method of measuring the formed double strand of labeled DNA and RNA by detecting a signal derived from the label.

When the expression level of a target gene or a nucleic acid derived therefrom is measured using the RT-PCR method, for example, first, cDNA is prepared from RNA derived from a biological sample according to a conventional method, and the target gene of the present invention is obtained using this as a template. A pair of primers prepared for amplification (the positive strand that binds to the above cDNA (− strand) and the reverse strand that binds to the + strand) is hybridized with this. After that, 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 for detecting the labeled double-stranded DNA produced by performing the above-mentioned PCR using primers previously labeled with RI, a fluorescent substance, etc. is used. can be done.

When measuring the expression level of a target gene or a 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. , mRNA expression level can be measured by binding labeled cDNA or cRNA prepared from mRNA onto a microarray and detecting the label on the microarray.
The nucleic acids immobilized on the array may be nucleic acids that hybridize specifically (that is, substantially only to the target nucleic acid) under stringent conditions. It may be a nucleic acid having a sequence or a nucleic acid consisting of a partial sequence. Here, the “partial sequence” includes nucleic acids consisting of at least 15 to 25 bases. Here, stringent conditions usually include washing conditions of about "1×SSC, 0.1% SDS, 37° C.", and more stringent hybridization conditions are "0.5×SSC, 0.1% SDS. % SDS, about 42° C.”, and a more stringent hybridization condition is about “0.1×SSC, 0.1% SDS, 65° C.”. Hybridization conditions are described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Thrd Edition, Cold Spring Harbor Laboratory Press (2001) and others.

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

Probes or primers used for the above measurements, that is, primers for specifically recognizing and amplifying the target gene of the present invention or nucleic acids derived therefrom, or for specifically detecting the RNA or nucleic acids derived therefrom Probes fall into this category, and they can be designed based on the nucleotide sequence that constitutes the target gene. Here, "specifically recognize" means that substantially only the target gene of the present invention or a nucleic acid derived therefrom can be detected, for example, in Northern blotting, and substantially only the nucleic acid in RT-PCR, for example. is amplified, it means that the detected product or product can be determined to be the gene or the nucleic acid derived therefrom.
Specifically, an oligonucleotide containing a certain number of nucleotides complementary to a DNA consisting of a nucleotide sequence constituting the target gene of the present invention or its complementary strand can be used. As used herein, the term "complementary strand" refers to one strand of a double-stranded DNA consisting of base pairs of A:T (U in the case of RNA) and G:C against the other strand. In addition, "complementary" is not limited to the case of a completely complementary sequence in the certain number of contiguous nucleotide regions, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, more preferably 80% or more, more preferably 90% or more More preferably, they should have 98% or more of nucleotide sequence identity. The identity of nucleotide sequences can be determined by algorithms such as BLAST.
When such oligonucleotides are used as primers, they only need to be capable of specific annealing and chain extension. Those having a chain length of preferably 50 bases or less, more preferably 35 bases or less are included. In addition, when used as a probe, it only needs to be capable of specific hybridization, and has a sequence of at least a part or the whole of DNA (or its complementary strand) consisting of the base sequence that constitutes the target gene of the present invention. Those having 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, more preferably 25 bases or less are used.
Here, "oligonucleotide" can be DNA or RNA, and may be synthetic or natural. Also, the probes used for hybridization are usually labeled ones.

In addition, protein chip analysis, immunoassay ( ELISA, etc.), mass spectrometry (e.g., LC-MS/MS, MALDI-TOF/MS), 1-hybrid method (PNAS 100, 12271-12276 (2003)) and 2-hybrid method (Biol. Reprod. 58 , 302-311 (1998)) can be used, and can be appropriately selected according to the subject.
For example, when a protein is used as a measurement target, an antibody that specifically recognizes the expression product of the present invention, specifically a structural characteristic site ( epitope) is brought into contact with a biological sample, the polypeptide or protein in the sample that binds to the antibody is detected, and the level is measured. For example, according to Western blotting, after using the above antibody as a primary antibody, an antibody that binds to the primary antibody labeled with a radioisotope, fluorescent substance, enzyme, etc. is used as a secondary antibody, and the primary antibody is Labeling is performed, and signals derived from these labeling substances are measured with a radiometer, a fluorescence detector, or the like.
The antibody against the translation product may be either a polyclonal antibody or a monoclonal antibody. These antibodies can be produced according to known methods. Specifically, a polyclonal antibody is obtained by immunizing a non-human animal such as a rabbit using a protein expressed in Escherichia coli or the like and purified according to a conventional method, or by synthesizing a partial polypeptide of the protein according to a conventional method, It can be obtained from the serum of the immunized animal according to a conventional method.
On the other hand, monoclonal antibodies are obtained by immunizing a non-human animal such as a mouse with a protein expressed in Escherichia coli or the like and purified according to a conventional method or a partial polypeptide of the protein, and fusing the obtained spleen cells with myeloma cells. It can be obtained from prepared hybridoma cells. Monoclonal antibodies may also be generated using phage display (Griffiths, AD; Duncan, AR, Current Opinion in Biotechnology, Volume 9, Number 1, February 1998, pp. 102-108(7)).

Thus, the expression level of the target gene of the present invention or its expression product in a biological sample collected from a subject is measured, and exacerbation of AD symptoms is detected based on the expression level. Specifically, 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 preset cutoff value (reference value).
When analyzing the expression levels of a plurality of target genes by sequencing, as described above, the read count value, which is the expression level data, and the RPM value obtained by correcting the difference in the total read number between samples , a value obtained by converting the RPM value to a base 2 logarithmic value (Log ₂ RPM value) or a base 2 logarithmic value obtained by adding an integer 1 (Log ₂ (RPM + 1) value), or DESeq2 (Love MI et al. Genome Biol 2014) or the base 2 logarithm (Log ₂ (Normalized count+1) value) obtained by adding the integer 1 is preferably used as an index. In addition, it is calculated by fragments per kilobase of exon per million reads mapped (FPKM), reads per kilobase of exon per million reads mapped (RPKM), transcripts per million (TPM), etc., which are commonly used as quantitative values for RNA-seq. can be a value. Alternatively, it may be a signal value obtained by a microarray method and its correction value. In addition, when only a specific target gene is analyzed by RT-PCR, etc., a method of converting the expression level of the target gene into a relative expression level based on the expression level of the housekeeping gene and analyzing it, or A method of quantifying the absolute copy number using a plasmid containing the region of the target gene (absolute quantification) and analyzing is preferred. It may be a copy number obtained by a digital PCR method.
Here, the "control level" includes the expression level of the target gene or its expression product in a patient population whose symptoms did not worsen for a certain period of time. The expression level may be a value determined with reference 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. A “reference value” can be determined in advance based on the relationship between whether or not the severity of the disease has deteriorated over a certain period of time and the expression level of the target marker. For example, divide a population into an exacerbated group and a non-exacerbated group based on whether the symptoms have worsened over a certain period of time. can be determined as a reference value for determining belonging to each group. When multiple types of genes are used as target genes, it is preferable to obtain control levels and reference values for each gene or its expression product.

Furthermore, measurement of the expression level of the target gene or its expression product derived from AD patients whose symptoms worsen within a certain period of time and the expression level of the target gene or its expression product derived from AD patients whose symptoms do not worsen within a certain period of time Using the value, construct a discriminant (prediction model) that divides a patient group with worsening symptoms and a patient group without worsening symptoms, and use the discriminant to detect worsening AD symptoms be able to. That is, measurement of the expression level of the target gene or its expression product derived from AD patients whose symptoms worsen within a certain period of time and the expression level of the target gene or its expression product derived from AD patients whose symptoms do not worsen within a certain period of time A discriminant (prediction model) that separates a group of patients whose symptoms worsen (aggravated group) and a group of patients whose symptoms do not worsen (non-aggravated group) is constructed using the values as a teacher sample, and the severity is based on the discriminant A cut-off value (reference value) for discriminating each patient group with different exacerbations is obtained.
Then, the level of the target gene or its expression product is similarly measured from the biological sample collected from the subject, the obtained measured value is substituted into the discriminant, and the result obtained from the discriminant is compared with the reference value. By doing so, exacerbation of AD in the subject can be detected.

As an algorithm for constructing the discriminant, a known algorithm such as an algorithm used for machine learning can be used. Examples of machine learning algorithms include Random forest, linear kernel support vector machine (SVM linear), rbf kernel support vector machine (SVM rbf), neural network, generalized linear model model), regularized linear discriminant analysis, regularized logistic regression, and the like. Input verification data into the constructed prediction model to calculate the prediction value, and select the model whose prediction value best matches the actual measurement value, for example, the model with the highest accuracy rate, as the optimum prediction model. can be done. In addition, the detection rate (Recall), the precision (Precision), and the F value, which is their harmonic average, are calculated from the predicted value and the measured value, and the model with the largest F value can be selected as the optimum prediction model. .

The method of determining the cutoff value (reference value) is not particularly limited, and can be determined according to a known method. For example, it can be obtained from an ROC (Receiver Operating Characteristic Curve) curve created using a discriminant. In the ROC curve, the vertical axis is the probability of positive results in positive patients (sensitivity), and the horizontal axis is the value obtained by subtracting the probability of negative results in negative patients (specificity) from 1 (false positive rate). be done. Regarding "true positive (sensitivity)" and "false positive (1-specificity)" shown in the ROC curve, "true positive (sensitivity)" - "false positive (1-specificity)" is the maximum value (Youden index) can be used as a cutoff value (reference value).

The test kit for detecting exacerbation of AD symptoms of the present invention contains test reagents for measuring the expression level of the target gene of the present invention or its expression product in a biological sample isolated from a patient. . Specifically, a reagent for nucleic acid amplification or hybridization containing an oligonucleotide (e.g., primer for PCR) that specifically binds (hybridizes) to the target gene of the present invention or a nucleic acid derived therefrom, or Reagents for immunoassays containing antibodies that recognize the expression product (protein) of the target gene of the present invention, and the like. Oligonucleotides, antibodies and the like included in the kit can be obtained by known methods as described above.
In addition to the above antibodies and nucleic acids, the test kit also contains labeling reagents, buffers, chromogenic substrates, secondary antibodies, blocking agents, tools necessary for testing, control reagents used as positive and negative controls, Equipment for collecting biological samples (eg, blotting film for collecting SSL, etc.) and the like can be included.

The following aspects are further disclosed in this invention regarding embodiment mentioned above.
<1> Atopic dermatitis of the subject, including the step of measuring the expression level of at least one gene or its expression product selected from two types of genes, NCLN and ZNF429, for a biological sample collected from the subject. A method for detecting exacerbation of symptoms.
<2> The method of <1>, further comprising comparing the measured expression level with a control level or a cutoff value (reference value).
<3> The method of <2>, further comprising the step of determining the control level or cutoff value (reference value).
<4> Further comprising the step of detecting aggravation of atopic dermatitis using a discriminant (prediction model) that separates a patient group with worsening symptoms and a patient group without worsening symptoms, <1> the method of.
<5> The method of <4>, further comprising the step of constructing the discriminant (prediction model).
<6> The method according to any one of <1> to <5>, wherein the exacerbation of symptoms is a condition after a period of 12 days or more and 16 days or less from the time of sample collection.
<7> The method according to any one of <1> to <6>, wherein the severity is the severity of atopic dermatitis for the whole body, corresponding to the Eczema Area and Severity Index.
<8> The method according to any one of <1> to <7>, wherein the expression level of the gene or its expression product is the measurement of the mRNA expression level.
<9> The method according to any one of <1> to <8>, wherein the gene or its expression product is RNA contained in the skin surface lipid of the subject.
<10> The method according to any one of <1> to <9>, wherein the subject is a human who needs or desires detection of exacerbation of atopic dermatitis.
<11> The subject is a human who is developing atopic dermatitis, a human suspected of developing atopic dermatitis, or a human genetically predisposed to atopic dermatitis, <1> to <9 > either method.
<12> Use of at least one gene or expression product thereof selected from two types of genes, NCLN and ZNF429, derived from a biological sample collected from a subject as a marker for detecting exacerbation of atopic dermatitis.
<13> The use of <12>, wherein the exacerbation of symptoms is a condition after a period of 12 days or more and 16 days or less from the time of sample collection.
<14> The use of <12> or <13>, which is the severity of atopic dermatitis for the whole body and whose severity corresponds to the Eczema Area and Severity Index.
<15> The use of any one of <12> to <14>, wherein the gene or its expression product is mRNA contained in the lipids on the skin surface of the subject.
<16> Atopy used in any of the methods <1> to <11>, which contains 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 A test kit for detecting exacerbation of dermatitis.
<17> A marker for detecting aggravation of atopic dermatitis, comprising at least one gene selected from two genes, NCLN and ZNF429, or an expression product thereof.
<18> The marker of <17>, wherein the severity corresponds to the Eczema Area and Severity Index and is the severity of atopic dermatitis targeting the whole body.

EXAMPLES The present invention will be described in more detail below based on examples, but the present invention is not limited to these.
Example 1 Search for expression-changed genes in exacerbated group versus non-exacerbated group using SSL-derived RNA (Case 1)
1) Acquisition of Severity Score and SSL Collection of AD Patients Twenty-five adults (23-56 years old, male) with AD were used as subjects. Subjects were AD patients who had been diagnosed with mild or moderate AD by a dermatologist at the time of the first measurement. Subjects underwent acquisition of AD severity scores and SSL sampling at the first measurement. Fourteen days after the first measurement, the subjects again underwent an AD severity score. Physician's systemic EASI score (Hanifin et al. Exp dermatol. 10, 2001, symptoms are scored from 0 to 72 based on systemic rash) was used as a score for AD severity. The EASI scores obtained at the time of the initial measurement and 14 days after that are referred to as the initial EASI score and the EASI score on the 14th day, respectively, and the SSL obtained at the time of the initial measurement is referred to as the initial SSL.

As mentioned above, SSL refers to lipids on the skin surface, and was collected from each subject's entire face using an oil removing film (5 x 8 cm, made of polypropylene, 3M). The blotting films were transferred to vials and stored at −80° C. for approximately one month before use for RNA extraction.

2) RNA Preparation and Sequencing The blotting film of 1) above was cut into pieces of appropriate size, and RNA was transferred to the aqueous layer using QIAzol Lysis Reagent (Qiagen) according to the attached protocol. RNA was extracted from the aqueous layer using a commercially available RNA extraction kit using a spin column for RNA extraction according to the attached protocol. The extracted RNA was reverse transcribed at 42° C. for 90 minutes using SuperScript VILO cDNA Synthesis kit (Life Technologies Japan) to synthesize cDNA. Random primers attached to the kit were used as primers for the reverse transcription reaction. A library containing DNA derived from the 20802 gene was prepared from the resulting cDNA by multiplex PCR. Multiplex PCR was performed using Ion AmpliSeq Transcriptome Human Gene Expression Kit (Life Technologies Japan Co., Ltd.) [99 ° C., 2 minutes → (99 ° C., 15 seconds → 62 ° C., 16 minutes) × 20 cycles → 4 ° C., Hold ]. The resulting PCR product was purified with Ampure XP (Beckman Coulter, Inc.) and then subjected to buffer reconstitution, primer sequence digestion, adapter ligation and purification, and amplification to prepare a library. The prepared library was loaded into the Ion 540 Chip and sequenced using the Ion S5/XL system (Life Technologies Japan). Each read sequence obtained by sequencing was genetically mapped using hg19 AmpliSeq Transcriptome ERCC v1, which is a reference sequence of the human genome, to determine the gene from which each read sequence was derived.

3) Usage data Compare the EASI score at the time of the first measurement (first time) of the 25 AD patients obtained in 1) above with the EASI score 14 days later (day 14), and how much the EASI score changed over the 14 days. calculated whether More specifically, a value (Δ value) was calculated by subtracting the initial EASI score from the same individual's EASI score on day 14. When the Δ value is a positive value, it is defined as “worse”, and when the value is negative or zero, it is defined as “non-worse”. grouped.

The read count of each read in the initial sequencing of the SSL-derived RNA of the subject measured in 2) above was used as data for the expression level of each RNA. A gene whose amplification region in sequencing spans at least two or more exons is the target gene for analysis, and the read count of the target gene is RPM (Reads per million mapped reads) in order to correct the difference in the total read count between samples. converted to a value. Among them, 5453 genes with a read count of 20 or more in 90% or more 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 logarithmic values (Log ₂ (RPM+1) values) with an integer of 1 added. Through the above procedure, expression level data (Log ₂ (RPM+1) values) of 5453 genes from 25 subjects were created.

4) Data analysis Based on the initial SSL-derived 5453 gene expression level data (Log ₂ (RPM + 1) value) created in 3) above, there is a difference in the expression level between the exacerbated group and the non-exacerbated group at the time of the initial measurement. searched for genes. First, genes with a p-value of less than 0.05 in Welch's t-test between the exacerbated group and the non-exacerbated group were selected. Next, {mean value of Log ₂ (RPM+1) values in the exacerbated group}−{mean value of Log ₂ (RPM+1) values in the non-exacerbated group} was calculated, and genes whose absolute value was greater than 1 were selected. Genes with a p-value of less than 0.05 and an absolute value of the difference in expression level data (Log ₂ (RPM+1) value) greater than 1 were extracted as expression-variable genes between the exacerbated group and the non-exacerbated group. , 78 genes corresponded.

Example 2 Exploration of expression-changed genes in exacerbated group versus non-exacerbated group using SSL-derived RNA (Case 2)
1) Acquisition of Score Relating to AD Patient's Symptom and SSL Extraction A similar test was conducted using 23 subjects (23 to 56 years old, male) out of the subjects of Example 1 as subjects. After 14 days from the first day in Example 1 (28 days from the first day in Example 1), the subject obtained a score related to the severity of AD and collected SSL in the same manner as in Example 1. received. After an additional 14 days after Day 28 (Day 42 from the first day of Example 1), the subjects were again scored for AD severity as in Example 1. The EASI scores obtained on Days 28 and 42 are referred to as Day 28 and Day 42 EASI scores, respectively, and the SSL taken on Day 28 is referred to as Day 28 SSL.

In the same manner as in Example 1, SSL was collected from the entire face of each subject using an oil removing film (5 x 8 cm, polypropylene, 3M company). The blotting films were transferred to vials and stored at −80° C. for approximately one month before use for RNA extraction.

2) RNA Preparation and Sequencing By the same method as in Example 1, RNA was prepared and sequenced from the blotting film collected in 1) above.

3) Use data Compare the EASI score on day 28 of the 23 AD patients obtained in 1) above with the EASI score 14 days later (day 42), and see how much the EASI score changed over the 14 days. Calculated. More specifically, a value (Δ value) was calculated by subtracting the EASI score on the 28th day from the EASI score on the 42nd day of the same individual. According to the definition of Example 1, 23 subject AD patients were grouped into a 7 exacerbation group and a 16 non-exacerbation group.

The read count of each read obtained by sequencing the SSL-derived RNA of the subject measured in 2) above on day 28 was used as data for the expression level of each RNA. A gene that spans at least two exons in which the region to be amplified by sequencing was used as the target gene for analysis. To correct for differences in total read counts between samples, the read counts of the analyzed gene were converted to RPM (Reads per million mapped reads) values. Among them, 5991 genes with a read count of 20 or more in 90% or more 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 logarithmic values (Log ₂ (RPM+1) values) with an integer of 1 added. Through the above procedure, expression level data (Log ₂ (RPM+1) values) of 5991 genes from 23 subjects were created.

4) Data analysis Based on the expression level data (Log ₂ (RPM + 1) value) of 5991 genes derived from SSL on day 28 created in 3) above, the expression levels in the exacerbated group and the non-exacerbated group at the time of measurement on day 28 We searched for genes with differences in First, genes with a p-value of less than 0.05 in Welch's t-test between the exacerbated group and the non-exacerbated group were selected. Next, {mean value of Log ₂ (RPM+1) values in the exacerbated group}−{mean value of Log ₂ (RPM+1) values in the non-exacerbated group} was calculated, and genes whose absolute value was greater than 1 were selected. Genes with a p-value of less than 0.05 and an absolute value of the difference in expression level data (Log ₂ (RPM+1) value) greater than 1 were extracted as expression-variable genes between the exacerbated group and the non-exacerbated group. , 33 genes corresponded.

Example 3 Comparison of Variable Expression Genes in Case 1 and Case 2 The 78 variable expression genes extracted in Example 1 (Case 1) above and the 33 variable expression genes extracted in Example 2 (Case 2) above. compared. As a result, as shown in Table 1, the expression levels of two genes, NCLN and ZNF429, were significantly elevated in the exacerbated group compared to the non-exacerbated group in both cases 1 and 2 (p value was less than 0.05 and the difference in expression level data was greater than 1). Therefore, the increase in the expression levels of SSL-derived NCLN and ZNF429 may be a sign of an increase in the EASI score 14 days after the SSL collection, or a worsening of systemic AD symptoms corresponding to the EASI score. was done.

 

Claims (9)

1. exacerbation of atopic dermatitis in a subject, comprising the step of measuring the expression level of at least one gene or its expression product selected from two genes, NCLN and ZNF429, in a biological sample collected from the subject detection method.
2. The method according to claim 1, wherein the exacerbation of symptoms is the state after a period of 12 days or more and 16 days or less from the time of sampling.
3. The method according to claim 1 or 2, wherein the severity is the severity of atopic dermatitis for the whole body corresponding to the Eczema Area and Severity Index.
4. The method according to any one of claims 1 to 3, wherein the expression level of the gene or its expression product is a measurement of the mRNA expression level.
5. The method according to any one of claims 1 to 4, wherein the gene or its expression product is RNA contained in the skin surface lipid of the subject.
6. Use of at least one gene or its expression product selected from two types of genes, NCLN and ZNF429, derived from a biological sample collected from a subject as a marker for detecting exacerbation of atopic dermatitis.
7. The use according to claim 6, wherein the gene or its expression product is mRNA contained in the skin surface lipid of the subject.
8. Atopic skin used in the method according to any one of claims 1 to 5, comprising an oligonucleotide that specifically hybridizes with said gene or a nucleic acid derived therefrom, or an antibody that recognizes an expression product of said gene. A test kit for detecting exacerbation of inflammation.
9. A marker for detecting exacerbation of atopic dermatitis, comprising at least one gene selected from two genes, NCLN and ZNF429, or an expression product thereof.