WO2022220298A1 - Method for sensing severity of diaper dermatitis in infants and toddlers - Google Patents

Method for sensing severity of diaper dermatitis in infants and toddlers Download PDF

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WO2022220298A1
WO2022220298A1 PCT/JP2022/017952 JP2022017952W WO2022220298A1 WO 2022220298 A1 WO2022220298 A1 WO 2022220298A1 JP 2022017952 W JP2022017952 W JP 2022017952W WO 2022220298 A1 WO2022220298 A1 WO 2022220298A1
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gene
infant
diaper dermatitis
severity
expression product
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PCT/JP2022/017952
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French (fr)
Japanese (ja)
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真恵子 岩村
高良 井上
哲矢 桑野
裕也 上原
恭子 志摩
優子 福田
倫子 高木
早苗 相良
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花王株式会社
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844Nucleic acid amplification reactions
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans

Definitions

  • Eczema is a general term for inflammation that occurs on the surface of the skin. Infants develop various types of eczema (dermatitis) as they grow. Typical examples include neonatal acne, seborrheic eczema, contact dermatitis (rash), and atopic dermatitis (AD).
  • Atopic dermatitis is a disease whose main lesion is itchy eczema with repeated exacerbations and remissions, and many patients have atopic predisposition (family history, medical history, or a predisposition to produce IgE antibodies). It is characterized (Non-Patent Document 1). In infancy, it has been reported that it is related to the subsequent allergic march (starting from one allergy onset and developing allergies one after another), and prevention of onset, early detection and therapeutic intervention are important. It is said that
  • Diaper dermatitis (diaper rash), which frequently occurs in infants, is dermatitis mainly characterized by erythema that occurs at the diaper wearing site, and is considered to be a non-allergic temporary irritant contact dermatitis.
  • Such temporary irritant contact dermatitis develops upon contact with an irritating causative agent, but is improved by removing the causative agent.
  • it may lead to the development of allergic diseases including atopic dermatitis due to the deterioration of skin barrier function, penetration of allergens, and establishment of sensitization due to long-term exposure to allergens. Therefore, like atopic dermatitis, it can be said that its onset prevention, early detection and therapeutic intervention are important (Non-Patent Document 2).
  • RNA contained in skin surface lipids can be used as a sample for biological analysis (Patent Document 1).
  • Patent Document 1 International Publication No. 2018/008319
  • Non-Patent Document 1 J Allenrgy Clin Immunol Pract. 2020; 8: 1721-1724
  • Non-Patent Document 2 J Allergy Clin Immunol. 2008; 121: 1331-6
  • the present invention relates to the following 1) to 4).
  • the present invention relates to providing a detection marker for detecting the severity of infant diaper dermatitis and a method for detecting the severity of infant diaper dermatitis using the detection marker.
  • the present inventor collected SSL from the abdomen, waist, left and right inguinal regions, and left and right buttocks of infants with diaper dermatitis, and comprehensively analyzed the expression state of RNA contained in SSL as sequence information.
  • the expression level of the gene was significantly correlated with the severity of diaper dermatitis in infants, and it was found that the severity of diaper dermatitis in infants can be detected using this as an index.
  • nucleic acid or “polynucleotide” means DNA or RNA.
  • DNA includes cDNA, genomic DNA, and synthetic DNA
  • RNA includes total RNA, mRNA, rRNA, tRNA, non-coding RNA, and synthetic RNA.
  • 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.
  • 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.
  • the names of the genes disclosed in this specification follow the Official Symbol described in NCBI ([www.ncbi.nlm.nih.gov/]).
  • 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.
  • diaper dermatitis refers to inflammation that occurs on the skin in contact with a diaper.
  • erythema, papules, maceration, and desquamation are seen.
  • erythema changes to erythema with edema
  • papules to pustules and maceration to erosions.
  • Areas where diaper dermatitis is likely to occur include the urination area, anus area, buttocks, groin area, waist area, abdomen area, thigh area, and the vicinity of these areas.
  • Infants broadly refers to "children” before the onset of secondary sex characteristics, specifically a concept including children under the age of 12, preferably from 0 years old to entering school, specifically Generally refers to infants from 0 to 5 years old.
  • diaper dermatitis score for example, for each evaluation site, 4 symptoms of erythema, papules, maceration and desquamation are 0: no symptoms, 1: slight, 2: mild, 3: mild to moderate, 4: moderate , 5: Moderate to severe, 6: Severe 7 grades are attached, and the value obtained by summing the scores of all evaluation sites (total value) can be mentioned (Pediatric Dermatology (2014) Vol. 31 No. 1 p. 1-7, and Dermatology (2000) 200 p.238-243).
  • the severity of diaper dermatitis is based on the total evaluation score, 0: no symptoms, 1 to 24. 25-48: mild, 49-72: mild to moderate, 73-96: moderate, 97-120: moderate to severe, 121-144: severe.
  • the diaper dermatitis score itself may be used as an indicator of the severity of infant diaper dermatitis.
  • “detection” of the severity of infant diaper dermatitis can also be rephrased with terms such as inspection, measurement, judgment, or evaluation support.
  • the terms “detection”, “examination”, “measurement”, “determination” or “evaluation” of the severity of infant diaper dermatitis in the present invention include diagnosis of the severity of infant diaper dermatitis by a doctor. is not.
  • the four genes of GALNT3, CMTM6, SLC35E1 and EID3 are the gene group shown in Table 2, the gene group shown in Table 3, and the gene group shown in Table 4. In, any two or more gene clusters are overlapping genes. In addition, among these, GALNT3 and CMTM6 are overlapping genes in all of these three gene clusters.
  • the four genes, GALNT3, CMTM6, SLC35E1, and EID3, are genes that have not been reported to be associated with infant diaper dermatitis (marked with * and shown in bold in each table).
  • the severity of infant diaper dermatitis is detected by a discriminant (diaper dermatitis score prediction model) using at least one or more genes selected from these genes as feature amount genes.
  • a discriminant diaper dermatitis score prediction model
  • at least one selected from genes selected from the four gene groups of GALNT3, CMTM6, SLC35E1 and EID3 or their expression products is used as a detection marker, and based on the expression level, symptoms of infant diaper dermatitis are detected. degree can be detected.
  • the expression level of one or more genes selected from the four gene groups or their expression products, and the 14 gene groups shown in Table 5 (GALNT3 from the genes shown in Tables 2 to 4) , CMTM6, SLC35E1 and EID3 genes) and one or more expression levels selected from genes or their expression products, based on the severity of diaper dermatitis in the test infant detection is possible.
  • the state of diaper dermatitis in infants for example, the presence or absence of onset of dermatitis, the degree of progression of dermatitis, the degree of healing of dermatitis, therapeutic or preventive effect on dermatitis, etc. can be grasped.
  • Each of the four genes, GALNT3, CMTM6, SLC35E1 and EID3, can be independently used as a detection marker for detecting the severity of infant diaper dermatitis.
  • a combination of three or more, more preferably four, is used. Among them, it is preferable to select two or more kinds including GALNT3 and CMTM6.
  • the gene that can be a detection marker for detecting the severity of infant diaper dermatitis includes, as long as it can be a biomarker for detecting the severity of infant diaper dermatitis,
  • a gene having a base sequence substantially identical to that of the DNA constituting the gene is also included.
  • target genes are selected from four gene groups, GALNT3, CMTM6, SLC35E1 and EID3, as one aspect, for biological samples collected from test infants. Measuring the level of expression of at least one gene or its expression product. As another aspect, the expression level of at least one gene or its expression product selected from the four gene groups and the 14 gene groups shown in Table 5 for the biological sample collected from the test infant and measuring the level of expression of at least one gene or its expression product.
  • the subject infants in the present invention include, for example, infants who desire or need detection of diaper dermatitis.
  • the infants to be tested are infants who have developed diaper dermatitis, infants suspected of developing diaper dermatitis, infants genetically predisposed to diaper dermatitis, or close relatives such as brothers and sisters who have diaper dermatitis. Infants who have or have had symptoms are included.
  • 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.
  • 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 or superficial skin lipids (SSL), more preferably skin superficial 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., preferably the site where infant diaper dermatitis is likely to occur.
  • any means used to collect or remove SSL from the skin can be used to collect SSL from the skin of the test infant.
  • an SSL absorbent material, an SSL adhesive material, or an instrument that scrapes the SSL off the skin 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.
  • an SSL absorbent material previously impregnated with a solvent having high fat solubility may be used.
  • 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.
  • RNA-containing SSL collected from test infants 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.
  • 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 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.
  • the expression level comprehensively means the expression level and activity of the gene or expression product.
  • SSL is used as a biological sample.
  • 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.
  • RNA from SSL 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.
  • the AGPC acid guanidinium thiocyanate-phenol-chloroform extraction
  • 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
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • PCR is performed according to a conventional method, and the resulting amplified double-stranded DNA is detected.
  • 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.
  • a DNA microarray 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.
  • the “partial sequence” includes nucleic acids consisting of at least 15 to 25 bases.
  • 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, Third Edition, Cold Spring Harbor Laboratory Press (2001) and others.
  • 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.
  • “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.
  • 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.
  • 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.
  • oligonucleotide can be DNA or RNA, and may be synthetic or natural.
  • the probes used for hybridization are usually labeled ones.
  • protein chip analysis e.g., 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.
  • mass spectrometry e.g., LC-MS/MS, MALDI-TOF/MS
  • 1-hybrid method PNAS 100, 12271-12276 (2003)
  • 2-hybrid method Biol. Reprod. 58 , 302-311 (1998)
  • a protein 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
  • 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.
  • 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)).
  • the expression level of the target gene of the present invention or its expression product in a biological sample collected from a test infant is measured, and the severity of diaper dermatitis in the test infant is detected based on the expression level.
  • the expression level of the target gene of the present invention or its expression product in a biological sample collected from a test infant is measured at least at two times, and the change in the expression level or the amount of change is used as an index, and the test infant is It is possible to detect the presence or absence of change in the degree of diaper dermatitis or the degree of change. Specifically, detection is performed by comparing the measured expression level of the target gene of the present invention or its expression product with a preset cutoff value (reference value).
  • 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 logarithmic value of base 2 log 2 RPM value
  • a logarithmic value of base 2 obtained by adding an integer 1 (log 2 (RPM + 1) value)
  • DESeq2 Love MI et al. Genome Biol 2014
  • the base 2 logarithm log 2 (Normalized count+1) value
  • RNA-seq 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 general 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.
  • 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.
  • the "cutoff value"("referencevalue”) can be determined in advance based on the relationship between the diaper dermatitis score and the expression level of the target gene of the present invention or its expression product. For example, a population is divided into diaper dermatitis status, i.e. no symptoms, mild, mild, mild-to-moderate, moderate, moderate-to-severe, severe groups according to the diaper dermatitis score, and the target in each group A value determined with reference to statistical values such as the average value and standard deviation of the expression level of a gene or its expression product can be determined as a cutoff value (reference value) for determining belonging to each group. When multiple types of genes are used as target genes, it is preferable to determine a cutoff value (reference value) for each gene or its expression product. Groups may be formed according to sex, race, and age.
  • the expression level of the target gene of the present invention or its expression product is similarly measured from the biological sample collected from the test infant, the obtained measured value is input into the discriminant (prediction model), and from the discriminant
  • the obtained result predicted value of diaper dermatitis score
  • the obtained result can be detected as the degree of diaper dermatitis in the test infant.
  • diaper skin with different severity A discriminant formula to distinguish a group of infants with inflammation (e.g., two or more groups selected from mild, mild, mild-to-moderate, moderate, moderate-to-severe, severe, etc.) and healthy infants (no symptoms)
  • a (prediction model) can be constructed and the discriminant can be used to detect the severity of infant diaper dermatitis.
  • the measured values of the expression levels of target genes or their expression products derived from a group of diaper dermatitis infants with different degrees of diaper dermatitis and the measured values of the expression levels of target genes or their expression products derived from healthy infants were used as teaching samples, and diapers with different degrees of severity were used.
  • a discriminant to divide infants with dermatitis e.g., two or more groups selected from mild, mild, mild-to-moderate, moderate, moderate-to-severe, severe, etc.
  • a (prediction model) is constructed, and a cutoff value (reference value) for discriminating diaper dermatitis infants with different degrees of severity is obtained based on the discriminant.
  • a known one such as an algorithm used for machine learning can be used.
  • 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, Lasso (Least Absolute Shrinkage and Selection Operator) regression, and the like.
  • Enter verification data into the constructed prediction model to calculate prediction values, and select the model that best matches the prediction values with the measured values, for example, the model with the highest accuracy rate as the optimal prediction model. can be done.
  • 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 root mean square error (RMSE) between the predicted value and the measured value can be used as an accuracy evaluation index of the prediction model, and the model with the smallest RMSE can be selected as the optimum prediction model.
  • the method of determining the cutoff 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 (prediction model).
  • ROC Receiveiver Operating Characteristic Curve
  • the vertical axis is the probability of a positive result in positive subjects (sensitivity)
  • the horizontal axis is the value obtained by subtracting the probability of a negative result in negative subjects (specificity) from 1 (false positive rate).
  • a test kit for detecting the severity of infant diaper dermatitis of the present invention contains a test reagent for measuring the expression level of the target gene of the present invention or its expression product in a biological sample isolated from a test infant. It is. 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.
  • an oligonucleotide e.g., primer for PCR
  • the present invention further discloses the following aspects.
  • ⁇ 2> preferably two or more, more preferably three or more, still more preferably two or more including GALNT3 and CMTM6, more preferably four, selected from the four gene groups of GALNT3, CMTM6, SLC35E1 and EID3
  • ⁇ 4> Preferably two or more, more preferably three or more, more preferably infant diaper dermatitis indicated in bold with * in Table 5 selected from the 14 gene groups shown in Table 5
  • the detection method according to ⁇ 3> wherein the expression level of at least one, preferably two or more, more preferably three or more genes selected from unreported genes or their expression products is measured.
  • ⁇ 5> Preferably, the detection method according to any one of ⁇ 1> to ⁇ 4>, wherein the expression level of the gene or its expression product described in Table 2, Table 3 or Table 4 is measured.
  • the biological sample is preferably an organ, skin, blood, urine, saliva, sweat, stratum corneum, superficial skin lipid (SSL), body fluids such as tissue exudate, serum prepared from blood, plasma , stool or hair, more preferably skin or superficial skin lipids (SSL), and still more preferably superficial skin lipids (SSL) ⁇ 1> to ⁇ 5>.
  • the object to be measured for the expression level of the gene or its expression product is preferably cDNA artificially synthesized from RNA, DNA encoding the RNA, a protein encoded by the RNA, and interacting with the protein.
  • SSL superficial skin lipids
  • the test infant is an infant who has developed infant diaper dermatitis, an infant suspected of developing infant diaper dermatitis, an infant genetically predisposed to infant diaper dermatitis, or a relative such as siblings
  • the site of the skin from which lipids on the skin surface (SSL) are collected may be either a rash area where infant diaper dermatitis develops or a non-rash area where infant diaper dermatitis does not develop.
  • ⁇ 12> The detection method according to any one of ⁇ 1> to ⁇ 11>, comprising detecting the degree of diaper dermatitis in the test infant based on the expression level of the gene or its expression product.
  • detecting the severity of infant diaper dermatitis using a discriminant (predictive model) based on the expression level of the gene or its expression product Any of ⁇ 1> to ⁇ 12>, wherein the discriminant (prediction model) is constructed by machine learning with the measured value of the expression level of the gene or its expression product as an explanatory variable and the diaper dermatitis score as an objective variable.
  • the severity of infant diaper dermatitis is 4 symptoms of erythema, papule, maceration and desquamation for each evaluation site: 0: no symptoms, 1: slight, 2: mild, 3: mild to moderate, 4: Moderate, 5: Moderate to severe, 6: Severe 7 grades are given, and the sum of the scores of all the evaluation sites (total value) is the degree of severity corresponding to ⁇ 1> to ⁇ 13.
  • the detection method according to any one of >. ⁇ 15> The detection method according to ⁇ 14>, wherein the evaluation sites are the abdomen, waist, left and right inguinal regions, and left and right buttocks of the infant.
  • Infant diaper skin used in the detection method of ⁇ 1> to ⁇ 15>, containing 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 the severity of inflammation.
  • a detection marker for detecting the severity of infant diaper dermatitis comprising at least one gene or its expression product selected from the gene group indicated in bold with * in Tables 2 to 4 above.
  • ⁇ 19> Infant diaper dermatitis of at least one gene or its expression product selected from the group of genes indicated in bold with * in Tables 2 to 4 above derived from biological samples collected from test infants Use as a marker for detection of disease severity.
  • ⁇ 20> Use as a detection marker according to ⁇ 19>, which is at least one gene or its expression product selected from the four gene groups of GALNT3, CMTM6, SLC35E1 and EID3.
  • RNA Preparation and Sequencing The blotting film of 2) above was cut into an appropriate size, and RNA was extracted using QIAzol Lysis Reagent (Qiagen) according to the attached protocol. Based on the extracted RNA, reverse transcription was performed at 42° C. for 90 minutes using SuperScript VILO cDNA Synthesis kit (Life Technologies Japan Co., Ltd.) 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.
  • RNA profile data for 48 children was used as Train data for model construction, and RNA profile data for the remaining 11 children was used as Test data for model accuracy evaluation.
  • Data partitioning was performed using the createDataPartition function of [R] so that the distribution of the objective variable was uniform between the Train data and the Test data.
  • Diaper dermatitis score prediction model creation using the caret package Top 8 genes highly correlated with diaper dermatitis score, 10 genes with high variable importance by random forest, and 7 genes selected by BORUTA as feature values. A diaper dermatitis score prediction model was constructed with the caret package. The expression level data (log 2 (RPM+1) value) of the feature gene selected from the SSL-derived RNA was used as an explanatory variable, and the value obtained by converting the diaper dermatitis score into a deviation value was used as an objective variable.
  • the caret package linear regression model (Lm), random forest (Rf), neural network (Nnet), Lasso regression (Lasso), rbf kernel support vector machine (SVM rbf ), a diaper dermatitis score prediction model was constructed with six algorithms of a linear kernel support vector machine (SVM linear). To reduce the impact of data bias on the model, model building was cross-validated 10 times. As an index of the best prediction model, RMSE (root mean square error) was calculated, and the model with the smallest value was selected as the best model.
  • SVM linear linear kernel support vector machine
  • the feature quantity gene expression level (log 2 (RPM+1) value) of the Test data was input to calculate the predicted value (predicted score) of the diaper dermatitis score.
  • the PEARSON correlation coefficient between the obtained prediction value (prediction score) and the actual diagnosis score was calculated, and the closer this value was to 1, the higher the prediction accuracy of the model.
  • Especially important genes among the plurality of feature amount genes are GALNT3, CMTM6, SLC35E1, and EID3, which are redundantly extracted by two or more of the three methods of extracting feature amount genes.
  • a diaper dermatitis score prediction model is constructed using two types of GALNT3 and CMTM6 selected from the four types or two types of SLC35E1 and EID3, the correlation coefficient between the prediction score and the diagnosis score based on the test data is 0, respectively.

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Abstract

The present invention provides: a sensing marker for sensing the severity of diaper dermatitis in infants and toddlers; and a method for sensing the severity of diaper dermatitis in infants and toddlers using said sensing marker. This method is for sensing the severity of diaper dermatitis in a subject infant or toddler, and comprises a step for measuring, in a biological sample collected from the infant or toddler, the expression level of at least one gene selected from a group of four genes, which are for GALNT3, CMTM6, SLC35E1, and EID3, or the expression level of an expression product thereof.

Description

乳幼児おむつ皮膚炎の症度の検出方法Method for detecting the severity of infant diaper dermatitis
 本発明は、乳幼児おむつ皮膚炎の症度検出マーカーを用いた乳幼児おむつ皮膚炎の症度の検出方法を提供することに関する。 The present invention relates to providing a method for detecting the severity of infant diaper dermatitis using an infant diaper dermatitis severity detection marker.
 湿疹(皮膚炎)は皮膚の表面にできる炎症の総称である。乳幼児は成長に伴い、様々な湿疹(皮膚炎)を発症する。代表的なものとしては、新生児ざ瘡、脂漏性湿疹、接触皮膚炎(かぶれ)、アトピー性皮膚炎(AD)等がある。アトピー性皮膚炎は、増悪緩解を繰り返す、掻痒のある湿疹を主病変とする疾患であり、患者の多くはアトピー素因(家族歴、既往歴、またはIgE抗体を産生しやすい素因)を持つことを特徴とする(非特許文献1)。乳幼児期においては、その後のアレルギーマーチ(1つのアレルギー発症を起点として次々とアレルギーを発症していくこと)に関係していることが報告されており、その発症予防、早期発見と治療介入が重要であるとされている。 Eczema (dermatitis) is a general term for inflammation that occurs on the surface of the skin. Infants develop various types of eczema (dermatitis) as they grow. Typical examples include neonatal acne, seborrheic eczema, contact dermatitis (rash), and atopic dermatitis (AD). Atopic dermatitis is a disease whose main lesion is itchy eczema with repeated exacerbations and remissions, and many patients have atopic predisposition (family history, medical history, or a predisposition to produce IgE antibodies). It is characterized (Non-Patent Document 1). In infancy, it has been reported that it is related to the subsequent allergic march (starting from one allergy onset and developing allergies one after another), and prevention of onset, early detection and therapeutic intervention are important. It is said that
 乳幼児に頻発するおむつ皮膚炎(おむつかぶれ)はおむつ着用部位にできる紅斑を主とした皮膚炎で、非アレルギー性の一時刺激性接触皮膚炎と考えられる。このような一時刺激性接触皮膚炎は刺激となる原因物質の接触により発症するが、それを除去することにより改善する。しかしながら、適切な処置がなされない状態が続くと、皮膚のバリア能低下、アレルゲンの侵入、アレルゲン長期暴露による感作の成立によりアトピー性皮膚炎を含むアレルギー疾患の発症にもつながる可能性もあることから、アトピー性皮膚炎と同様、その発症予防、早期発見と治療介入が重要であるといえる(非特許文献2)。 Diaper dermatitis (diaper rash), which frequently occurs in infants, is dermatitis mainly characterized by erythema that occurs at the diaper wearing site, and is considered to be a non-allergic temporary irritant contact dermatitis. Such temporary irritant contact dermatitis develops upon contact with an irritating causative agent, but is improved by removing the causative agent. However, if the condition continues without appropriate treatment, it may lead to the development of allergic diseases including atopic dermatitis due to the deterioration of skin barrier function, penetration of allergens, and establishment of sensitization due to long-term exposure to allergens. Therefore, like atopic dermatitis, it can be said that its onset prevention, early detection and therapeutic intervention are important (Non-Patent Document 2).
 湿疹状態の把握は皮膚科医の診断が重要であるが、医療機関の受診の判断は保護者主観に委ねられており、その価値基準は個々人で異なることから、早期かつ適切な治療のタイミングを逃してしまう可能性を秘めている。そのため、より簡便に湿疹状態を判定できる技術は、乳幼児の適切なスキンケアをもたらし、健全な成長をサポートする技術になると考えられる。 Diagnosis by a dermatologist is important for understanding the eczema condition, but the decision to see a medical institution is left to the subjective judgment of the guardian, and the value standard differs from individual to individual. It has the potential to be missed. Therefore, a technology that can more easily determine the eczema state is considered to be a technology that brings about appropriate skin care for infants and supports their healthy growth.
 近年、生体試料中のDNAやRNA等の核酸の解析によりヒトの生体内の現在さらには将来の生理状態を調べる技術が開発されている。生体由来の核酸は、血液等の体液、分泌物、組織等から抽出することができる。さらに最近、皮膚表上脂質(skin surface lipids;SSL)に含まれるRNAを生体の解析用の試料として利用可能であることが報告されている(特許文献1)。 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).
  (特許文献1)国際公開公報第2018/008319号 (Patent Document 1) International Publication No. 2018/008319
  (非特許文献1)J Allenrgy Clin Immunol Pract.2020;8:1721-1724
  (非特許文献2)J Allergy Clin Immunol.2008;121:1331-6
(Non-Patent Document 1) J Allenrgy Clin Immunol Pract. 2020; 8: 1721-1724
(Non-Patent Document 2) J Allergy Clin Immunol. 2008; 121: 1331-6
 本発明は、以下の1)~4)に係るものである。
 1)被験乳幼児から採取された生体試料について、GALNT3、CMTM6、SLC35E1及びEID3の4種の遺伝子群より選択される少なくとも1つの遺伝子又はその発現産物の発現レベルを測定する工程を含む、当該乳幼児のおむつ皮膚炎の症度を検出する方法。
 2)前記遺伝子又はそれに由来する核酸と特異的にハイブリダイズするオリゴヌクレオチド、又は前記遺伝子の発現産物を認識する抗体を含有する、1)の方法に用いられる乳幼児おむつ皮膚炎の症度を検出するための検査用キット。
 3)表1に示す14種の遺伝子群より選択される少なくとも1つの遺伝子又はその発現産物からなる、乳幼児おむつ皮膚炎の症度を検出するための検出マーカー。
 4)被験乳幼児から採取された生体試料に由来する表1に示す14種の遺伝子群より選択される少なくとも1つの遺伝子又はその発現産物の、乳幼児おむつ皮膚炎皮膚炎の症度の検出マーカーとしての使用。
The present invention relates to the following 1) to 4).
1) For biological samples collected from test infants, including the step of measuring the expression level of at least one gene or its expression product selected from the four gene groups GALNT3, CMTM6, SLC35E1 and EID3 of the infants A method for detecting the severity of diaper dermatitis.
2) detecting the severity of infant diaper 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
3) A detection marker for detecting the severity of infant diaper dermatitis, comprising at least one gene selected from the 14 gene groups shown in Table 1 or an expression product thereof.
4) At least one gene or its expression product selected from the 14 gene groups shown in Table 1 derived from a biological sample collected from a test infant as a marker for detecting the severity of infant diaper dermatitis dermatitis use.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
おむつ皮膚炎スコア予測モデルにより得られたおむつ皮膚炎スコアの予測スコアと診断スコアとの関係を示すグラフ。A graph showing the relationship between the prediction score of the diaper dermatitis score obtained by the diaper dermatitis score prediction model and the diagnostic score.
発明の詳細な説明Detailed description of the invention
 本発明は、乳幼児おむつ皮膚炎の症度を検出するための検出マーカー、及び当該検出マーカーを用いた乳幼児おむつ皮膚炎の症度の検出方法を提供することに関する。 The present invention relates to providing a detection marker for detecting the severity of infant diaper dermatitis and a method for detecting the severity of infant diaper dermatitis using the detection marker.
 本発明者は、おむつ皮膚炎が見られる乳幼児の腹部、腰部、左右鼠径部及び左右臀部からSSLを採取し、SSL中に含まれるRNAの発現状態をシーケンス情報として網羅的に解析した結果、特定の遺伝子の発現レベルが乳幼児おむつ皮膚炎の症度と有意に相関し、これを指標として乳幼児おむつ皮膚炎の症度を検出できることを見出した。 The present inventor collected SSL from the abdomen, waist, left and right inguinal regions, and left and right buttocks of infants with diaper dermatitis, and comprehensively analyzed the expression state of RNA contained in SSL as sequence information. The expression level of the gene was significantly correlated with the severity of diaper dermatitis in infants, and it was found that the severity of diaper dermatitis in infants can be detected using this as an index.
 本発明によれば、簡便且つ非侵襲的に、乳幼児のおむつ皮膚炎の症度を検出することが可能である。これにより、保護者は、乳幼児のおむつ皮膚炎の状態を客観的指標に基づいて把握できるため、当該おむつ皮膚炎に対する適切な対策を講じることができる。 According to the present invention, it is possible to simply and non-invasively detect the severity of infant diaper dermatitis. As a result, parents can grasp the state of diaper dermatitis in infants and toddlers based on the objective index, and can take appropriate measures against the diaper dermatitis.
 本明細書中で引用された全ての特許文献、非特許文献、及びその他の刊行物は、その全体が本明細書中において参考として援用される。 All patent documents, non-patent documents, and other publications cited in this specification are hereby incorporated by reference in their entirety.
 本発明において、「核酸」又は「ポリヌクレオチド」と云う用語は、DNA又はRNAを意味する。DNAには、cDNA、ゲノムDNA、及び合成DNAのいずれもが含まれ、「RNA」には、total RNA、mRNA、rRNA、tRNA、non-coding RNA及び合成のRNAのいずれもが含まれる。 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.
 本発明において「遺伝子」とは、ヒトゲノムDNAを含む2本鎖DNAの他、cDNAを含む1本鎖DNA(正鎖)、当該正鎖と相補的な配列を有する1本鎖DNA(相補鎖)、及びこれらの断片を包含するものであって、DNAを構成する塩基の配列情報の中に、何らかの生物学的情報が含まれているものを意味する。
 また、本発明における「遺伝子」には、特定の塩基配列で表される「遺伝子」だけではなく、その同族体(すなわち、ホモログもしくはオーソログ)、遺伝子多型等の変異体、及び誘導体が包含される。
 ここで、本明細書中に開示される遺伝子の名称は、NCBI([www.ncbi.nlm.nih.gov/])に記載のあるOfficial Symbolに従う。
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.
Here, the names of the genes disclosed in this specification follow the Official Symbol described in NCBI ([www.ncbi.nlm.nih.gov/]).
 本発明において、遺伝子の「発現産物」とは、遺伝子の転写産物及び翻訳産物を包含する概念である。「転写産物」とは、遺伝子(DNA)から転写されて生じるRNAであり、「翻訳産物」とは、RNAに基づき翻訳合成される、遺伝子にコードされたタンパク質を意味する。 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.
 本発明において、「おむつ皮膚炎」とは、おむつと接触した皮膚に起きる炎症を指す。臨床的には、紅斑、丘疹、浸軟、落屑が見られる。症状が強くなると、紅斑は浮腫を伴う紅斑に、丘疹は膿疱(うみ)をもち、浸軟は糜爛(びらん)へと変化が見られる。
 おむつ皮膚炎が生じやすい部位としては、排尿部、肛門部、臀部、鼠径部、腰部、腹部、大腿部、これらの近傍部等が挙げられる。
 「乳幼児」は、広義には第2次性徴が開始する前の「小児」、具体的には12歳以下の小児を含めた概念であり、好ましくは0歳から学童に入るまでの年齢、具体的には0歳から5歳までの乳幼児を指す。
In the present invention, "diaper dermatitis" refers to inflammation that occurs on the skin in contact with a diaper. Clinically, erythema, papules, maceration, and desquamation are seen. As the symptoms worsen, erythema changes to erythema with edema, papules to pustules, and maceration to erosions.
Areas where diaper dermatitis is likely to occur include the urination area, anus area, buttocks, groin area, waist area, abdomen area, thigh area, and the vicinity of these areas.
"Infants" broadly refers to "children" before the onset of secondary sex characteristics, specifically a concept including children under the age of 12, preferably from 0 years old to entering school, specifically Generally refers to infants from 0 to 5 years old.
 乳幼児おむつ皮膚炎の「症度」とは、乳幼児おむつ皮膚炎の症状の悪さの程度を指し、例えば、症状なし、軽微、軽度、軽度から中等度、中等度、中等度から重度、重度のように分類される。
 乳幼児おむつ皮膚炎の症度は、おむつ皮膚炎の症状を評価する公知の評価スコア(以下、「おむつ皮膚炎スコア」ともいう)に基づいて決定することができる。おむつ皮膚炎スコアとしては、例えば、評価部位毎に、紅斑、丘疹、浸軟及び落屑の4症状について0:症状なし、1:軽微、2:軽度、3:軽度から中等度、4:中等度、5:中等度から重度、6:重度の7段階のスコアを付し、評価部位全てのスコアを合算した値(合算値)が挙げられる(Pediatric Dermatology (2014) Vol. 31 No. 1 p.1-7、及びDermatology (2000) 200 p.238-243参照)。評価部位が6箇所(腹部、腰部、左右鼠径部、左右臀部)の場合を例に取れば、おむつ皮膚炎の症度は、評価スコアの合算値にもとづいて、0:症状なし、1~24:軽微、25~48:軽度、49~72:軽度から中等度、73~96:中等度、97~120:中等度から重度、121~144:重度と決定することができる。本発明では、当該おむつ皮膚炎スコア自体を乳幼児おむつ皮膚炎の症度を示す指標としてもよい。
The “severity” of infant diaper dermatitis refers to the severity of the symptoms of infant diaper dermatitis, such as no symptoms, mild, mild, mild to moderate, moderate, moderate to severe, and severe. are categorized.
The severity of infant diaper dermatitis can be determined based on a known evaluation score for evaluating symptoms of diaper dermatitis (hereinafter also referred to as "diaper dermatitis score"). As the diaper dermatitis score, for example, for each evaluation site, 4 symptoms of erythema, papules, maceration and desquamation are 0: no symptoms, 1: slight, 2: mild, 3: mild to moderate, 4: moderate , 5: Moderate to severe, 6: Severe 7 grades are attached, and the value obtained by summing the scores of all evaluation sites (total value) can be mentioned (Pediatric Dermatology (2014) Vol. 31 No. 1 p. 1-7, and Dermatology (2000) 200 p.238-243). Taking the case of six evaluation sites (abdomen, waist, left and right inguinal regions, left and right buttocks) as an example, the severity of diaper dermatitis is based on the total evaluation score, 0: no symptoms, 1 to 24. 25-48: mild, 49-72: mild to moderate, 73-96: moderate, 97-120: moderate to severe, 121-144: severe. In the present invention, the diaper dermatitis score itself may be used as an indicator of the severity of infant diaper dermatitis.
 本発明において、乳幼児おむつ皮膚炎の症度の「検出」は、検査、測定、判定又は評価支援等の用語で言い換えることもできる。なお、本発明における乳幼児おむつ皮膚炎の症度の「検出」、「検査」、「測定」、「判定」又は「評価」という用語は、医師による乳幼児おむつ皮膚炎の症度の診断を含むものではない。 In the present invention, "detection" of the severity of infant diaper dermatitis can also be rephrased with terms such as inspection, measurement, judgment, or evaluation support. The terms "detection", "examination", "measurement", "determination" or "evaluation" of the severity of infant diaper dermatitis in the present invention include diagnosis of the severity of infant diaper dermatitis by a doctor. is not.
 後述する実施例に示すように、おむつ皮膚炎が見られる乳幼児の腹部、腰部、左右鼠径部、左右臀部からSSLを採取し、該SSL由来RNAの発現量のデータ(リードカウント値)を取得し、以下の手順1)、2)又は3)により、表2に示す8種の遺伝子、表3に示す10種の遺伝子及び表4に示す7種の遺伝子が特徴量遺伝子として選択された。そして、該特徴量遺伝子を用いた判別式(おむつ皮膚炎スコアの予測モデル)で、乳幼児おむつ皮膚炎の症度の検出が可能であることが示された。したがって、表2~表4に示された遺伝子群より選択される遺伝子又はその発現産物を検出マーカーとし、その発現レベルに基づいて、乳幼児おむつ皮膚炎の症度を検出することができる。さらに、表2~表4において*を付し太字で表示した遺伝子は、これまでに乳幼児おむつ皮膚炎との関連が報告されていない遺伝子であり、これら遺伝子群より選択される遺伝子又はその発現産物は、乳幼児おむつ皮膚炎の症度を検出するための新規な検出マーカーである。
1)リードカウント値をサンプル被験乳幼児間の総リード数の違いを補正したRPM値に変換し、これに整数1を加算し底2の対数値に変換した値((log(RPM+1)値)と、おむつ皮膚炎スコアについてスピアマンの順位相関係数を求める。スピアマンの順位相関係数(ρ)の大きな遺伝子(上位遺伝子)を特徴量遺伝子として選択する。
2)リードカウント値をサンプル被験乳幼児間の総リード数の違いを補正したRPM値に変換し、これに整数1を加算した底2の対数値(log(RPM+1)値)を説明変数、おむつ皮膚炎スコアを目的変数とした機械学習(機械学習アルゴリズム;ランダムフォレスト)を行い、ランダムフォレスト関数で算出されるジニ係数に基づく変数重要度上位の遺伝子を特徴量遺伝子として選択する。
3)対数値(log(RPM+1)値)を説明変数、おむつ皮膚炎スコアを目的変数とした機械学習(機械学習アルゴリズム;BORUTA法)を行い、‘‘tentative’’あるいは‘‘confirmed’’と判定された遺伝子を、特徴量遺伝子として選択する。
As shown in Examples described later, SSL was collected from the abdomen, waist, left and right inguinal regions, and left and right buttocks of infants with diaper dermatitis, and the data (read count value) of the expression level of the SSL-derived RNA was obtained. 8 genes shown in Table 2, 10 genes shown in Table 3, and 7 genes shown in Table 4 were selected as feature amount genes by the following procedures 1), 2), or 3). It was also shown that the degree of infant diaper dermatitis can be detected by a discriminant (diaper dermatitis score prediction model) using the feature gene. Therefore, a gene or its expression product selected from the gene group shown in Tables 2 to 4 can be used as a detection marker, and the severity of infant diaper dermatitis can be detected based on the expression level. Furthermore, the genes indicated in bold with * in Tables 2 to 4 are genes that have not been reported to be related to infant diaper dermatitis so far, and genes selected from these gene groups or expression products thereof is a novel detectable marker for detecting the severity of infant diaper dermatitis.
1) Convert the read count value to an RPM value corrected for the difference in the total number of reads between the sample test infants, add an integer 1 to this, and convert it to a base 2 logarithmic value ((log 2 (RPM + 1) value) Then, the Spearman's rank correlation coefficient of the diaper dermatitis score is obtained, and the gene (upper gene) with the large Spearman's rank correlation coefficient (ρ) is selected as the feature amount gene.
2) Convert the read count value to an RPM value corrected for the difference in the total number of reads between the sample tested infants, and add an integer 1 to the base 2 logarithm (log 2 (RPM + 1) value) as an explanatory variable, diaper Machine learning (machine learning algorithm: random forest) is performed with the dermatitis score as the objective variable, and genes with high variable importance based on the Gini coefficient calculated by the random forest function are selected as feature amount genes.
3) Perform machine learning (machine learning algorithm; BORUTA method) with the logarithmic value (log 2 (RPM + 1) value) as an explanatory variable and the diaper dermatitis score as the objective variable, and ``tentative'' or ``confirmed'' The determined gene is selected as a feature gene.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
 表2~表4に示された遺伝子のうち、GALNT3、CMTM6、SLC35E1及びEID3の4種の遺伝子は、表2に記載の遺伝子群と表3に記載の遺伝子群と表4に記載の遺伝子群において、いずれか2以上の遺伝子群において重複する遺伝子である。また、このうち、GALNT3及びCMTM6は、これら3つの遺伝子群全てにおいて重複する遺伝子である。なお、GALNT3、CMTM6、SLC35E1及びEID3の4種の遺伝子は、これまでに乳幼児おむつ皮膚炎との関連が報告されていない遺伝子(各表において*を付し太字で表示)である。
 後述する実施例に示すように、これら遺伝子から選択される少なくとも1種以上の遺伝子を特徴量遺伝子として用いた判別式(おむつ皮膚炎スコアの予測モデル)で、乳幼児おむつ皮膚炎の症度の検出が可能であることが示された。したがって、GALNT3、CMTM6、SLC35E1及びEID3の4種の遺伝子群より選択される遺伝子又はその発現産物から選択される少なくとも1種以上を検出マーカーとし、その発現レベルに基づいて、乳幼児おむつ皮膚炎の症度を検出することができる。さらに当該4種の遺伝子群より選択される遺伝子又はその発現産物から選択される1種以上の発現レベルと、表5に示す14種の遺伝子群(表2~表4に示された遺伝子からGALNT3、CMTM6、SLC35E1及びEID3の4種の遺伝子を除いた遺伝子)より選択される遺伝子又はその発現産物から選択される1種以上の発現レベルとに基づいて、被験乳幼児のおむつ皮膚炎の症度の検出が可能である。
 乳幼児おむつ皮膚炎の症度の検出により乳幼児のおむつ皮膚炎の状態、例えば、皮膚炎の発症の有無、皮膚炎の病態の進行度、皮膚炎の治癒の程度、皮膚炎に対する治療もしくは予防効果等を把握することができる。
Among the genes shown in Tables 2 to 4, the four genes of GALNT3, CMTM6, SLC35E1 and EID3 are the gene group shown in Table 2, the gene group shown in Table 3, and the gene group shown in Table 4. In, any two or more gene clusters are overlapping genes. In addition, among these, GALNT3 and CMTM6 are overlapping genes in all of these three gene clusters. The four genes, GALNT3, CMTM6, SLC35E1, and EID3, are genes that have not been reported to be associated with infant diaper dermatitis (marked with * and shown in bold in each table).
As shown in Examples described later, the severity of infant diaper dermatitis is detected by a discriminant (diaper dermatitis score prediction model) using at least one or more genes selected from these genes as feature amount genes. was shown to be possible. Therefore, at least one selected from genes selected from the four gene groups of GALNT3, CMTM6, SLC35E1 and EID3 or their expression products is used as a detection marker, and based on the expression level, symptoms of infant diaper dermatitis are detected. degree can be detected. Furthermore, the expression level of one or more genes selected from the four gene groups or their expression products, and the 14 gene groups shown in Table 5 (GALNT3 from the genes shown in Tables 2 to 4) , CMTM6, SLC35E1 and EID3 genes) and one or more expression levels selected from genes or their expression products, based on the severity of diaper dermatitis in the test infant detection is possible.
By detecting the severity of diaper dermatitis in infants, the state of diaper dermatitis in infants, for example, the presence or absence of onset of dermatitis, the degree of progression of dermatitis, the degree of healing of dermatitis, therapeutic or preventive effect on dermatitis, etc. can be grasped.
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
 GALNT3、CMTM6、SLC35E1及びEID3の4種の遺伝子は、それぞれ単独で乳幼児おむつ皮膚炎の症度を検出するための検出マーカーとなり得るが、当該4種のうち、好ましくは2種以上、より好ましくは3種以上、さらに好ましくは4種の組み合わせを用いる。なかでも、GALNT3及びCMTM6を含む2種以上を選択することが好ましい。
 また、GALNT3、CMTM6、SLC35E1及びEID3の4種の遺伝子と組みわせる、表5から選択される遺伝子としては、少なくとも1種以上であれば良いが、好ましくは2種以上、より好ましくは3種以上である。さらに表中*を付し太字で表示した乳幼児おむつ皮膚炎との関連が報告されていない遺伝子から少なくとも1種以上、好ましくは2種以上、より好ましくは3種以上選択されるのが好ましい。本発明においては、前記表2、表3又は表4に記載された遺伝子の組み合わせを選択することが好ましい。
Each of the four genes, GALNT3, CMTM6, SLC35E1 and EID3, can be independently used as a detection marker for detecting the severity of infant diaper dermatitis. A combination of three or more, more preferably four, is used. Among them, it is preferable to select two or more kinds including GALNT3 and CMTM6.
In addition, at least one or more, preferably two or more, and more preferably three genes selected from Table 5 to be combined with the four genes of GALNT3, CMTM6, SLC35E1, and EID3. That's it. Furthermore, it is preferable to select at least one, preferably two or more, more preferably three or more genes from the genes marked with * in bold that have not been reported to be related to infant diaper dermatitis. In the present invention, it is preferable to select combinations of genes listed in Table 2, Table 3 or Table 4 above.
 上記の乳幼児おむつ皮膚炎の症度を検出するための検出マーカーとなり得る遺伝子(以下、「標的遺伝子」とも称す)には、乳幼児おむつ皮膚炎の症度を検出するためのバイオマーカーとなり得る限り、当該遺伝子を構成するDNAの塩基配列と実質的に同一の塩基配列を有する遺伝子も包含される。ここで、実質的に同一の塩基配列とは、例えば、相同性計算アルゴリズムNCBI BLASTを用い、期待値=10;ギャップを許す;フィルタリング=ON;マッチスコア=1;ミスマッチスコア=-3の条件にて検索をした場合、当該遺伝子を構成するDNAの塩基配列と90%以上、好ましくは95%以上、より好ましく98%以上、さらに好ましくは99%以上の同一性があることを意味する。 The gene that can be a detection marker for detecting the severity of infant diaper dermatitis (hereinafter also referred to as "target gene") includes, as long as it can be a biomarker for detecting the severity of infant diaper dermatitis, A gene having a base sequence substantially identical to that of the DNA constituting the gene is 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 .
 本発明の乳幼児おむつ皮膚炎の症度を検出する方法は、被験乳幼児から採取された生体試料について、標的遺伝子、一態様として、GALNT3、CMTM6、SLC35E1及びEID3の4種の遺伝子群より選択される少なくとも1つの遺伝子又はその発現産物の発現レベルを測定する工程を含む。
 別の一態様として、被験乳幼児から採取された生体試料について、当該4種の遺伝子群より選択される少なくとも1つの遺伝子又はその発現産物の発現レベルと、表5に示す14種の遺伝子群より選択される少なくとも1つの遺伝子又はその発現産物の発現レベルとを測定する工程を含む。
In the method for detecting the severity of infant diaper dermatitis of the present invention, target genes are selected from four gene groups, GALNT3, CMTM6, SLC35E1 and EID3, as one aspect, for biological samples collected from test infants. Measuring the level of expression of at least one gene or its expression product.
As another aspect, the expression level of at least one gene or its expression product selected from the four gene groups and the 14 gene groups shown in Table 5 for the biological sample collected from the test infant and measuring the level of expression of at least one gene or its expression product.
 本発明における被験乳幼児は、例えば、おむつ皮膚炎の検出を所望するか又は必要とする乳幼児が挙げられる。例えば、該被験乳幼児は、おむつ皮膚炎を発症している乳幼児、おむつ皮膚炎の発症が疑われる乳幼児、遺伝的におむつ皮膚炎の素因を有する乳幼児又は兄弟姉妹等の近親者がおむつ皮膚炎を発症している若しくは発症していた乳幼児が挙げられる。 The subject infants in the present invention include, for example, infants who desire or need detection of diaper dermatitis. For example, the infants to be tested are infants who have developed diaper dermatitis, infants suspected of developing diaper dermatitis, infants genetically predisposed to diaper dermatitis, or close relatives such as brothers and sisters who have diaper dermatitis. Infants who have or have had symptoms are included.
 本発明において用いられる生体試料としては、本発明の遺伝子が発現変化する細胞、組織及び生体材料であればよい。具体的には臓器、皮膚、血液、尿、唾液、汗、角層、皮膚表上脂質(SSL)、組織浸出液等の体液、血液から調製された血清、血漿、その他、便、毛髪等が挙げられ、好ましくは皮膚又は皮膚表上脂質(SSL)、より好ましくは皮膚表上脂質(SSL)が挙げられる。SSLが採取される皮膚の部位としては、特に限定されず、頭、顔、首、体幹、手足等の身体の任意の部位の皮膚が挙げられるが、好ましくは乳幼児おむつ皮膚炎が生じやすい部位、例えば、排尿部、肛門部、臀部、鼠径部、腰部、腹部、大腿部、これらの近傍部等が挙げられる。SSLが採取される皮膚の部位は、乳幼児おむつ皮膚炎が発症している皮疹部であっても、発症していない無疹部であってもいずれでもよいが、好ましくは皮疹部又は皮疹部近傍の無疹部である。ここで皮疹部近傍とは、皮疹部に隣接する10cm以内の範囲を指す。 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. 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 or superficial skin lipids (SSL), more preferably skin superficial 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., preferably the site where infant diaper dermatitis is likely to occur. , for example, the urinary part, the anal part, the buttocks, the groin, the waist, the abdomen, the thigh, the vicinity of these parts, and the like. The site of the skin from which SSL is collected may be an erupted area where infant diaper dermatitis has developed or an erupted area where infant diaper dermatitis has not developed, but preferably the rashed area or the vicinity of the rashed area. is the non-erupted part of the Here, the vicinity of the rash refers to a range within 10 cm adjacent to the rash.
 ここで、「皮膚表上脂質(SSL)」とは、皮膚の表上に存在する脂溶性画分をいい、皮脂と呼ばれることもある。一般に、SSLは、皮膚にある皮脂腺等の外分泌腺から分泌された分泌物を主に含み、皮膚表面を覆う薄い層の形で皮膚表上に存在している。SSLは、皮膚細胞で発現したRNAを含む。(前記特許文献1参照)。また本発明において、「皮膚」とは、特に限定しない限り、角層、表皮、真皮、毛包、ならびに汗腺、皮脂腺及びその他の腺等の組織を含む領域の総称である。 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.
 被験乳幼児の皮膚からのSSLの採取には、皮膚からのSSLの回収又は除去に用いられているあらゆる手段を採用することができる。好ましくは、後述するSSL吸収性素材、SSL接着性素材、又は皮膚からSSLをこすり落とす器具を使用することができる。SSL吸収性素材又はSSL接着性素材としては、SSLに親和性を有する素材であれば特に限定されず、例えばポリプロピレン、パルプ等が挙げられる。皮膚からのSSLの採取手順のより詳細な例としては、あぶら取り紙、あぶら取りフィルム等のシート状素材へSSLを吸収させる方法、ガラス板、テープ等へSSLを接着させる方法、スパーテル、スクレイパー等によりSSLをこすり落として回収する方法、等が挙げられる。SSLの吸着性を向上させるため、脂溶性の高い溶媒を予め含ませたSSL吸収性素材を用いてもよい。一方、SSL吸収性素材は、水溶性の高い溶媒や水分を含んでいるとSSLの吸着が阻害されるため、水溶性の高い溶媒や水分の含有量が少ないことが好ましい。SSL吸収性素材は、乾燥した状態で用いることが好ましい。 Any means used to collect or remove SSL from the skin can be used to collect SSL from the skin of the test infant. 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.
 被験乳幼児から採取されたRNA含有SSLは一定期間保存されてもよい。採取されたSSLは、含有するRNAの分解を極力抑えるために、採取後できるだけ速やかに低温条件で保存することが好ましい。本発明における該RNA含有SSLの保存の温度条件は、0℃以下であればよく、好ましくは-20±20℃~-80±20℃、より好ましくは-20±10℃~-80±10℃、さらに好ましくは-20±20℃~-40±20℃、さらに好ましくは-20±10℃~-40±10℃、さらに好ましくは-20±10℃、さらに好ましくは-20±5℃である。該RNA含有SSLの該低温条件での保存の期間は、特に限定されないが、好ましくは12か月以下、例えば6時間以上12ヶ月以下、より好ましくは6ヶ月以下、例えば1日間以上6ヶ月以下、さらに好ましくは3ヶ月以下、例えば3日間以上3ヶ月以下である。  RNA-containing SSL collected from test infants 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.
 本発明において、標的遺伝子又はその発現産物の発現レベルの測定対象としては、RNAから人工的に合成されたcDNA、そのRNAをエンコードするDNA、そのRNAにコードされるタンパク質、該タンパク質と相互作用をする分子、そのRNAと相互作用する分子、又はそのDNAと相互作用する分子等が挙げられる。ここで、RNA、DNA又はタンパク質と相互作用する分子としては、DNA、RNA、タンパク質、多糖、オリゴ糖、単糖、脂質、脂肪酸、及びこれらのリン酸化物、アルキル化物、糖付加物等、及び上記いずれかの複合体が挙げられる。また、発現レベルとは、当該遺伝子又は発現産物の発現量や活性を包括的に意味する。 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.
 本発明の方法においては、好ましい態様として、生体試料としてSSLが用いられるが、この場合にはSSLに含まれるRNAの発現レベルが解析され、具体的にはRNAを逆転写によりcDNAに変換した後、該cDNA又はその増幅産物が測定される。
 SSLからのRNAの抽出には、生体試料からのRNAの抽出又は精製に通常使用される方法、例えば、フェノール/クロロホルム法、AGPC(acid guanidinium thiocyanate-phenol-chloroform extraction)法、又はTRIzol(登録商標)、RNeasy(登録商標)、QIAzol(登録商標)等のカラムを用いた方法、シリカをコーティングした特殊な磁性体粒子を用いる方法、Solid Phase Reversible Immobilization磁性体粒子を用いる方法、ISOGEN等の市販のRNA抽出試薬による抽出等を用いることができる。
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.
 該逆転写には、解析したい特定のRNAを標的としたプライマーを用いてもよいが、より包括的な核酸の保存及び解析のためにはランダムプライマーを用いることが好ましい。該逆転写には、一般的な逆転写酵素又は逆転写試薬キットを使用することができる。好適には、正確性及び効率性の高い逆転写酵素又は逆転写試薬キットが用いられ、その例としては、M-MLV Reverse Transcriptase及びその改変体、あるいは市販の逆転写酵素又は逆転写試薬キット、例えばPrimeScript(登録商標)Reverse Transcriptaseシリーズ(タカラバイオ社)、SuperScript(登録商標)Reverse Transcriptaseシリーズ(Thermo Scientific社)等が挙げられる。SuperScript(登録商標)III Reverse Transcriptase、SuperScript(登録商標)VILO cDNA Synthesis kit(いずれもThermo Scientific社)等が好ましく用いられる。
 該逆転写における伸長反応は、温度を好ましくは42℃±1℃、より好ましくは42℃±0.5℃、さらに好ましくは42℃±0.25℃に調整し、一方、反応時間を好ましくは60分間以上、より好ましくは80~120分間に調整するのが好ましい。
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.
 発現レベルを測定する方法は、RNA、cDNA又はDNAを対象とする場合、これらにハイブリダイズするDNAをプライマーとしたPCR法、リアルタイムRT-PCR法、マルチプレックスPCR、SmartAmp法、LAMP法等に代表される核酸増幅法、これらにハイブリダイズする核酸をプローブとして用いるハイブリダイゼーション法(DNAチップ、DNAマイクロアレイ、ドットブロットハイブリダイゼーション、スロットブロットハイブリダイゼーション、ノーザンブロットハイブリダイゼーション等)、塩基配列を決定する方法(シーケンシング)、又はこれらを組み合わせた方法から選ぶことができる。 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.
 PCRでは、解析したい特定のDNAを標的としたプライマーペアを用いて該特定の1種のDNAのみを増幅してもよいが、複数のプライマーペアを用いて同時に複数の特定のDNAを増幅してもよい。好ましくは、該PCRはマルチプレックスPCRである。マルチプレックスPCRは、PCR反応系に複数のプライマー対を同時に使用することで、複数の遺伝子領域を同時に増幅する方法である。マルチプレックスPCRは、市販のキット(例えば、Ion AmpliSeqTranscriptome Human Gene Expression Kit;ライフテクノロジーズジャパン株式会社等)を用いて実施することができる。
 該PCRにおけるアニーリング及び伸長反応の温度は、使用するプライマーに依存するため一概には言えないが、上記のマルチプレックスPCRキットは用いる場合、好ましくは62℃±1℃、より好ましくは62℃±0.5℃、さらに好ましくは62℃±0.25℃である。したがって、該PCRでは、好ましくはアニーリング及び伸長反応が1ステップで行われる。該アニーリング及び伸長反応のステップの時間は、増幅すべきDNAのサイズ等に依存して調整され得るが、好ましくは14~18分間である。該PCRにおける変性反応の条件は、増幅すべきDNAに依存して調整され得るが、好ましくは95~99℃で10~60秒間である。上記のような温度及び時間での逆転写及びPCRは、一般的にPCRに使用されるサーマルサイクラーを用いて実行することができる。
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.
 当該PCRで得られた反応産物の精製は、反応産物のサイズ分離によって行われることが好ましい。サイズ分離により、目的のPCR反応産物を、PCR反応液中に含まれるプライマーやその他の不純物から分離することができる。DNAのサイズ分離は、例えば、サイズ分離カラムや、サイズ分離チップ、サイズ分離に利用可能な磁気ビーズ等によって行うことができる。サイズ分離に利用可能な磁気ビーズの好ましい例としては、Ampure XP等のSolid Phase Reversible Immobilization(SPRI)磁性ビーズが挙げられる。 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.
 精製したPCR反応産物に対して、その後の定量解析を行うために必要なさらなる処理を施してもよい。例えば、DNAのシーケンシングのために、精製したPCR反応産物を、適切なバッファー溶液へと調製したり、PCR増幅されたDNAに含まれるPCRプライマー領域を切断したり、増幅されたDNAにアダプター配列をさらに付加したりしてもよい。例えば、精製したPCR反応産物をバッファー溶液へと調製し、増幅DNAに対してPCRプライマー配列の除去及びアダプターライゲーションを行い、得られた反応産物を、必要に応じて増幅して、定量解析のためのライブラリーを調製することができる。これらの操作は、例えば、SuperScript(登録商標)VILO cDNA Synthesis kit(ライフテクノロジーズジャパン株式会社)に付属している5×VILO RT Reaction Mix、及びIon AmpliSeq Transcriptome Human Gene Expression Kit(ライフテクノロジーズジャパン株式会社)に付属している5×Ion AmpliSeq HiFi Mix、及びIon AmpliSeq Transcriptome Human Gene Expression Core Panelを用いて、各キット付属のプロトコルに従って行うことができる。 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.
 ノーザンブロットハイブリダイゼーション法を利用して標的遺伝子又はそれに由来する核酸の発現量を測定する場合は、例えば、まずプローブDNAを放射性同位元素、蛍光物質等で標識し、次いで、得られた標識DNAを、常法に従ってナイロンメンブレン等にトランスファーした生体試料由来のRNAとハイブリダイズさせる。その後、形成された標識DNAとRNAとの二重鎖を、標識物に由来するシグナルを検出することにより測定する方法が挙げられる。 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.
 RT-PCR法を用いて標的遺伝子又はそれに由来する核酸の発現量を測定する場合は、例えば、まず生体試料由来のRNAから常法に従ってcDNAを調製し、これを鋳型として本発明の標的遺伝子が増幅できるように調製した一対のプライマー(上記cDNA(-鎖)に結合する正鎖、+鎖に結合する逆鎖)をこれとハイブリダイズさせる。その後、常法に従ってPCR法を行い、得られた増幅二本鎖DNAを検出する。増幅された二本鎖DNAの検出には、予めRI、蛍光物質等で標識しておいたプライマーを用いて上記PCRを行うことによって産生される標識二本鎖DNAを検出する方法等を用いることができる。 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.
 DNAマイクロアレイを用いて標的遺伝子又はそれに由来する核酸の発現量を測定する場合は、例えば、支持体に本発明の標的遺伝子由来の核酸(cDNA又はDNA)の少なくとも1種を固定化したアレイを用い、mRNAから調製した標識化cDNA又はcRNAをマイクロアレイ上に結合させ、マイクロアレイ上の標識を検出することによって、mRNAの発現量を測定することができる。
 前記アレイに固定化される核酸としては、ストリンジェントな条件下に特異的(すなわち、実質的に目的の核酸のみに)にハイブリダイズする核酸であればよく、例えば、本発明の標的遺伝子の全配列を有する核酸であってもよく、部分配列からなる核酸であってもよい。ここで、「部分配列」とは、少なくとも15~25塩基からなる核酸が挙げられる。ここでストリンジェントな条件は、通常「1×SSC、0.1%SDS、37℃」程度の洗浄条件を挙げることができ、より厳しいハイブリダイズ条件としては「0.5×SSC、0.1%SDS、42℃」程度、さらに厳しいハイブリダイズ条件としては「0.1×SSC、0.1%SDS、65℃」程度の条件を挙げることができる。ハイブリダイズ条件は、J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press (2001)等に記載されている。
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, Third Edition, Cold Spring Harbor Laboratory Press (2001) and others.
 シーケンシングによって標的遺伝子又はそれに由来する核酸の発現量を測定する場合は、例えば、次世代シーケンサー(例えばIon S5/XLシステム、ライフテクノロジーズジャパン株式会社)が用いて解析することが挙げられる。シーケンシングで作成されたリードの数(リードカウント)に基づいて、RNA発現を定量することができる。 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).
 上記の測定に用いられるプローブ又はプライマー、すなわち、本発明の標的遺伝子又はそれに由来する核酸を特異的に認識し増幅するためのプライマー、又は該RNA又はそれに由来する核酸を特異的に検出するためのプローブがこれに該当するが、これらは、当該標的遺伝子を構成する塩基配列に基づいて設計することができる。ここで「特異的に認識する」とは、例えばノーザンブロット法において、実質的に本発明の標的遺伝子又はそれに由来する核酸のみを検出できること、また例えばRT-PCR法において、実質的に当該核酸のみが増幅される如く、当該検出物又は生成物が当該遺伝子又はそれに由来する核酸であると判断できることを意味する。
 具体的には、本発明の標的遺伝子を構成する塩基配列からなるDNA又はその相補鎖に相補的な一定数のヌクレオチドを含むオリゴヌクレオチドを利用することができる。ここで「相補鎖」とは、A:T(RNAの場合はU)、G:Cの塩基対からなる2本鎖DNAの一方の鎖に対する他方の鎖を指す。また、「相補的」とは、当該一定数の連続したヌクレオチド領域で完全に相補配列である場合に限られず、好ましくは80%以上、より好ましくは90%以上、さらに好ましくは95%以上、よりさらに好ましくは98%以上の塩基配列上の同一性を有すればよい。塩基配列の同一性は、前記BLAST等のアルゴリズムにより決定することができる。
 斯かるオリゴヌクレオチドは、プライマーとして用いる場合には、特異的なアニーリング及び鎖伸長ができればよく、通常、例えば10塩基以上、好ましくは15塩基以上、より好ましくは20塩基以上、かつ例えば100塩基以下、好ましくは50塩基以下、より好ましくは35塩基以下の鎖長を有するものが挙げられる。また、プローブとして用いる場合には、特異的なハイブリダイゼーションができればよく、本発明の標的遺伝子を構成する塩基配列からなるDNA(又はその相補鎖)の少なくとも一部若しくは全部の配列を有し、例えば10塩基以上、好ましくは15塩基以上、かつ例えば100塩基以下、好ましくは50塩基以下、より好ましくは25塩基以下の鎖長のものが用いられる。
 なお、ここで、「オリゴヌクレオチド」は、DNAあるいはRNAであることができ、合成されたものでも天然のものでもよい。又、ハイブリダイゼーションに用いるプローブは、通常標識したものが用いられる。
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.
 また、本発明の標的遺伝子の翻訳産物(タンパク質)、当該タンパク質と相互作用する分子、RNAと相互作用する分子、又はDNAと相互作用する分子を測定する場合は、プロテインチップ解析、免疫測定法(例えば、ELISA等)、質量分析(例えば、LC-MS/MS、MALDI-TOF/MS)、1-ハイブリッド法(PNAS 100, 12271-12276(2003))や2-ハイブリッド法(Biol. Reprod. 58, 302-311 (1998))のような方法を用いることができ、対象に応じて適宜選択できる。
 例えば、測定対象としてタンパク質が用いられる場合は、本発明の発現産物を特異的に認識する抗体、具体的には発現産物であるタンパク質を他のタンパク質から識別することが可能な構造的特徴部位(エピトープ)を認識する抗体を生体試料と接触させ、当該抗体に結合した試料中のポリペプチド又はタンパク質を検出し、そのレベルを測定することによって実施される。例えば、ウェスタンブロット法によれば、一次抗体として上記の抗体を用いた後、二次抗体として放射性同位元素、蛍光物質又は酵素等で標識した一次抗体に結合する抗体を用いて、その一次抗体を標識し、これら標識物質由来のシグナルを放射線測定器、蛍光検出器等で測定することが行われる。
 尚、上記翻訳産物に対する抗体は、ポリクローナル抗体であっても、モノクローナル抗体であってもよい。これらの抗体は、公知の方法に従って製造することができる。具体的には、ポリクローナル抗体は、常法に従って大腸菌等で発現し精製したタンパク質を用いて、あるいは常法に従って当該タンパク質の部分ポリペプチドを合成して、家兎等の非ヒト動物に免疫し、該免疫動物の血清から常法に従って得ることが可能である。
 一方、モノクローナル抗体は、常法に従って大腸菌等で発現し精製したタンパク質又は該タンパク質の部分ポリペプチドをマウス等の非ヒト動物に免疫し、得られた脾臓細胞と骨髄腫細胞とを細胞融合させて調製したハイブリドーマ細胞から得ることができる。また、モノクローナル抗体は、ファージディスプレイを用いて作製してもよい(Griffiths, A.D.; Duncan, A.R., Current Opinion in Biotechnology, Volume 9, Number 1, February 1998, pp.102-108(7))。
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)).
 斯くして、被験乳幼児から採取された生体試料中の本発明の標的遺伝子又はその発現産物の発現レベルが測定され、当該発現レベルに基づいて当該被験乳幼児のおむつ皮膚炎の症度が検出される。
 また本発明では、被験乳幼児から採取された生体試料中の本発明の標的遺伝子又はその発現産物の発現レベルを少なくとも2つの時期に測定し、発現レベルの変化、又は変化量を指標に、被験乳幼児におけるおむつ皮膚炎の症度の変化の有無、又は変化の程度を検出することができる。
 検出は、具体的には、測定された本発明の標的遺伝子又はその発現産物の発現レベルを予め設定したカットオフ値(参照値)と比較することによって行われる。
 シーケンシングにより複数の標的遺伝子の発現レベルの解析を行う場合は、上記したように、発現量のデータであるリードカウント値、当該リードカウント値をサンプル間の総リード数の違いを補正したRPM値、当該RPM値を底2の対数値に変換した値(logRPM値)又は整数1を加算した底2の対数値(log(RPM+1)値)、あるいはDESeq2(Love MI et al. Genome Biol. 2014)を用いて補正されたカウント値(Normalized count値)又は整数1を加算した底2の対数値(log(Normalized count+1)値)を指標として用いるのが好ましい。また、RNA-seqの定量値として一般的な、fragments per kilobase of exon per million reads mapped(FPKM)、reads per kilobase of exon per million reads mapped(RPKM)、transcripts per million(TPM)等によって算出される値であってもよい。また、マイクロアレイ法によって得られるシグナル値、及びその補正値であってもよい。また、RT-PCR等により特定の標的遺伝子のみの解析を行う場合には、対象遺伝子の発現量をハウスキーピング遺伝子の発現量を基準とする相対的な発現量に変換して解析する方法、又は標的遺伝子の領域を含むプラスミドを用いて絶対的なコピー数を定量(絶対定量)して解析する方法が好ましい。デジタルPCR法によって得られるコピー数であってもよい。
Thus, the expression level of the target gene of the present invention or its expression product in a biological sample collected from a test infant is measured, and the severity of diaper dermatitis in the test infant is detected based on the expression level. .
Further, in the present invention, the expression level of the target gene of the present invention or its expression product in a biological sample collected from a test infant is measured at least at two times, and the change in the expression level or the amount of change is used as an index, and the test infant is It is possible to detect the presence or absence of change in the degree of diaper dermatitis or the degree of change.
Specifically, detection is performed by comparing the measured expression level of the target gene of the present invention or its expression product with a preset cutoff value (reference value).
When analyzing the expression level of multiple 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 logarithmic value of base 2 (log 2 RPM value) or a logarithmic value of base 2 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 general 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.
 ここで、「カットオフ値」(「参照値」)は、おむつ皮膚炎スコアと、本発明の標的遺伝子又はその発現産物の発現レベルの関係に基づき、予め決定することができる。例えば、ある集団を、おむつ皮膚炎の状態、すなわちおむつ皮膚炎スコアより症状なし、軽微、軽度、軽度から中等度、中等度、中等度から重度、重度の群に分け、それぞれの群における当該標的遺伝子又はその発現産物の発現レベルの平均値や標準偏差等の統計値を参考に決定した値を、それぞれの群への属否を判別するカットオフ値(参照値)として決定することができる。
 標的遺伝子として複数種の遺伝子を用いる場合は、それぞれ各々の遺伝子又はその発現産物についてカットオフ値(参照値)を求めることが好ましい。
 集団としては、性別、人種、年齢毎に集団を形成してもよい。
Here, the "cutoff value"("referencevalue") can be determined in advance based on the relationship between the diaper dermatitis score and the expression level of the target gene of the present invention or its expression product. For example, a population is divided into diaper dermatitis status, i.e. no symptoms, mild, mild, mild-to-moderate, moderate, moderate-to-severe, severe groups according to the diaper dermatitis score, and the target in each group A value determined with reference to statistical values such as the average value and standard deviation of the expression level of a gene or its expression product can be determined as a cutoff value (reference value) for determining belonging to each group.
When multiple types of genes are used as target genes, it is preferable to determine a cutoff value (reference value) for each gene or its expression product.
Groups may be formed according to sex, race, and age.
 さらに、本発明の標的遺伝子又はその発現産物の発現レベルの測定値を利用して、判別式(予測モデル)を構築し、当該判別式を利用して、おむつ皮膚炎の症度を検出することができる。例えば、当該標的遺伝子又はその発現産物の発現レベルの測定値を説明変数とし、おむつ皮膚炎スコア(例えば、スコアを偏差値に変換した値)を目的変数とした機械学習により、おむつ皮膚炎の症度を検出するための最適な判別式(予測モデル)を構築することができる。
 そして、被験乳幼児から採取された生体試料から本発明の標的遺伝子又はその発現産物の発現レベルを同様に測定し、得られた測定値を当該判別式(予測モデル)に入力し、当該判別式から得られた結果(おむつ皮膚炎スコアの予測値)を、被験乳幼児におけるおむつ皮膚炎の症度として検出できる。
Furthermore, a discriminant (prediction model) is constructed using the measured value of the expression level of the target gene of the present invention or its expression product, and the degree of diaper dermatitis is detected using the discriminant. can be done. For example, the measured value of the expression level of the target gene or its expression product is used as an explanatory variable, and the diaper dermatitis score (e.g., the value obtained by converting the score into a deviation value) is used as the objective variable by machine learning. An optimal discriminant (prediction model) can be constructed to detect the degree.
Then, the expression level of the target gene of the present invention or its expression product is similarly measured from the biological sample collected from the test infant, the obtained measured value is input into the discriminant (prediction model), and from the discriminant The obtained result (predicted value of diaper dermatitis score) can be detected as the degree of diaper dermatitis in the test infant.
 さらに、症度が異なるおむつ皮膚炎乳幼児由来の標的遺伝子又はその発現産物の発現レベルと、健常乳幼児由来の標的遺伝子又はその発現産物の発現レベルの測定値を利用して、症度が異なるおむつ皮膚炎乳幼児群(例えば、軽微、軽度、軽度から中等度、中等度、中等度から重度、重度等の症度から選択される、2以上の群)及び健常乳幼児(症状なし)とを分ける判別式(予測モデル)を構築し、当該判別式を利用して、乳幼児おむつ皮膚炎の症度を検出することができる。すなわち、症度が異なるおむつ皮膚炎乳幼児群由来の標的遺伝子又はその発現産物の発現レベルと、健常乳幼児由来の標的遺伝子又はその発現産物の発現レベルの測定値を教師サンプルとして、症度が異なるおむつ皮膚炎乳児群(例えば、軽微、軽度、軽度から中等度、中等度、中等度から重度、重度等の症度から選択される、2以上の群)及び健常乳幼児(症状なし)を分ける判別式(予測モデル)を構築し、当該判別式に基づいて症度が異なる各おむつ皮膚炎乳幼児群を判別するカットオフ値(参照値)を求める。
 そして、被験乳幼児から採取された生体試料から標的遺伝子又はその発現産物の発現レベルを同様に測定し、得られた測定値を当該判別式に代入し、当該判別式から得られた結果をカットオフ値(参照値)と比較することによって、当該被検乳幼児におけるおむつ皮膚炎の症度を検出できる。
Furthermore, by using the expression level of the target gene or its expression product derived from diaper dermatitis infants with different severity and the expression level of the target gene or its expression product derived from healthy infants, diaper skin with different severity A discriminant formula to distinguish a group of infants with inflammation (e.g., two or more groups selected from mild, mild, mild-to-moderate, moderate, moderate-to-severe, severe, etc.) and healthy infants (no symptoms) A (prediction model) can be constructed and the discriminant can be used to detect the severity of infant diaper dermatitis. That is, the measured values of the expression levels of target genes or their expression products derived from a group of diaper dermatitis infants with different degrees of diaper dermatitis and the measured values of the expression levels of target genes or their expression products derived from healthy infants were used as teaching samples, and diapers with different degrees of severity were used. A discriminant to divide infants with dermatitis (e.g., two or more groups selected from mild, mild, mild-to-moderate, moderate, moderate-to-severe, severe, etc.) and healthy infants (no symptoms) A (prediction model) is constructed, and a cutoff value (reference value) for discriminating diaper dermatitis infants with different degrees of severity is obtained based on the discriminant.
Then, similarly measure the expression level of the target gene or its expression product from the biological sample collected from the test infant, substitute the obtained measured value into the discriminant, and cut off the result obtained from the discriminant By comparing with the value (reference value), the degree of diaper dermatitis in the subject infant can be detected.
 判別式(予測モデル)の構築におけるアルゴリズムとしては、機械学習に用いるアルゴリズム等の公知のものを利用することができる。機械学習アルゴリズムの例としては、ランダムフォレスト(Random forest)、線形カーネルのサポートベクターマシン(SVM linear)、rbfカーネルのサポートベクターマシン(SVM rbf)、ニューラルネットワーク(Nerural net)、一般線形モデル(Generalized linear model)、正則化線形判別分析(Regularized linear discriminant analysis)、正則化ロジスティック回帰(Regularized logistic regression)、ラッソ(Least Absolute Shrinkage and Selection Operator)回帰等が挙げられる。構築した予測モデルに検証用のデータを入力して予測値を算出し、当該予測値が実測値と最も適合するモデル、例えば正解率(Accuracy)が最も大きいモデルを最適な予測モデルとして選抜することができる。また、予測値と実測値から検出率(Recall)、精度(Precision)、及びそれらの調和平均であるF値を計算し、そのF値が最も大きいモデルを最適な予測モデルとして選抜することができる。また、予測値と実測値の二乗平均平方根誤差(RMSE)を予測モデルの精度評価指標として用い、そのRMSEの最も小さいモデルを最適な予測モデルとして選抜することができる。 As an algorithm for constructing the discriminant (prediction model), a known one 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, Lasso (Least Absolute Shrinkage and Selection Operator) regression, and the like. Enter verification data into the constructed prediction model to calculate prediction values, and select the model that best matches the prediction values with the measured values, for example, the model with the highest accuracy rate as the optimal 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. . Also, the root mean square error (RMSE) between the predicted value and the measured value can be used as an accuracy evaluation index of the prediction model, and the model with the smallest RMSE can be selected as the optimum prediction model.
 カットオフ値(参照値)の決定方法は特に制限されず、公知の手法に従って決定することができる。例えば、判別式(予測モデル)を使用して作成されたROC(Receiver Operating Characteristic Curve)曲線より求めることができる。ROC曲線では、縦軸に陽性被験者において陽性の結果がでる確率(感度)と、横軸に陰性被験者において陰性の結果がでる確率(特異度)を1から減算した値(偽陽性率)がプロットされる。ROC曲線に示される「真陽性(感度)」及び「偽陽性(1-特異度)」に関し、「真陽性(感度)」-「偽陽性(1-特異度)」が最大となる値(Youden index)をカットオフ値(参照値)とすることができる。 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 (prediction model). In the ROC curve, the vertical axis is the probability of a positive result in positive subjects (sensitivity), and the horizontal axis is the value obtained by subtracting the probability of a negative result in negative subjects (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).
 本発明の乳幼児おむつ皮膚炎の症度を検出するための検査用キットは、被験乳幼児から分離した生体試料における本発明の標的遺伝子又はその発現産物の発現レベルを測定するための検査試薬を含有するものである。具体的には、本発明の標的遺伝子又はそれに由来する核酸と特異的に結合(ハイブリダイズ)するオリゴヌクレオチド(例えば、PCR用のプライマー)を含む、核酸増幅、ハイブリダイゼーションのための試薬、或いは、本発明の標的遺伝子の発現産物(タンパク質)を認識する抗体を含む免疫学的測定のための試薬等が挙げられる。当該キットに包含されるオリゴヌクレオチド、抗体等は、上述したとおり公知の方法により得ることができる。
 また、当該検査用キットには、上記抗体や核酸の他、標識試薬、緩衝液、発色基質、二次抗体、ブロッキング剤や、試験に必要な器具やポジティブコントロールやネガティブコントロールとして使用するコントロール試薬、生体試料を採取するための用具(例えば、SSLを採取するためのあぶら取りフィルム等)等を含むことができる。
A test kit for detecting the severity of infant diaper dermatitis of the present invention contains a test reagent for measuring the expression level of the target gene of the present invention or its expression product in a biological sample isolated from a test infant. It is. 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.
 上述した実施形態に関し、本発明は以下の態様をさらに開示する。 Regarding the above-described embodiments, the present invention further discloses the following aspects.
<1>被験乳幼児から採取された生体試料について、GALNT3、CMTM6、SLC35E1及びEID3の4種の遺伝子群より選択される少なくとも1つの遺伝子又はその発現産物の発現レベルを測定する工程を含む、当該乳幼児のおむつ皮膚炎の症度を検出する方法。 <1> For biological samples collected from test infants, the infants comprising the step of measuring the expression level of at least one gene or its expression product selected from the four gene groups of GALNT3, CMTM6, SLC35E1 and EID3 method for detecting the severity of diaper dermatitis in children.
<2>前記GALNT3、CMTM6、SLC35E1及びEID3の4種の遺伝子群より選択される好ましくは2種以上、より好ましくは3種以上、さらに好ましくはGALNT3及びCMTM6を含む2種以上、さらに好ましくは4種全ての遺伝子又はその発現産物の発現レベルが測定される<1>記載の検出方法。
<3>好ましくは、さらに前記表5に示す14種の遺伝子群より選択される少なくとも1つの遺伝子又はその発現産物の発現レベルを測定する工程を含む、<1>又は<2>記載の検出方法。
<4>前記表5に示す14種の遺伝子群より選択される好ましくは2種以上、より好ましくは3種以上、さらに好ましくは表5中*を付し太字で表示した乳幼児おむつ皮膚炎との関連が報告されていない遺伝子から選択される少なくとも1つ、好ましくは2種以上、より好ましくは3種以上の遺伝子又はその発現産物の発現レベルが測定される<3>記載の検出方法。
<5>好ましくは、前記表2、表3又は表4に記載の遺伝子又はその発現産物の発現レベルが測定される<1>~<4>のいずれかに記載の検出方法。
<6>前記生体試料が、被験乳幼児の好ましくは臓器、皮膚、血液、尿、唾液、汗、角層、皮膚表上脂質(SSL)、組織浸出液等の体液、血液から調製された血清、血漿、便又は毛髪であり、より好ましくは皮膚又は皮膚表上脂質(SSL)であり、さらに好ましくは皮膚表上脂質(SSL)である<1>~<5>のいずれかに記載の検出方法。
<7>前記遺伝子又はその発現産物の発現レベルの測定対象が、好ましくはRNAから人工的に合成されたcDNA、そのRNAをエンコードするDNA、そのRNAにコードされるタンパク質、該タンパク質と相互作用をする分子、そのRNAと相互作用する分子、又はそのDNAと相互作用する分子である<1>~<6>のいずれかに記載の検出方法。
<8>前記遺伝子又はその発現産物の発現レベルが、好ましくは被験乳幼児の皮膚表上脂質(SSL)から採取されたmRNAの発現量である<1>~<5>のいずれかに記載の検出方法。
<9>前記被験乳幼児が、乳幼児おむつ皮膚炎を発症している乳幼児、乳幼児おむつ皮膚炎の発症が疑われる乳幼児、遺伝的に乳幼児おむつ皮膚炎の素因を有する乳幼児又は兄弟姉妹等の近親者が乳幼児おむつ皮膚炎を発症している若しくは発症していた乳幼児である<8>記載の検出方法。
<10>前記被験乳幼児の皮膚が、好ましくは排尿部、肛門部、臀部、鼠径部、腰部、腹部又は大腿部の皮膚である<8>又は<9>記載の検出方法。
<11>皮膚表上脂質(SSL)が採取される皮膚の部位は、乳幼児おむつ皮膚炎が発症している皮疹部であっても、発症していない無疹部であってもいずれでもよいが、好ましくは皮疹部又は皮疹部近傍の無疹部である<8>~<10>のいずれかに記載の検出方法。
<12>前記遺伝子又はその発現産物の発現レベルに基づいて前記被験乳幼児のおむつ皮膚炎の症度を検出することを含む<1>~<11>のいずれかに記載の検出方法。
<13>好ましくは、前記遺伝子又はその発現産物の発現レベルに基づく判別式(予測モデル)を用いて乳幼児おむつ皮膚炎の症度を検出することを含み、
 前記判別式(予測モデル)が、前記遺伝子又はその発現産物の発現レベルの測定値を説明変数とし、おむつ皮膚炎スコアを目的変数とした機械学習により構築される<1>~<12>のいずれかに記載の検出方法。
<14>前記乳幼児おむつ皮膚炎の症度が、評価部位毎に、紅斑、丘疹、浸軟及び落屑の4症状について0:症状なし、1:軽微、2:軽度、3:軽度から中等度、4:中等度、5:中等度から重度、6:重度の7段階のスコアを付し、評価部位全てのスコアを合算した値(合算値)に対応する症度である<1>~<13>のいずれかに記載の検出方法。
<15>前記評価部位が、乳幼児の腹部、腰部、左右鼠径部及び左右臀部の6箇所である<14>記載の検出方法。
<16>前記遺伝子又はそれに由来する核酸と特異的にハイブリダイズするオリゴヌクレオチド、又は前記遺伝子の発現産物を認識する抗体を含有する、<1>~<15>の検出方法に用いられる乳幼児おむつ皮膚炎の症度を検出するための検査用キット。
<17>前記表2~表4において*を付し太字で表示した遺伝子群より選択される少なくとも1つの遺伝子又はその発現産物からなる、乳幼児おむつ皮膚炎の症度を検出するための検出マーカー。
<18>GALNT3、CMTM6、SLC35E1及びEID3の4種の遺伝子群より選択される少なくとも1つの遺伝子又はその発現産物である、<17>記載の乳幼児おむつ皮膚炎の症度を検出するための検出マーカー。
<19>被験乳幼児から採取された生体試料に由来する前記表2~表4において*を付し太字で表示した遺伝子群より選択される少なくとも1つの遺伝子又はその発現産物の、乳幼児おむつ皮膚炎の症度の検出マーカーとしての使用。
<20>GALNT3、CMTM6、SLC35E1及びEID3の4種の遺伝子群より選択される少なくとも1つの遺伝子又はその発現産物である、<19>記載の検出マーカーとしての使用。
<21>遺伝子又はその発現産物が前記被験乳幼児の皮膚表上脂質に含まれるmRNAである、<19>又は<20>記載の使用。
<2> preferably two or more, more preferably three or more, still more preferably two or more including GALNT3 and CMTM6, more preferably four, selected from the four gene groups of GALNT3, CMTM6, SLC35E1 and EID3 The detection method according to <1>, wherein the expression level of the gene or its expression product is measured for all species.
<3> Preferably, the detection method according to <1> or <2>, further comprising the step of measuring the expression level of at least one gene selected from the 14 gene groups shown in Table 5 or its expression product. .
<4> Preferably two or more, more preferably three or more, more preferably infant diaper dermatitis indicated in bold with * in Table 5 selected from the 14 gene groups shown in Table 5 The detection method according to <3>, wherein the expression level of at least one, preferably two or more, more preferably three or more genes selected from unreported genes or their expression products is measured.
<5> Preferably, the detection method according to any one of <1> to <4>, wherein the expression level of the gene or its expression product described in Table 2, Table 3 or Table 4 is measured.
<6> The biological sample is preferably an organ, skin, blood, urine, saliva, sweat, stratum corneum, superficial skin lipid (SSL), body fluids such as tissue exudate, serum prepared from blood, plasma , stool or hair, more preferably skin or superficial skin lipids (SSL), and still more preferably superficial skin lipids (SSL) <1> to <5>.
<7> The object to be measured for the expression level of the gene or its expression product is preferably cDNA artificially synthesized from RNA, DNA encoding the RNA, a protein encoded by the RNA, and interacting with the protein. The detection method according to any one of <1> to <6>, wherein the molecule interacts with the RNA, the molecule interacts with the RNA, or the molecule interacts with the DNA.
<8> The detection according to any one of <1> to <5>, wherein the expression level of the gene or its expression product is preferably the expression level of mRNA collected from superficial skin lipids (SSL) of the test infant. Method.
<9> The test infant is an infant who has developed infant diaper dermatitis, an infant suspected of developing infant diaper dermatitis, an infant genetically predisposed to infant diaper dermatitis, or a relative such as siblings The detection method according to <8>, wherein the infant is developing or has developed infant diaper dermatitis.
<10> The detection method according to <8> or <9>, wherein the skin of the subject infant is preferably the skin of the urinary region, anus, buttocks, groin, waist, abdomen, or thigh.
<11> The site of the skin from which lipids on the skin surface (SSL) are collected may be either a rash area where infant diaper dermatitis develops or a non-rash area where infant diaper dermatitis does not develop. , The detection method according to any one of <8> to <10>, which is preferably an erupted area or a non-erupted area near the erupted area.
<12> The detection method according to any one of <1> to <11>, comprising detecting the degree of diaper dermatitis in the test infant based on the expression level of the gene or its expression product.
<13> Preferably, detecting the severity of infant diaper dermatitis using a discriminant (predictive model) based on the expression level of the gene or its expression product,
Any of <1> to <12>, wherein the discriminant (prediction model) is constructed by machine learning with the measured value of the expression level of the gene or its expression product as an explanatory variable and the diaper dermatitis score as an objective variable. The detection method according to .
<14> The severity of infant diaper dermatitis is 4 symptoms of erythema, papule, maceration and desquamation for each evaluation site: 0: no symptoms, 1: slight, 2: mild, 3: mild to moderate, 4: Moderate, 5: Moderate to severe, 6: Severe 7 grades are given, and the sum of the scores of all the evaluation sites (total value) is the degree of severity corresponding to <1> to <13. The detection method according to any one of >.
<15> The detection method according to <14>, wherein the evaluation sites are the abdomen, waist, left and right inguinal regions, and left and right buttocks of the infant.
<16> Infant diaper skin used in the detection method of <1> to <15>, containing 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 the severity of inflammation.
<17> A detection marker for detecting the severity of infant diaper dermatitis, comprising at least one gene or its expression product selected from the gene group indicated in bold with * in Tables 2 to 4 above.
<18> A detection marker for detecting the severity of infant diaper dermatitis according to <17>, which is at least one gene selected from the four gene groups of GALNT3, CMTM6, SLC35E1 and EID3 or an expression product thereof. .
<19> Infant diaper dermatitis of at least one gene or its expression product selected from the group of genes indicated in bold with * in Tables 2 to 4 above derived from biological samples collected from test infants Use as a marker for detection of disease severity.
<20> Use as a detection marker according to <19>, which is at least one gene or its expression product selected from the four gene groups of GALNT3, CMTM6, SLC35E1 and EID3.
<21> The use according to <19> or <20>, wherein the gene or its expression product is mRNA contained in the lipids on the skin surface of the test infant.
実施例1 SSLから抽出されたRNAを用いたおむつ皮膚炎の症度の検出
1)被験乳幼児及びおむつ皮膚炎スコアの測定
 生後3~8ヵ月の日本人男女乳幼児59名を被験乳幼児とした。被験乳幼児の腹部、腰部、左右鼠径部、左右臀部の計6箇所を対象に、紅斑、丘疹、浸軟及び落屑の4症状について表6に示す基準に基づき皮膚科医が診察によりスコアを付け、計6箇所のスコアを合算した値(合算値)をおむつ皮膚炎スコアとした。
 その結果、症状なし(スコア0)は0名、軽微(スコア1~24)は45名、軽度(スコア25~48)は13名、軽度から中等度(スコア49~72)は1名、中等度(スコア73~96)は0名、中等度から重度(スコア97~120)は0名、重度(スコア121~144)は0名であった。
Example 1 Detection of Diaper Dermatitis Severity Using RNA Extracted from SSL 1) Test Infants and Measurement of Diaper Dermatitis Score Fifty-nine male and female infants aged 3 to 8 months were used as test infants. A dermatologist scored the 4 symptoms of erythema, papules, maceration and desquamation based on the criteria shown in Table 6 by examining the abdomen, waist, left and right inguinal regions, and left and right buttocks of the infants to be tested. A value (total value) obtained by summing up the scores of a total of six points was defined as the diaper dermatitis score.
As a result, 0 patients had no symptoms (score 0), 45 patients had mild symptoms (scores 1 to 24), 13 patients had mild symptoms (scores 25 to 48), 1 patient had mild to moderate symptoms (scores 49 to 72), and 1 patient had moderate symptoms. None were severe (scores 73-96), 0 were moderate to severe (scores 97-120), and 0 were severe (scores 121-144).
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
2)SSL採取
 各被験乳幼児の腹部、腰部、左右鼠径部及び左右臀部からあぶら取りフィルム(5×8cm、ポリプロピレン製、3M社)を用いて皮脂を回収後、該あぶら取りフィルムをバイアルに移し、RNA抽出に使用するまで-80℃にて保存した。
2) SSL collection After collecting sebum from the abdomen, waist, left and right inguinal regions, and left and right buttocks of each test infant using an oil blotting film (5 x 8 cm, made of polypropylene, 3M company), the oil blotting film was transferred to a vial, Stored at -80°C until used for RNA extraction.
3)RNA調製及びシーケンシング
 上記2)のあぶら取りフィルムを適当な大きさに切断し、QIAzol Lysis Reagent(Qiagen)を用いて、付属のプロトコルに準じてRNAを抽出した。抽出されたRNAを元に、SuperScript VILO cDNA Synthesis kit(ライフテクノロジーズジャパン株式会社)を用いて42℃、90分間逆転写を行いcDNAの合成を行った。逆転写反応のプライマーには、キットに付属しているランダムプライマーを使用した。得られたcDNAから、マルチプレックスPCRにより20802遺伝子に由来するDNAを含むライブラリーを調製した。マルチプレックスPCRは、Ion AmpliSeqTranscriptome Human Gene Expression Kit(ライフテクノロジーズジャパン株式会社)を用いて、[99℃、2分→(99℃、15秒→62℃、16分)×20サイクル→4℃、Hold]の条件で行った。得られたPCR産物は、Ampure XP(ベックマン・コールター株式会社)で精製した後に、バッファーの再構成、プライマー配列の消化、アダプターライゲーションと精製、増幅を行い、ライブラリーを調製した。調製したライブラリーをIon 540 Chipにローディングし、Ion S5/XLシステム(ライフテクノロジーズジャパン株式会社)を用いてシーケンシングした。
3) RNA Preparation and Sequencing The blotting film of 2) above was cut into an appropriate size, and RNA was extracted using QIAzol Lysis Reagent (Qiagen) according to the attached protocol. Based on the extracted RNA, reverse transcription was performed at 42° C. for 90 minutes using SuperScript VILO cDNA Synthesis kit (Life Technologies Japan Co., Ltd.) 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).
4)データ解析
(1)使用データ
 上記3)で測定したSSL由来RNAの発現量のデータ(リードカウント値)を取得し、サンプル被験乳幼児間の総リード数の違いを補正したRPM値に変換した。特徴量遺伝子の選択及び機械学習モデルの構築には、負の二項分布に従うRPM値を正規分布に近似するため、RPM値に整数1を加算した底2の対数値(log(RPM+1)値)を用いた。なお、全サンプル被験乳幼児の発現量データのうち90%以上のサンプル被験乳幼児で欠損値ではない発現量データが得られている5809遺伝子のみ以下の解析に使用した。
4) Data analysis (1) Data used The data (read count value) of the expression level of SSL-derived RNA measured in 3) above was obtained and converted into an RPM value corrected for the difference in the total number of reads between sample infants tested. . For the selection of feature genes and the construction of the machine learning model, in order to approximate the RPM value following the negative binomial distribution to the normal distribution, the base 2 logarithmic value (log 2 (RPM + 1) value obtained by adding an integer 1 to the RPM value ) was used. Only 5809 genes for which non-missing expression level data were obtained in 90% or more of the sample tested infants among the expression level data of all the sample tested infants were used for the following analysis.
(2)データセット分割
 被験乳幼児59名のデータセットのうち、48名のRNAプロファイルデータをモデル構築のTrainデータとし、残り11名分のRNAプロファイルデータをモデル精度評価に使用するTestデータとした。
 データ分割は[R]のcreateDataPartition関数を用い、TrainデータとTestデータ間で目的変数の分布が均一になるように実施した。
(2) Data Set Division Of the data set of 59 test infants, RNA profile data for 48 children was used as Train data for model construction, and RNA profile data for the remaining 11 children was used as Test data for model accuracy evaluation.
Data partitioning was performed using the createDataPartition function of [R] so that the distribution of the objective variable was uniform between the Train data and the Test data.
(3)特徴量遺伝子の選択
 (2)で分割されたTrainデータを用いて、分類予測モデルに適すると推測される特徴量遺伝子を、以下1)~3)の3種の手法を用いて抽出した。
1)スピアマンの相関係数に基づく抽出
 Trainデータの由来元である48名の被験乳幼児のおむつ皮膚炎スコアと、全ての遺伝子RNA発現データの組み合わせについてスピアマンの順位相関係数を求めた。
 その結果、表7に示すスピアマンの順位相関係数(ρ)の大きな遺伝子上位8遺伝子を特徴量遺伝子として選択した。表中*を付し太字で表示した6遺伝子は、これまでに乳幼児おむつ皮膚炎との関連が報告されていない遺伝子であった。
(3) Selection of feature amount genes Using the train data divided in (2), feature amount genes that are presumed to be suitable for the classification prediction model are extracted using the following three methods 1) to 3). did.
1) Extraction Based on Spearman's Correlation Coefficients Spearman's rank correlation coefficients were determined for combinations of diaper dermatitis scores of 48 test infants from whom the Train data were derived and all gene RNA expression data.
As a result, the top 8 genes with a large Spearman's rank correlation coefficient (ρ) shown in Table 7 were selected as feature amount genes. The 6 genes indicated in bold with * in the table have not been reported to be related to infant diaper dermatitis.
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000010
2)変数重要度に基づく抽出
 [R]のcaretパッケージを用い、ランダムフォレスト関数で算出されるジニ係数に基づく各遺伝子の重要度を算出し、表8に示すモデル作成に寄与率が高かった遺伝子の上位にくるもの10遺伝子を特徴量遺伝子として選択した。表中*を付し太字で表示した9遺伝子は、これまでに乳幼児おむつ皮膚炎との関連が報告されていない遺伝子であった。
2) Extraction based on variable importance [R] Caret package was used to calculate the importance of each gene based on the Gini coefficient calculated by the random forest function. The top 10 genes were selected as feature genes. The 9 genes indicated in bold with * in the table were genes that had not been reported to be related to infant diaper dermatitis.
Figure JPOXMLDOC01-appb-T000011
Figure JPOXMLDOC01-appb-T000011
3)BORUTAによる特徴量遺伝子の抽出
 [R]のBORUTAパッケージをもとにしたプログラムを利用した。リードカウント値をRPM補正し、log(RPM+1)の値を用いた。正解ラベルとしておむつ皮膚炎スコアを用い、‘‘tentative’’あるいは‘‘confirmed’’と判定された表9に示す7遺伝子を、特徴量遺伝子として選択した。表中*を付し太字で表示した5遺伝子は、これまでに乳幼児おむつ皮膚炎との関連が報告されていない遺伝子であった。
3) Extraction of feature amount genes by BORUTA A program based on the BORUTA package of [R] was used. Read count values were RPM corrected and log 2 (RPM+1) values were used. Using the diaper dermatitis score as a correct label, the 7 genes shown in Table 9 that were judged to be ``tentative'' or ``confirmed'' were selected as feature amount genes. The 5 genes marked with * in the table and shown in bold are genes that have not been reported to be related to infant diaper dermatitis.
Figure JPOXMLDOC01-appb-T000012
Figure JPOXMLDOC01-appb-T000012
(4)caretパッケージ用いたおむつ皮膚炎スコア予測モデル作成
 おむつ皮膚炎スコアと相関が高い遺伝子上位8遺伝子、ランダムフォレストによる変数重要度の高い10遺伝子、BORUTAにより選ばれた7遺伝子をそれぞれ特徴量として選抜し、caretパッケージでおむつ皮膚炎スコア予測モデルを構築した。SSL由来RNAから選択された前記特徴量遺伝子の発現量のデータ(log(RPM+1)値)を説明変数とし、おむつ皮膚炎スコアを偏差値に変換した値を目的変数として用いた。
 上記で選抜した特徴量を用い、Trainデータで、caretパッケージで線形回帰モデル(Lm)、ランダムフォレスト(Rf)、ニューラルネットワーク(Nnet)、ラッソ回帰(Lasso)、rbfカーネルのサポートベクターマシーン(SVM rbf)、線形カーネルのサポートベクターマシーン(SVM linear)の6つのアルゴリズムでおむつ皮膚炎スコア予測モデルを構築した。データの偏りがモデルに与える影響を抑えるために、モデル作成は10回の交差検証を行った。最良の予測モデルの指標として、RMSE(二乗平均平方根誤差)を算出し、最も値が小さかったモデルを最良モデルに選択した。次いで、最良モデルに対し、Testデータの特徴量遺伝子発現量(log(RPM+1)値)を入力して、おむつ皮膚炎スコアの予測値(予測スコア)を計算した。得られた予測値(予測スコア)と実際の診断スコアとの間のPEARSON相関係数を算出し、この値が1に近いほど、予測精度が高いモデルであるとした。
(4) Diaper dermatitis score prediction model creation using the caret package Top 8 genes highly correlated with diaper dermatitis score, 10 genes with high variable importance by random forest, and 7 genes selected by BORUTA as feature values. A diaper dermatitis score prediction model was constructed with the caret package. The expression level data (log 2 (RPM+1) value) of the feature gene selected from the SSL-derived RNA was used as an explanatory variable, and the value obtained by converting the diaper dermatitis score into a deviation value was used as an objective variable.
Using the features selected above, with Train data, the caret package linear regression model (Lm), random forest (Rf), neural network (Nnet), Lasso regression (Lasso), rbf kernel support vector machine (SVM rbf ), a diaper dermatitis score prediction model was constructed with six algorithms of a linear kernel support vector machine (SVM linear). To reduce the impact of data bias on the model, model building was cross-validated 10 times. As an index of the best prediction model, RMSE (root mean square error) was calculated, and the model with the smallest value was selected as the best model. Next, for the best model, the feature quantity gene expression level (log 2 (RPM+1) value) of the Test data was input to calculate the predicted value (predicted score) of the diaper dermatitis score. The PEARSON correlation coefficient between the obtained prediction value (prediction score) and the actual diagnosis score was calculated, and the closer this value was to 1, the higher the prediction accuracy of the model.
(5)結果
 図1に示すように、おむつ皮膚炎スコアとの相関係数が上位の8遺伝子を特徴量に用いた場合、ランダムフォレストによるモデル(モデル1)がRMSE=5.90で最も精度が高かった。その時のTestデータによる予測スコアと診断スコアの相関係数は0.902(p<0.01)であった。
 ランダムフォレストによる変数重要度の高い10遺伝子を特徴量に用いた場合、SVMrbfにより構築したモデル(モデル2)がRMSE=6.32で最も精度が高く、Testデータによる予測スコアと診断スコアの相関係数は0.706(p=0.015)であった。
 BORUTAにより抽出した9遺伝子を特徴量に用いた場合、ランダムフォレストにより構築したモデル(モデル3)がRMSE=6.39で最も精度が高く、その時のTestデータによる予測スコアと診断スコアの相関係数は0.754(p<0.01)であった。
 このことから、モデルに使用した遺伝子は、乳幼児おむつ皮膚炎スコアの回帰予測に重要な遺伝子であり、乳幼児おむつ皮膚炎の症度の検出に有用であることが明らかとなった。
 複数の特徴量遺伝子の中でも特に重要な遺伝子として、3種の手法による特徴量遺伝子の抽出のうち2種以上の手法において重複して抽出された、GALNT3、CMTM6、SLC35E1及びEID3の4種が挙げられ、当該4種を用いておむつ皮膚炎スコア予測モデルを構築した場合、ニューラルネットワークにより構築したモデルがRMSE=6.445で最も精度が高く、その時のTestデータによる予測スコアと診断スコアの相関係数は0.698(p<0.05)であった。
 また、4種から選択したGALNT3とCMTM6の2種又はSLC35E1とEID3の2種を用いておむつ皮膚炎スコア予測モデルを構築した場合、Testデータによる予測スコアと診断スコアの相関係数はそれぞれ、0.623(rbfカーネルのサポートベクターマシン、RMSE=7.023)(p<0.05)、0.614(ニューラルネットワーク、RMSE=7.072)(p<0.05)であり、重要な遺伝子であることが確認された。
 さらに、GALNT3又はEID3のいずれか1種のみを用いて、おむつ皮膚炎スコア予測モデルを構築したところ、Testデータによる予測スコアと診断スコアの相関係数はそれぞれ、0.705(rbfカーネルのサポートベクターマシン、RMSE=6.724)(p<0.05)、0.649(線形回帰モデル、RMSE=7.201)(p<0.05)であり、特に重要な遺伝子であることが確認された。
(5) Results As shown in FIG. 1, when the 8 genes with the highest correlation coefficients with the diaper dermatitis score are used as feature values, the random forest model (model 1) is the most accurate with RMSE = 5.90. was high. The correlation coefficient between the predicted score and the diagnostic score based on the Test data at that time was 0.902 (p<0.01).
When 10 genes with high variable importance by random forest are used as feature values, the model (model 2) constructed by SVMrbf has the highest accuracy with RMSE = 6.32, and the correlation between the prediction score and the diagnosis score by Test data The number was 0.706 (p=0.015).
When the 9 genes extracted by BORUTA are used as feature values, the model (model 3) constructed by random forest has the highest accuracy with RMSE = 6.39, and the correlation coefficient between the prediction score and the diagnosis score based on the test data at that time. was 0.754 (p<0.01).
From this, it was clarified that the gene used in the model is an important gene for regression prediction of the infant diaper dermatitis score, and is useful for detecting the severity of infant diaper dermatitis.
Especially important genes among the plurality of feature amount genes are GALNT3, CMTM6, SLC35E1, and EID3, which are redundantly extracted by two or more of the three methods of extracting feature amount genes. When a diaper dermatitis score prediction model is constructed using the four types, the model constructed by the neural network has the highest accuracy with RMSE = 6.445, and the correlation between the prediction score and the diagnosis score based on the test data at that time The number was 0.698 (p<0.05).
In addition, when a diaper dermatitis score prediction model is constructed using two types of GALNT3 and CMTM6 selected from the four types or two types of SLC35E1 and EID3, the correlation coefficient between the prediction score and the diagnosis score based on the test data is 0, respectively. 0.623 (support vector machine for rbf kernel, RMSE=7.023) (p<0.05), 0.614 (neural network, RMSE=7.072) (p<0.05), with significant genes It was confirmed that
Furthermore, when a diaper dermatitis score prediction model was constructed using only one of GALNT3 or EID3, the correlation coefficient between the prediction score and the diagnosis score by Test data was 0.705 (rbf kernel support vector Machine, RMSE = 6.724) (p < 0.05), 0.649 (linear regression model, RMSE = 7.201) (p < 0.05), confirming it as a particularly important gene. rice field.

Claims (12)

  1.  被験乳幼児から採取された生体試料について、GALNT3、CMTM6、SLC35E1及びEID3の4種の遺伝子群より選択される少なくとも1つの遺伝子又はその発現産物の発現レベルを測定する工程を含む、当該乳幼児のおむつ皮膚炎の症度を検出する方法。 Diaper skin of the test infant, comprising the step of measuring the expression level of at least one gene or its expression product selected from the four gene groups of GALNT3, CMTM6, SLC35E1 and EID3 for a biological sample collected from the test infant. A method for detecting the severity of inflammation.
  2.  さらに表1に示す14種の遺伝子群より選択される少なくとも1つの遺伝子又はその発現産物の発現レベルを測定する工程を含む、請求項1記載の検出方法。
    Figure JPOXMLDOC01-appb-T000001
    2. The detection method according to claim 1, further comprising the step of measuring the expression level of at least one gene selected from the 14 gene groups shown in Table 1 or its expression product.
    Figure JPOXMLDOC01-appb-T000001
  3.  前記遺伝子又はその発現産物の発現レベルがmRNAの発現量である請求項1又は2記載の検出方法。 The detection method according to claim 1 or 2, wherein the expression level of the gene or its expression product is the expression level of mRNA.
  4.  前記生体試料が被験乳幼児の皮膚表上脂質である請求項1~3のいずれか1項記載の検出方法。 The detection method according to any one of claims 1 to 3, wherein the biological sample is skin surface lipids of the test infant.
  5.  前記被験乳幼児の皮膚が排尿部、肛門部、臀部、鼠径部、腰部、腹部又は大腿部の皮膚である請求項4記載の検出方法。 The detection method according to claim 4, wherein the skin of the subject infant is the skin of the urinary region, anus, buttocks, groin, waist, abdomen, or thigh.
  6.  前記遺伝子又はその発現産物の発現レベルに基づいて前記被験乳幼児のおむつ皮膚炎の症度を検出することを含む請求項1~5のいずれか1項記載の検出方法。 The detection method according to any one of claims 1 to 5, comprising detecting the severity of diaper dermatitis in the test infant based on the expression level of the gene or its expression product.
  7.  前記遺伝子又はそれに由来する核酸と特異的にハイブリダイズするオリゴヌクレオチド、又は前記遺伝子の発現産物を認識する抗体を含有する、請求項1~6の検出方法に用いられる乳幼児おむつ皮膚炎の症度を検出するための検査用キット。 The degree of infant diaper dermatitis used in the detection method according to any one of claims 1 to 6, 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 detection.
  8.  表2に示す14種の遺伝子群より選択される少なくとも1つの遺伝子又はその発現産物からなる、乳幼児おむつ皮膚炎の症度を検出するための検出マーカー。
    Figure JPOXMLDOC01-appb-T000002
    A detection marker for detecting the severity of infant diaper dermatitis, comprising at least one gene or its expression product selected from the 14 gene groups shown in Table 2.
    Figure JPOXMLDOC01-appb-T000002
  9.  GALNT3、CMTM6、SLC35E1及びEID3の4種の遺伝子群より選択される少なくとも1つの遺伝子又はその発現産物である、請求項8記載の乳幼児おむつ皮膚炎の症度を検出するための検出マーカー。 The detection marker for detecting the severity of infant diaper dermatitis according to claim 8, which is at least one gene or its expression product selected from the four gene groups of GALNT3, CMTM6, SLC35E1 and EID3.
  10.  被験乳幼児から採取された生体試料に由来する表3に示す14種の遺伝子群より選択される少なくとも1つの遺伝子又はその発現産物の、乳幼児おむつ皮膚炎皮膚炎の症度の検出マーカーとしての使用。
    Figure JPOXMLDOC01-appb-T000003
    Use of at least one gene or its expression product selected from the 14 gene groups shown in Table 3 derived from a biological sample collected from a test infant as a marker for detecting the severity of infant diaper dermatitis dermatitis.
    Figure JPOXMLDOC01-appb-T000003
  11.  GALNT3、CMTM6、SLC35E1及びEID3の4種の遺伝子群より選択される少なくとも1つの遺伝子又はその発現産物である、請求項10記載の検出マーカーとしての使用。 Use as a detection marker according to claim 10, which is at least one gene or its expression product selected from the four gene groups of GALNT3, CMTM6, SLC35E1 and EID3.
  12.  遺伝子又はその発現産物が前記被験乳幼児の皮膚表上脂質に含まれるmRNAである、請求項10又は11記載の使用。 The use according to claim 10 or 11, wherein the gene or its expression product is mRNA contained in the lipids on the skin surface of the test infant.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024111617A1 (en) * 2022-11-24 2024-05-30 花王株式会社 Internal standard gene in skin surface lipid specimen

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170241989A1 (en) * 2014-03-31 2017-08-24 Laboratoires Expanscience Method for evaluating the harmful effects of urine on children's skin

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170241989A1 (en) * 2014-03-31 2017-08-24 Laboratoires Expanscience Method for evaluating the harmful effects of urine on children's skin

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE Nucleotide 14 September 2006 (2006-09-14), ANONYMOUS: "Homo sapiens cDNA FLJ25832 fis, clone TST08178", XP055976551, retrieved from Genbank Database accession no. AK098698 *
DATABASE Nucleotide 25 July 2016 (2016-07-25), ANONYMOUS: "Homo sapiens mRNA; cDNA DKFZp686H05207 (from clone DKFZp686H05207)", XP055976548, retrieved from Genbank Database accession no. BX640756 *
DATABASE Nucleotide 7 October 2008 (2008-10-07), ANONYMOUS: "Homo sapiens mRNA for UDP-GalNAc:polypeptide N-acetylgalactosaminyl transferase (GalNAc-T3)", XP055976544, retrieved from Genbank Database accession no. X92689 *
DATABASE Nucleotide 8 April 2009 (2009-04-08), ANONYMOUS: "Homo sapiens chemokine-like factor super family 6 (CKLFSF6) mRNA, complete cds", XP055976546, retrieved from Genbank Database accession no. AF479261 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024111617A1 (en) * 2022-11-24 2024-05-30 花王株式会社 Internal standard gene in skin surface lipid specimen

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