WO2022220298A1 - 乳幼児おむつ皮膚炎の症度の検出方法 - Google Patents

乳幼児おむつ皮膚炎の症度の検出方法 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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French (fr)
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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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Kao Corp
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Kao Corp
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING 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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING 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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING 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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides 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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WO2024111617A1 (ja) * 2022-11-24 2024-05-30 花王株式会社 皮膚表上脂質検体内部標準遺伝子

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

Family Cites Families (1)

* Cited by examiner, † Cited by third party
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EP3372693B1 (en) * 2016-07-08 2020-02-12 Kao Corporation Method for preparing nucleic acid sample

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 (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024111617A1 (ja) * 2022-11-24 2024-05-30 花王株式会社 皮膚表上脂質検体内部標準遺伝子
JP2024076370A (ja) * 2022-11-24 2024-06-05 花王株式会社 皮膚表上脂質検体内部標準遺伝子
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