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WO2019225972A1 - Composition de diagnostic de dermatite atopique et procédé de détection utilisant un polymorphisme mononucléotidique du gène dock8 - Google Patents

Composition de diagnostic de dermatite atopique et procédé de détection utilisant un polymorphisme mononucléotidique du gène dock8 Download PDF

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WO2019225972A1
WO2019225972A1 PCT/KR2019/006151 KR2019006151W WO2019225972A1 WO 2019225972 A1 WO2019225972 A1 WO 2019225972A1 KR 2019006151 W KR2019006151 W KR 2019006151W WO 2019225972 A1 WO2019225972 A1 WO 2019225972A1
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base
atopic dermatitis
polynucleotide
seq
dock8
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Korean (ko)
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허원일
서성준
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Industry Academic Cooperation Foundation of Chung Ang University
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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
    • C12Q1/6876—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
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a marker composition and detection method for diagnosing atopic dermatitis using DOCK8 gene single nucleotide polymorphism, and more specifically, 289th base is A in the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, and the 289th base Detecting polymorphism of DOCK8 gene and atopic dermatitis diagnostic composition comprising a polynucleotide consisting of 20 to 100 contiguous nucleotide sequences and one or more polynucleotide detection agents selected from the group consisting of complementary polynucleotides thereof It's about how to do it.
  • AD Atopic dermatitis
  • AD is a chronic skin inflammatory disease with major symptoms of eczema damage and dry, itchy skin.
  • AD is thought to be caused by a combination of genetic predisposition and environmental factors.
  • AD is caused by a large number of genes, inherited from generation to generation, and develops in childhood. Cohort studies in the AD family have been shown to be inherited in an autosomal dominant fashion.
  • AD occurs in about 20% of children (Nutten S, Annals of nutrition & metabolism 2015, 66 Suppl 1: 8-16), Infant atopic dermatitis did not heal itself before and after puberty, so that people who continue to suffer from adulthood or who did not have any atopy before puberty become adults.
  • AD Alzheimer's disease
  • WES Whole-exome sequencing
  • SNPs -nucleotide polymorphisms
  • CD-CV common disease-common variant hypothesis
  • GWASs genome-wide association studies
  • the present inventors performed WES on Korean atopic patients, patients with asthma or rhinitis, and controls to identify genetic variants that cause adult atopic dermatitis (AD), and compared alleles.
  • AD adult atopic dermatitis
  • DOCK8 gene was confirmed that the mutation is closely related to hereditary AD to complete the present invention.
  • an object of the present invention is a polynucleotide comprising a base sequence of SEQ ID NO: 1, the 289th base is A, 20 to 100 consecutive base sequence comprising the 289th base, and their complementary It is to provide a composition for diagnosing atopic dermatitis comprising a detection agent of at least one polynucleotide selected from the group consisting of polynucleotides.
  • Another object of the present invention is to provide a third object of the present invention.
  • It provides a method for detecting a polymorphism of the DOCK8 gene comprising a marker.
  • Another object of the present invention is a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 for preparing a diagnostic agent for atopic dermatitis, the 289th base is A, 20 to 100 consecutive base sequence comprising the 289th base
  • Another object of the present invention is to provide a third object of the present invention.
  • a polynucleotide consisting of 20 to 100 consecutive nucleotide sequences including the 289th base A and the 289th base in the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 by analyzing the nucleotide sequence of the sample; Detecting at least one polynucleotide selected from the group consisting of their complementary polynucleotides; And
  • c) detecting the polynucleotide is to provide a method for diagnosing atopic dermatitis comprising determining that the polynucleotide is atopic dermatitis.
  • the present invention is a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 289 base A is a poly, consisting of 20 to 100 consecutive base sequence comprising the 289 th base It provides a composition for diagnosing atopic dermatitis comprising a detection agent of one or more polynucleotides selected from the group consisting of nucleotides and their complementary polynucleotides.
  • It provides a method for detecting a polymorphism of the DOCK8 gene comprising a marker.
  • the present invention is a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 for the preparation of a diagnostic agent for atopic dermatitis, the 289th base is A, 20 to 20 comprising the 289th base
  • a detection agent of at least one polynucleotide selected from the group consisting of polynucleotides consisting of 100 consecutive base sequences and their complementary polynucleotides.
  • a polynucleotide consisting of 20 to 100 consecutive nucleotide sequences including the 289th base A and the 289th base in the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 by analyzing the nucleotide sequence of the sample; Detecting at least one polynucleotide selected from the group consisting of their complementary polynucleotides; And
  • detecting the polynucleotide provides a method for diagnosing atopic dermatitis comprising determining that the polynucleotide is atopic dermatitis.
  • the present invention is a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, the 289th base is A, polynucleotide consisting of 20 to 100 consecutive base sequence comprising the 289th base and their complementary polynucleotides It provides a composition for diagnosing atopic dermatitis comprising a detection agent of at least one polynucleotide selected from the group consisting of.
  • the present invention is a polynucleotide comprising the base sequence of SEQ ID NO: 1 289 base is A, polynucleotide consisting of 20 to 100 consecutive base sequence comprising the 289 base and their complementary polynucleotide It provides a composition for diagnosing atopic dermatitis consisting of a detection agent of one or more polynucleotides selected from the group consisting of.
  • the present invention is a polynucleotide comprising the base sequence of SEQ ID NO: 1 289 base is A, polynucleotide consisting of 20 to 100 consecutive base sequence comprising the 289 base and their complementary polynucleotide It provides a composition for diagnosing atopic dermatitis consisting essentially of a detection agent of one or more polynucleotides selected from the group consisting of.
  • 'polynucleotide' or 'nucleic acid' refers to deoxyribonucleotides (DNA) or ribonucleotides (RNA) in the form of single- or double-strands.
  • DNA deoxyribonucleotides
  • RNA ribonucleotides
  • known analogues of natural nucleotides that hybridize to nucleic acids in a similar manner to naturally occurring nucleotides are also included.
  • DNA is composed of four bases: adenine (A), guanine (G), cytosine (C), and thymine (T), and RNA is uracil (Uracil, U) instead of thymine.
  • gDNA gDNA
  • 'c.' Denotes a mutation of a protein coding region
  • '(base position) (base character) (symbol>) (base character)' denotes a corresponding base position. It means that the base indicated earlier is substituted with the base indicated later.
  • 'protein' is used interchangeably with 'polypeptide' or 'peptide', and refers to a polymer of amino acid residues, for example, as commonly found in natural proteins.
  • the one letter (three letter) of amino acid means the following amino acids according to standard abbreviation definitions in the field of biochemistry: A (Ala): alanine; C (Cys): cysteine; D (Asp): aspartic acid; E (Glu): glutamic acid; F (Phe): phenylalanine; G (Gly): glycine; H (His): histidine; I (IIe): isoleucine; K (Lys): lysine; L (Leu): leucine; M (Met): methionine; N (Asn): asparagine; O (Ply): pyrrolysine; P (Pro): proline; Q (Gln): glutamine; R (Arg): arginine; S (Ser): serine; T (Thr): threonine; U (Sec): selenocysteine; V (Val): valine;
  • amino acid letter amino acid position
  • amino acid letter amino acid letter
  • 'Atopy' refers to a series of allergic symptoms that occur in the skin, respiratory mucosa, eye mucosa, and intestinal mucosa in individuals with atopy predisposition. These atopic predispositions (allergic constitutions) are inherited and transmitted to the next generation of the household. Allergic diseases caused by atopy predisposition include allergic dermatitis, allergic rhinitis, asthma, allergic conjunctivitis, atopic urticaria, and these diseases may be present alone or in various diseases.
  • 'Atopic dermatitis' or 'atopic dermatitis' is a representative skin disorder in people with allergic atopic dermatitis. Red spots, swelling, dry skin and itching of the skin are the main symptoms, with immunological properties that may be accompanied by other allergic diseases such as urticaria, metal allergy, asthma or allergic rhinitis (atopic dermatitis).
  • Diagnosis herein includes all types of analyzes used to predict or develop disease risk and determine or derive risk or susceptibility to development.
  • the diagnosis means diagnosis of atopic dermatitis.
  • Atopic dermatitis in the present invention is preferably hereditary atopic dermatitis caused by a genetic factor (genetic factor), the symptoms may be aggravated by the environmental impact added to the genetic predisposition.
  • atopic dermatitis in the present invention may be adult atopic dermatitis.
  • the term 'adult atopic dermatitis' specifically refers to the first occurrence of atopic symptoms after adolescent infants do not heal themselves before or after puberty and continue to suffer from adulthood, or 2 who have not had any atopy before puberty.
  • polymorphism of the DOCK8 gene is detected as a diagnostic marker in order to provide information necessary for the diagnosis of atopic dermatitis in a subject.
  • 'DOCK8' is a 'Dedicator of cytokinesis 8' that is involved in intracellular signal networks. Located at 9p24.3, it has been reported that mutations in DOCK8 are associated with Hyper-IgE syndrome. Karin R et al., J Allergy Clin Immunol. 2009; 1234 (6): 1289.
  • DOCK8 is most preferably derived from human, and sequence information of mRNA and protein of human DOCK8 gene is known as an accession number of NM_203447.3 in the GenBank database. (SEQ ID NO: 2 and SEQ ID NO: 3)
  • 'polymorphism' refers to the occurrence of two or more alternative sequences or alleles (or alleles) within a genetically determined population
  • 'single nucleotide polymorphism (SNP)' refers to a single polynucleotide.
  • a single base (A, T, C or G) refers to the diversity of DNA sequences occurring between members of a species or between individual pairs of chromosomes. For example, it contains differences in a single base such as three DNA fragments from different individuals (eg AAGT [A / A] AG, AAGT [A / G] AG, AAGT [G / G] AG).
  • SNPs Two alleles (A or G), and generally all SNPs have two alleles.
  • SNPs are genetically closely associated with a particular disease, the SNPs may have resulted in mutations of one base at a particular position compared to the normal or wild-type (WT) individuals or alleles identified. It also means.
  • the SNP region of DOCK8, a diagnostic marker for atopic dermatitis according to the present invention, is located in a protein coding region of DOCK8, and the SNP allele associated with atopic dermatitis may cause damage to DOCK8 protein function.
  • Specific associations between DOCK8 SNP and atopic dermatitis are as follows:
  • SEQ ID NO: 1 is the nucleotide sequence of SNP rs529208, which corresponds to the 286,593 base on chromosome 9. Exome sequencing results for atopic dermatitis and atopic dermatitis patients were selected as candidate mutations, and the mutations related to atopic dermatitis were 289 bases from C to A in the protein coding region of the DOCK8 gene (c.289C>). A), in the DOCK8 protein, the 97th amino acid proline is substituted with threonine (Pro97Thr or P97T). The mRNA sequencing of the DOCK8 gene (sequence from the start codon to the stop codon) and the amino acid sequence of the protein are described herein as SEQ ID NO: 2 and SEQ ID NO: 3, respectively.
  • the diagnostic composition for atopic dermatitis includes an agent capable of detecting the DOCK8 SNP. More specifically, in the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, the 289th base is A, and the polynucleotide consisting of 20 to 100 consecutive base sequences including the 289th base and their complementary polynucleotides. It comprises a detection agent of one or more polynucleotides selected from the group consisting of.
  • the detection agent may be a probe capable of identifying a SNP base by hybridizing with a polynucleotide of a site including the DOCK8 SNP. That is, it is a probe capable of detecting the 289th base of SEQ ID NO: 1.
  • Probes are fragments of polynucleotides, such as RNA or DNA, ranging from short to several hundred bases in length that can specifically bind mRNA, cDNA (complementary DNA), DNA, etc. It means that the presence of the target mRNA or cDNA to be bound and the amount of expression is labeled (labeled).
  • the selection and hybridization conditions of the probe can be appropriately selected according to techniques known in the art.
  • the probe may be used in diagnostic methods for detecting alleles (or alleles).
  • the diagnostic method includes detection methods based on hybridization of nucleic acids such as Southern blot and the like, and may be provided in a form that is pre-bound to the substrate of the DNA chip in a method using a DNA chip.
  • the hybridization may be carried out conventionally under stringent conditions, for example, salt concentrations of 1 M or less and temperatures of 25 ° C. or more.
  • stringent conditions for example, salt concentrations of 1 M or less and temperatures of 25 ° C. or more.
  • conditions of 5XSSPE 750 mM NaCl, 50 mM sodium phosphate, 5 mM EDTA, pH 7.4 and 25-30 ° C. may be suitable for allele-specific probe hybridization.
  • the detection agent may be a pair of primers capable of amplifying a polynucleotide of a site including a DOCK8 SNP. That is, it is a pair of primers capable of amplifying the 289th base of SEQ ID NO: 1.
  • a 'primer' is a single stranded oligonucleotide that acts as a starting point for DNA synthesis.
  • the primer specifically binds to a polynucleotide that is a template at appropriate buffer and temperature conditions, and the DNA polymerase adds nucleoside triphosphate having a base complementary to the template DNA to the primer.
  • DNA is synthesized by ligation.
  • the primer is generally composed of 15 to 30 base sequences, and the melting temperature (T m ) of binding to the template strand varies depending on the base composition and length.
  • the sequence of the primer does not need to have a sequence that is completely complementary to some nucleotide sequences of the template, and it is sufficient to have sufficient complementarity within a range capable of hybridizing with the template to perform the primer-specific function.
  • the primer for the polymerase chain reaction (PCR) is opposite to the template (or sense) of both ends (5 'end or 3' end) of a specific section of the polynucleotide to be amplified (antisense). It consists of a pair (set) each complementary to. Primers can easily design primer pairs for diagnosing atopic dermatitis according to the present invention with reference to the base sequence of cDNA or genomic DNA of the SNP region of DOCK8 described above. PCR cycle conditions for detecting the SNP of DOCK8 according to the present invention are described in detail in the experimental method of the Examples.
  • the primer pair may be a pair of base sequences represented by SEQ ID NO: 4 and SEQ ID NO: 5.
  • Primers or probes of the present invention can be synthesized chemically using phosphoramidite solid support synthesis or other well known methods.
  • the primer or probe can be variously modified according to methods known in the art within a range that does not prevent hybridization with the polynucleotide to be detected. Examples of such modifications include methylation, encapsulation, substitution of one or more homologs of natural nucleotides and modifications between nucleotides, such as uncharged linkages such as methyl phosphonate, phosphoester, phosphoramidate, carbamate, etc. Or charged linkages (eg, phosphorothioate, phosphorodithioate, etc.), and binding of labeling materials using fluorescence or enzymes.
  • the diagnostic composition according to the present invention may be manufactured as a diagnostic kit or a diagnostic microarray (microarray) capable of detecting DOCK8 SNP.
  • the probe of the present invention is an allele specific probe of DOCK8 SNP, in which a polymorphic site exists in nucleic acid fragments derived from two members of the same species, and hybridizes to a DNA fragment derived from one member, but not to a fragment derived from another member. Do not hybridize. In this case, hybridization conditions show significant differences in hybridization strength between alleles, and should be sufficiently stringent to hybridize to only one of the alleles. This can lead to clear hybridization differences between different alleles. Hybridization conditions are as described above.
  • the process of immobilizing the polynucleotide on a substrate can be easily carried out using conventional techniques.
  • the hybridization of nucleic acids on microarrays and detection of hybridization results are well known in the art.
  • the detection is accomplished by labeling a nucleic acid sample with a labeling substance capable of generating a detectable signal comprising, for example, a fluorescent substance such as Cy3 and Cy5, and then hybridizing onto a microarray and generating from the labeling substance.
  • the hybridization result can be detected by detecting the signal.
  • It provides a method for detecting a polymorphism of the DOCK8 gene comprising a marker.
  • a sample of a subject may be used without limitation as long as it can separate nucleic acid samples such as blood, whole blood, plasma, sputum, saliva, ocular fluid, semen, cells and tissues (for example, skin, etc.) separated from the subject.
  • nucleic acid samples such as blood, whole blood, plasma, sputum, saliva, ocular fluid, semen, cells and tissues (for example, skin, etc.) separated from the subject.
  • blood can be used.
  • the sample can be subjected to pretreatment such as filtration, distillation, extraction, concentration, inactivation of interference components, addition of reagents, and the like before use for detection. Methods of separating nucleic acid samples are well known in the art.
  • 'nucleic acid sample' refers to a sample which can be separated from blood, whole blood, plasma, sputum, saliva, ocular fluid, semen, cells and tissues (for example, skin, etc.) separated from a subject, and specifically, May be DNA or RNA.
  • 'detection' refers to analyzing, imaging, identifying or establishing the presence or absence of a specific base of a diagnostic marker according to the present invention, specifically, DOCK8 SNP.
  • the monobasic polymorphism (SNP) site of DOCK8 is as described above. Specifically, it is characterized by the 289th base of SEQ ID NO: 1.
  • the single nucleotide polymorphism site is the 289th base of SEQ ID NO: 1, it is diagnosed as atopic dermatitis, or it is determined that the risk or sensitivity of developing atopic dermatitis is high.
  • Determining the base may be used without limitation as long as it is conventionally used to analyze and determine the base sequence in the art. More specifically, sequencing, exome sequencing, microarray hybridization, allele specific PCR, dynamic allele-specific hybridization, It can be performed by any method selected from the group consisting of PCR extension analysis and Taqman technique.
  • the present invention is a polynucleotide comprising a base sequence of SEQ ID NO: 1 for the preparation of the diagnostic agent for atopic dermatitis, the 289th base is A, 20 to 100 consecutive base sequence comprising the 289th base And their complementary polynucleotides, the use of a detection agent for one or more polynucleotides selected from the group consisting of.
  • the present invention is a.
  • a polynucleotide consisting of 20 to 100 consecutive nucleotide sequences including the 289th base A and the 289th base in the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 by analyzing the nucleotide sequence of the sample; Detecting at least one polynucleotide selected from the group consisting of their complementary polynucleotides; And
  • a step of providing a sample from an individual or a potential patient may be performed before step a).
  • the "individual" of the present invention may be an animal, preferably an animal including a mammal, especially a human, and includes the animal-derived cells, tissues, organs, and the like. More preferably it may be a human or patient in need of diagnosis.
  • the subject may be a human suspected of developing atopic dermatitis, may be a patient with atopic dermatitis (such as for confirmation), or to find an appropriate treatment as a patient with atopic dermatitis (to find the cause)
  • a patient may need to confirm the development of atopic dermatitis.
  • the term 'diagnosis' refers to identifying (identifying) the presence or characteristic of an abnormal state, a hazard state, a pathological state for an individual.
  • the diagnosis may be identifying any individual (suspect individual) atopic dermatitis.
  • the term 'comprising' is used in the same way as 'containing' or 'featured', and does not exclude an additional component element or method step that is not mentioned in the composition or method. .
  • the term “consisting of” or “consisting of” means to exclude additional elements, steps, or components, etc., unless otherwise specified.
  • the term “consisting essentially of” means within the scope of the composition or method, including the component elements or steps described, as well as the component elements or steps that do not substantially affect its basic properties, and the like.
  • the present invention provides a method for diagnosing and treating atopic dermatitis comprising the following steps:
  • a polynucleotide consisting of 20 to 100 consecutive nucleotide sequences including the 289th base A and the 289th base in the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 by analyzing the nucleotide sequence of the sample; Detecting at least one polynucleotide selected from the group consisting of their complementary polynucleotides;
  • Step d) is a step of performing the treatment of the disease in a subject diagnosed in step c) by means of administering a therapeutic drug or surgery.
  • 'treatment' refers generically to ameliorating symptoms of atopic dermatitis or atopic dermatitis, which may include treating, substantially preventing, or ameliorating atopic dermatitis, resulting from atopic dermatitis It includes, but is not limited to, alleviating, healing or preventing one or most symptoms.
  • the therapeutic drug is not particularly limited as long as it is a type of drug commonly used for the treatment of atopic dermatitis, and in one embodiment is selected from the group consisting of steroids (typically dexamethasone, etc.), immunomodulators and antihistamines. It may be to treat one or more drugs, but is not limited thereto.
  • the therapeutic drug is administered to the subject in a 'therapeutically effective amount', wherein the therapeutically effective amount is determined by the person skilled in the art as well as the specificity of the drug, the route of administration, and the number of treatments, as well as the age, weight, health condition, sex and disease of the patient. Various factors, such as severity, diet, and excretion rate, can be considered to determine the effective dosage for a patient.
  • the route of administration of the therapeutic drug is not particularly limited and may be administered orally or parenterally, and includes both systemic routes as well as topical administration.
  • the parenteral administration is not limited thereto, but may be, for example, intranasal drug application, subcutaneous injection, or the like, and may be another method using intramuscular injection or intravenous injection.
  • the present invention provides a method for detecting the atopic dermatitis diagnostic composition
  • SNP single nucleotide polymorphism
  • the present inventors have analyzed the protein coding region of genomic DNA of atopic dermatitis patients with severe symptoms and found that the specific base mutation of the SNP of the DOCK8 gene is closely related to the increase in IgE in patients with atopic dermatitis and marching patients.
  • the present invention can be used to develop genetic diagnostic reagents for diagnosing atopic dermatitis.
  • Figure 1 is a schematic diagram of the filtering process of the variants calculated in exome sequencing.
  • Figure 2 shows the relationship between the total IgE level and DOCK8 mutation, clinical information of atopic patients.
  • Genomic DNA was isolated from the peripheral blood obtained from atopic patients and atopic dermatitis patients using a QIAamp DNA mini kit (Qiagen Inc, Valencia, CA, USA). The quantity and quality of DNA was determined by Nanodrop spectrometer (Nanodrop Technologies, Wilmington, DE, USA) and Qubit Fluorometer (Life Technologies, Grand Island, NY, USA). WES was performed using SureSelect Human All Exon V4 + UTR 71 Mb (Agilent, CA, USA) according to the manufacturer's standard protocol. Genomic DNA was sheared using Covaris (Covaris, Woburn, Mass., USA).
  • Paired-end DNA sequencing libraries were prepared by shearing, end-repair, A-tailing, peak detection, PE adapter ligation, and amplification.
  • the prepared library was hybridized with a bait sequence for 24 hours, separated, and amplified using an index index barcode tag to determine quantity and quality.
  • Exome library was sequenced in 100-bp paired-end mode of HiSeq SBS kit.
  • Sequence reads in the FASTQ format are generated using the Burrows-Wheeler Aligner (BWA, v0.7.7) program to create a SAM file with the correct mate pair information. Mapping (Li H et al., Bioinformatics 2009, 25 (14): 1754-1760). The read group tag contains the sample name.
  • the SAM file was converted to a compressed BAM file using Picard (v1.92) and the BAM file was sorted according to chromosome coordinates.
  • the Genome Analysis Toolkit (v2.3.9 Lite) was used to reorder BAM files locally at intervals that could include alignment errors in insertion / deletion (indel) (DePristo MA et al., Nature genetics 2011 , 43 (5): 491-498).
  • Insertions and deletions were identified using Mutect (Cingolani P et al., Fly 2012, 6 (2): 80-92) and GATK Somatic Indel Detector, respectively.
  • Single base mutations and indels are 'synonymous' and 'non-like' using snpEff (v3.6c) (Cibulskis K et al., Nature biotechnology 2013, 31 (3): 213-219). synonymous, '' missense ',' frameshift point mutation ', and' frameshift indel ', etc.
  • Filter 1 SnpEff (http://snpeff.sourceforge.net/index.html) is a program that annotates variants such as amino acid substitutions of various genes and predicts the effects of the variants. Genetic mutations cause a variety of effects (eg, 'Non_Synonymous_coding', 'Stop gained', 'Insertion', 'Deletion', etc.).
  • the SIFT score predicts the effect of amino acid substitutions on protein function.
  • the prediction of SIFT is based on the degree of conservation of amino acids found in the alignment with closely related sequences collected through PSI-BLAST. The range is from 0 to 1, and substitutions with scores less than 0.05 are impaired in function (the lower the value, the greater the degree of damage), and the higher the score, the more likely the substitution is likely to be tolerated.
  • Polyphen2 HDIV scores are based on HumDiv (hdiv_prob). The score ranges from 0 to 1, with a score between 0.957 and 1 probably damaging, between 0.453 and 0.956 'possibly damaging' and between 0 and 0.452 'good' (benign) '(higher score indicates higher damage).
  • Filter 4 PhyloP in dbNSFP finds a position where a positive or negative selection is in progress, while allowing for differences in the rate of evolution over branches of the phylogenetic tree. Higher PhyloP scores indicate conserved positions (http://varianttools.sourceforge.net/Annotation/DbNSFP).
  • PhastCons measures the intensity of purifying selectons that act on DNA sequences. A high PhastCons score (0.2) is an important evidence that the genomic site is functionally important.
  • Filter 6 The 1000 Genome allele frequency of dbNSFP selects gene mutations with frequencies less than 0.01 or unknown. Filter 6 eliminates the universal variation of more than 0.01 minor allele frequencies (MAFs) observed in races around the world.
  • MAFs minor allele frequencies
  • Filter 7 The Korean Personal Genome Project (KPGP) is part of an international personal genome project. KPGP selects MAF frequency less than 0.002 or unknown variation from the KPGP dataset (http://kpgp.kr/). In other words, Filter 7 removes the universal variation of Korean MAF 0.002 or more.
  • PCR conditions for verifying SNPs selected by WES by the Sanger method are as follows: 95 ° C., 10 min (1 cycle); 95 ° C., 30 s; 55-58 ° C., 30 s; 72 ° C., 40 s (35 cycles); 72 ° C., 1 min 30 s (1 cycle).
  • the PCR reaction solution (total volume 50 ⁇ L) is 2 ⁇ L containing 25 ⁇ L 2 ⁇ EF-Taq premix (SolGent, Seoul, South Korea), 2.5 ⁇ L oligonucleotide primer (10 pmol/ ⁇ L), 18 ⁇ L distilled water, and 20ng genomic DNA I have included a template for.
  • PCR products were isolated and purified using a PCR purification kit (Favorgen, Pingtung, Taiwan) and sequenced using Applied Biosystems 3500 DNA sequencer (Foster City, CA, USA) according to the manufacturer's manual.
  • WES Whole-exome sequencing
  • WES data obtained from each individual were filtered stepwise as shown in FIG . 1 to screen for AD candidate mutation candidates, and detected candidate gene mutation DOCK8.
  • PhastCons predicts the possibility that a nucleotide belongs to a conserved element in the phylogenetic tree (value> 0.2).
  • Sanger sequencing correlates the mutations identified with WES and atopic dermatitis for the universal variant DOCK8 gene with a significant functional prediction score in relation to atopic dermatitis.
  • Primers used for Sanger seqeuncing are as described in Table 3 .
  • the relationship between total IgE and DOCK8 mutations in patients with atopic dermatitis was compared with 154 patients with atopic dermatitis and atopic dermatitis. As shown in FIG. 2 , the increased total IgE levels in the blood of most allergic patients showed a correlation with DOCK8 mutation. In the AA genotype, the total IgE level was found to be higher than the CC and CA genotypes.
  • the present invention can be usefully used to develop atopic dermatitis gene diagnostic reagents and diagnostic chips for early detection of atopic dermatitis so that it can be effectively treated early and appropriately.

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Abstract

La présente invention concerne une composition de marqueur de diagnostic de dermatite atopique et un procédé de détection utilisant un polymorphisme mononucléotidique du gène DOCK8 et, plus précisément, une composition destinée à diagnostiquer une dermatite atopique, contenant une préparation pour détecter un ou plusieurs polynucléotides choisis dans le groupe constitué par un polynucléotide comprenant la séquence nucléotidique de SEQ ID NO: 1, la 289ème base étant A et le polynucléotide étant constitué de 20 à 100 séquences nucléotidiques consécutives comprenant la 289ème base, et des polynucléotides qui lui sont complémentaires ; et un procédé de détection du polymorphisme du gène DOCK8 à l'aide d'un marqueur.
PCT/KR2019/006151 2018-05-23 2019-05-23 Composition de diagnostic de dermatite atopique et procédé de détection utilisant un polymorphisme mononucléotidique du gène dock8 Ceased WO2019225972A1 (fr)

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KR1020180058661A KR102072504B1 (ko) 2018-05-23 2018-05-23 Dock8 유전자 단일염기다형성을 이용한 아토피 피부염 진단용 조성물과 검출 방법

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20160118691A (ko) * 2015-04-03 2016-10-12 연세대학교 산학협력단 아토피 피부염 또는 면역력 감소 여부를 진단하는 dock8 단백질
WO2017146227A1 (fr) * 2016-02-25 2017-08-31 国立大学法人九州大学 Animal modèle de dermatite atopique et son utilisation

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* Cited by examiner, † Cited by third party
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KR20130041961A (ko) * 2010-07-23 2013-04-25 프레지던트 앤드 펠로우즈 오브 하바드 칼리지 체액에서 질환 또는 상태의 특징을 검출하는 방법
KR102095017B1 (ko) * 2016-08-23 2020-03-30 주식회사 큐어젠 Col6a6 유전자 단일염기다형성을 이용한 아토피 피부염 진단용 조성물과 검출 방법

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20160118691A (ko) * 2015-04-03 2016-10-12 연세대학교 산학협력단 아토피 피부염 또는 면역력 감소 여부를 진단하는 dock8 단백질
WO2017146227A1 (fr) * 2016-02-25 2017-08-31 国立大学法人九州大学 Animal modèle de dermatite atopique et son utilisation

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE NCBI 19 October 2000 (2000-10-19), "TSC0818900", Database accession no. ss2102793 *
KIENZLER, A.-K. %: "Hypomorphic function and somatic reversion of DOCKS cause combined immunodeficiency without hyper-IgE", CLINICAL IMMUNOLOGY, vol. 163, 2016, pages 17 - 21, XP029413378, DOI: 10.1016/j.clim.2015.12.003 *
KIRISTI, D ET AL: "Whole-exome sequencing in patients with ichthyosis reveals modifiers associated with increased IgE levels and allergic sensitizations", JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY, vol. 5, no. 1, 13 January 2015 (2015-01-13), pages 280 - 283, XP055657734 *

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