KR102095017B1 - Composition and method for diagnosing atopic dermatitis using COL6A6 SNP - Google Patents

Abstract

The present invention relates to a marker composition and a detection method for diagnosing atopic dermatitis using the COL6A6 gene single base polymorphism, and more specifically, detecting the 26th base mutation of rs16830494, rs200963433, rs59021909 of the single base polymorphism (SNP) region of the COL6A6 gene. It relates to a composition for diagnosing atopic dermatitis comprising a preparation capable of and a method for detecting the SNP of the COL6A6 gene as a marker.
By using the present invention, it is possible to develop a gene diagnosis reagent and diagnostic chip for atopic dermatitis for early detection of atopic dermatitis and appropriate treatment in an early stage to effectively treat it.

Description

Composition and method for diagnosing atopic dermatitis using COL6A6 gene single-base polymorphism COL6A6 SNP}

The present invention relates to a marker composition and a detection method for diagnosing atopic dermatitis using the COL6A6 gene single base polymorphism, and more specifically, the 26th base is A in the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, and the 26th base is A polynucleotide consisting of 20 to 100 consecutive nucleotide sequences, the 26th base is T in the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2, and 20 to 100 consecutive nucleotide sequences comprising the 26th base. Polynucleotide consisting of, in the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 3 is the 26th base is T, polynucleotide consisting of 20 to 100 consecutive nucleotide sequences comprising the 26th base and their complementary One or more polynucleotides selected from the group consisting of polynucleotides To a method for detecting a polymorphism of the COL6A6 gene and atopic dermatitis diagnostic composition comprising a formulation as markers.

Atopic dermatitis (AD) is a chronic skin inflammatory disease in which eczema damage and dry, itchy skin are the main symptoms and easily recurs. AD is thought to be caused by a combination of genetic predisposition and environmental factors. AD is caused by a number of genes, inherited from generation to generation by family history, and is characterized by onset in childhood. A cohort study of AD family history revealed that it was inherited in an autosomal dominant manner. AD occurs in about 20% of children (Nutten S, Annals of nutrition & metabolism 2015, 66 Suppl 1: 8-16), therefore it is important to understand the genetic background for AD and develop early diagnosis and treatment. In addition, in order to diagnose AD early, it is necessary to identify mutations that cause complex and complicated phenomena such as AD.

Whole-exome sequencing (WES) is a technique that analyzes a base sequence of about 3 x 10 ⁷ bp that encodes all proteins known as exomes in the genome. Although the proportion of exomes in the human genome is less than 2%, mutations in this region can have far more serious consequences than those occurring in the remaining 98%. The goal of WES is single-base polymorphism that increases the risk of developing disease-causing mutations or common and complex diseases that are inherited in a Mendelian way through a filtering process in big data that includes all protein coding sites. It is to find genetic variations such as -nucleotide polymorphisms (SNP).

The common disease-common variant hypothesis (CD-CV) hypothesis can be most effectively tested through genome-wide association studies (GWASs). On the other hand, from the problem of loss of heritability in CD-CV, the hypothesis that a rare genetic variation occurred in an individual plays a more important role. Recently, a common genetic variation of a common disease has been reported to be common among different races (Marigorta UM et al., PLoS) genetics 2013, 9 (6): e1003566). The importance of the rareness or commonality of genetic variation is not yet known precisely, so it is necessary to identify not only rare variations, but also common and universal variations.

The present inventors conducted WES on all three family members of Koreans with AD family history who have severe symptoms to identify genetic mutations that cause early-onset atopic dermatitis (AD), and alleles Compared. As a result of the analysis, a number of genetic variations commonly found in common or rare in three families were observed, and among these, the variation of the COL6A6 gene was confirmed to be closely related to hereditary AD, thereby completing the present invention.

Therefore, the object of the present invention

In the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, the 26th base is A, and the polynucleotide consisting of 20 to 100 consecutive nucleotide sequences containing the 26th base, poly comprising the nucleotide sequence of SEQ ID NO: 2 The 26th base is T in the nucleotide, the polynucleotide consisting of 20 to 100 contiguous base sequences containing the 26th base, and the 26th base is T in the polynucleotide comprising the base sequence of SEQ ID NO: 3, It is to provide a composition for diagnosing atopic dermatitis comprising a detection agent for at least one polynucleotide selected from the group consisting of polynucleotides consisting of 20 to 100 consecutive nucleotide sequences comprising the 26th base and complementary polynucleotides thereof.

Another object of the present invention

To provide information necessary for diagnosing atopic dermatitis

(1) obtaining a nucleic acid sample from a subject; And

(2) analyzing the base sequence of the nucleic acid sample to determine the base of the single nucleotide polymorphism (SNP) region of the COL6A6 gene

It provides a method for detecting a polymorphism of the COL6A6 gene containing a marker.

In order to achieve the above object, the present invention

In the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, the 26th base is A, and the polynucleotide consisting of 20 to 100 consecutive nucleotide sequences containing the 26th base, poly comprising the nucleotide sequence of SEQ ID NO: 2 The 26th base is T in the nucleotide, the polynucleotide consisting of 20 to 100 contiguous base sequences containing the 26th base, and the 26th base is T in the polynucleotide comprising the base sequence of SEQ ID NO: 3, Provided is a composition for diagnosing atopic dermatitis, comprising a detection agent for at least one polynucleotide selected from the group consisting of polynucleotides consisting of 20 to 100 contiguous base sequences comprising a 26th base and complementary polynucleotides thereof.

In order to achieve another object of the present invention, the present invention

To provide information necessary for diagnosing atopic dermatitis

(1) obtaining a nucleic acid sample from a subject; And

(2) analyzing the base sequence of the nucleic acid sample to determine the base of the single nucleotide polymorphism (SNP) region of the COL6A6 gene

It provides a method for detecting a polymorphism of the COL6A6 gene containing a marker.

Hereinafter, the present invention will be described in detail.

The present invention

In the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, the 26th base is A, and is composed of 20 to 100 consecutive nucleotide sequences containing the 26th base Poly In the nucleotide, a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2, the 26th base is T, and the polynucleotide consisting of 20 to 100 consecutive nucleotide sequences containing the 26th base, including the nucleotide sequence of SEQ ID NO: 3 In the polynucleotide, the 26th base is T, and the detection agent for one or more polynucleotides selected from the group consisting of polynucleotides consisting of 20 to 100 consecutive base sequences containing the 26th base and their complementary polynucleotides It provides a composition for diagnosing atopic dermatitis comprising a.

As used herein, 'polynucleotide' or 'nucleic acid' refers to deoxyribonucleotide (DNA) or ribonucleotide (RNA) in a single- or double-stranded form. Known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides are also included, unless otherwise limited. In general, DNA is composed of four bases: adenine (A), guanine (G), cytosine (C), and thymine (T), and RNA is uracil (U) instead of thymine. Have In the double-stranded nucleic acid, A is T or U, C is hydrogen bond with G base, and the relationship between these bases is called 'complementary'. Also, in the present specification, “genomic DNA (gDNA)” refers to DNA that contains almost complete genetic information as the total sequencing of an individual gene.

On the other hand, when referring to the variation of a gene in the present specification, 'c.' Means a variation of a protein coding region, and '(base position) (base letter) (symbol>) (base date letter)' is the corresponding base position. It means that the previously indicated base is replaced with the following indicated base.

As used herein, 'protein' is used interchangeably with 'polypeptide' or 'peptide', and refers to a polymer of amino acid residues, such as is commonly found in proteins in nature.

As used herein, one letter (three letters) of an amino acid means the following amino acid according to standard abbreviation rules 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; W (Trp): tryptophan; Y (Tyr): Tyrosine.

'(Amino acid single character) (amino acid position) (amino acid single character)' used in the present specification means that an amino acid previously indicated in a corresponding amino acid position of a wild-type protein is replaced with an amino acid indicated later.

“Atopy” refers to a series of allergic symptoms that appear on the skin, respiratory mucosa, ocular mucosa, and mesothelioma in individuals with atopic predisposition, and these atopy predispositions (allergic constitution) are inherited and transmitted to the next generation in the household. Allergic diseases caused by atopic predisposition include allergic dermatitis, allergic rhinitis, asthma, allergic conjunctivitis, and atopic urticaria. These diseases may occur alone or in multiple diseases simultaneously.

'Atopic dermatitis' or 'atopic dermatitis' is a typical skin disease that occurs in people with atopic allergies, and is a chronic skin disease commonly referred to as fever. Red spots, edema, dry skin, and itching of the skin are the main symptoms. They show immunological characteristics and may be accompanied by hives, metal allergies, asthma or allergic rhinitis, which are allergic diseases.

“Diagnosis” as used herein includes all types of analysis used to predict disease outbreaks and to determine or derive an onset risk or susceptibility.

For the purposes of the present invention, the diagnosis refers to the diagnosis of atopic dermatitis. Atopic dermatitis in the present invention may be hereditary atopic dermatitis, which is preferably caused by a genetic factor, and an environmental effect may be added to the genetic predisposition, thereby increasing symptoms. In addition, atopic dermatitis in the present invention may be early onset. The term 'early-onset' means that the disease occurs before becoming an adult, and most preferably, it occurs within 2 years after birth.

In the present invention, in order to provide information necessary for the diagnosis of atopic dermatitis in a subject, polymorphism of the COL6A6 gene is detected as a diagnostic marker. 'COL6A6' is an alpha 6 chain of collagen type VI, located at the 3q21 position, and consists of about 43 exons. It is expressed in various tissues and cells in humans, and is particularly important for skeletal muscle and skin function. COL6A6 in the present invention is most preferably derived from human, and the sequence information of the mRNA and protein of the human COL6A6 gene is known to the GenBank database as the accession numbers of NM_001102608.1 and NP_001096078.1, respectively.

As used herein, the term 'polymorphism' refers to the generation of two or more alternative sequences or alleles (or alleles) within a genetically determined population, and 'single nucleotide polymorphism (SNP)' is one It means the polymorphism of the base. Specifically, in the genome, a single base (A, T, C or G) refers to the diversity of DNA sequences occurring between members of a species or between a pair of chromosomes of an individual. For example, when a single base contains differences, such as three DNA fragments of different individuals (eg AAGT [A / A] AG, AAGT [A / G] AG, AAGT [G / G] AG). , Called two alleles (A or G), and in general almost all SNPs have two alleles. In addition, when SNP is genetically closely related to a specific disease, SNP has a mutation in one base at a specific position compared to the identified normal or wild-type (WT) individual or allele. It also means

The SNP region of COL6A6, a marker for diagnosing atopic dermatitis according to the present invention, is located in the protein coding region of COL6A6, and the SNP allele associated with atopic dermatitis may cause damage to COL6A6 protein function. The specific association between COL6A6 SNP and atopic dermatitis is as follows:

1) 26th base in the polynucleotide constituting the nucleotide sequence of SEQ ID NO: 1

SEQ ID NO: 1 is the base sequence of SNP rs16830494, and shows the allele at the 26th base. The 26th base of SEQ ID NO: 1 corresponds to the 130,361,856th base on chromosome 3 (see Table 2). Patients with atopic dermatitis in the family Atopic Dermatitis were identified as having the 'adenine (A)' base as the SNP allele. The mutation related to atopic dermatitis is the 5216th base in the protein coding region of the COL6A6 gene substituted with G to A (c.5216G> A), and the 1739th amino acid arginine is the glutamine in the COL6A6 protein. Substituted (Arg1739Gln or R1739Q). The mRNA base sequence of the COL6A6 gene (from the start codon to the stop codon) and the amino acid sequence of the protein are described herein as SEQ ID NO: 10 and SEQ ID NO: 11, respectively.

2) 26th base from the polynucleotide constituting the nucleotide sequence of SEQ ID NO: 2

SEQ ID NO: 2 is the base sequence of SNP rs200963433, and shows the allele at the 26th base. The 26th base of SEQ ID NO: 2 corresponds to the 130,289,976th base on chromosome 3 (see Table 4). Patients with atopic dermatitis in the family Atopic Dermatitis have been identified as having the 'thymine (T)' base as the SNP allele. The mutation associated with atopic dermatitis is the 2716th base in the protein coding region of the COL6A6 gene (C.2716C> T) in normal humans (c.2716C> T). Substituted (Arg906Cys or R906C).

3) 26th base in the polynucleotide constituting the nucleotide sequence of SEQ ID NO: 3

SEQ ID NO: 3 is the base sequence of SNP rs59021909, and shows the allele at the 26th base. The 26 th base of SEQ ID NO: 3 corresponds to the 130,285929 th base of chromosome 3 (see Table 3). Patients with atopic dermatitis in the family Atopic Dermatitis have been identified as having the 'thymine (T)' base as the SNP allele. The mutation related to atopic dermatitis is that the 1666th base in the protein coding region of the COL6A6 gene is replaced with C to T of normal humans (c.1666C> T), and the 556th amino acid proline is the cysteine to the COL6A6 protein. Substituted (Pro556Ser or P556S).

Accordingly, the composition for diagnosing atopic dermatitis according to the present invention includes an agent capable of detecting the COL6A6 SNP. More specifically, in the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, the 26th base is A, and the polynucleotide consisting of 20 to 100 consecutive nucleotide sequences including the 26th base, The 26th base is T in the polynucleotide constituting the polynucleotide, and the 26th base is T in the polynucleotide constituting the nucleotide sequence of SEQ ID NO: 3, which is composed of 20 to 100 consecutive nucleotide sequences containing the 26th base. , And includes a detection agent for at least one polynucleotide selected from the group consisting of polynucleotides consisting of 20 to 100 contiguous base sequences comprising the 26th base and complementary polynucleotides thereof.

Specifically, the detection agent may be a probe capable of confirming the SNP base by hybridizing with a polynucleotide at a site containing the COL6A6 SNP. That is, it is a probe capable of detecting any one or more bases selected from the group consisting of the 26th base of SEQ ID NO: 1, the 26th base of SEQ ID NO: 2, and the 26th base of SEQ ID NO: 3.

A 'probe' is a fragment of a polynucleotide such as RNA or DNA of a few to several hundred base lengths, which can specifically bind to a specific gene mRNA, cDNA (complementary DNA), DNA, etc. It means, the presence of the target mRNA or cDNA to be bound because it is labeled (labeling), it is possible to confirm the expression amount. Probe selection and hybridization conditions may be appropriately selected according to techniques known in the art. The probe may be used in a diagnostic method for detecting alleles (or alleles). The diagnostic methods include detection methods based on hybridization of nucleic acids, such as Southern blots, and may be provided in a form pre-bonded to a substrate of a DNA chip in a method using a DNA chip. The hybridization may be performed under strict conditions, for example, a salt concentration of 1 M or less and a temperature of 25 ° C. or more. For example, conditions of 5 × SSPE (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.

In addition, the detection agent may be a primer pair capable of amplifying a polynucleotide at a site containing COL6A6 SNP. That is, it is a primer pair capable of amplifying any one or more bases selected from the group consisting of the 26th base of SEQ ID NO: 1, the 26th base of SEQ ID NO: 2, and the 26th base of SEQ ID NO: 3.

A primer is a short single-stranded oligonucleotide that acts as a starting point for DNA synthesis. The primer specifically binds to the template polynucleotide at a suitable buffer and temperature condition, and DNA polymerase adds a nucleoside triphosphate having a base complementary to the template DNA in the primer. DNA is synthesized by linking. Primers generally consist of 15 to 30 base sequences, and the temperature (melting temperature, T _m ) that binds to the template strand varies depending on the base composition and length. The sequence of the primer need not have a sequence completely complementary to some of the base sequence of the template, and it is sufficient to have sufficient complementarity within a range capable of hybridizing with the template and working with the primer. The primer for the polymerase chain reaction (PCR) is a template (or sense, sense) and the opposite side (antisense, antisense) of both ends (5 'end or 3' end) of a specific section of a polynucleotide to be amplified. It consists of a pair (set) that are complementary to each other. Primers can easily design a primer pair for the diagnosis of atopic dermatitis according to the present invention by referring to the base sequence of cDNA or genomic DNA of the SNP region of COL6A6 described above. PCR cycle conditions for detecting SNP of COL6A6 according to the present invention are specifically described in the experimental method of the Examples.

More specifically, the primer pair is a pair of base sequences represented by SEQ ID NO: 4 and SEQ ID NO: 5, a pair of base sequences represented by SEQ ID NO: 6 and SEQ ID NO: 7, and a base represented by SEQ ID NO: 8 and SEQ ID NO: 9 It may be any one primer pair selected from the group consisting of a pair of sequences.

The primer or probe of the present invention can be chemically synthesized by using a phosphoramidite solid support synthesis method or other well-known methods. In addition, the primer or probe can be variously modified according to methods known in the art within a range that does not interfere with hybridization with the targeted polynucleotide to be detected. Examples of such modifications are methylation, encapsulation, substitution with one or more homologs of natural nucleotides, and modification between nucleotides, such as uncharged linkages (eg methyl phosphonate, phosphotriester, phosphoroamidate, carbamate, etc.) ) Or charged linkages (eg phosphorothioate, phosphorodithioate, etc.), and binding of fluorescent or enzymatic labeling materials.

In addition, the diagnostic composition according to the present invention can be prepared as a diagnostic kit or a diagnostic microarray capable of detecting COL6A6 SNP.

Methods of manufacturing a microarray by immobilizing a probe on a substrate are well known in the art. The probe of the present invention is an allele-specific probe of COL6A6 SNP, wherein a polymorphic site is present in a nucleic acid fragment derived from two members of the same species, hybridizing to a DNA fragment derived from one member, but to a fragment derived from another member It does not hybridize. In this case, the hybridization conditions show a significant difference in the hybridization strength between alleles, and must be sufficiently strict to hybridize to only one of the alleles. This can cause a clear hybridization difference between different alleles. Hybridization conditions are as described above.

The process of immobilizing the polynucleotide on the substrate can be easily carried out using conventional techniques. In addition, hybridization of nucleic acids on microarrays and detection of hybridization results are well known in the art. The detection is, for example, by labeling a nucleic acid sample with a labeling substance capable of generating a detectable signal comprising a fluorescent substance such as Cy3 and Cy5, then hybridizing on a microarray and generating from the labeling substance. The hybridization result can be detected by detecting the signal to be performed.

In addition, the present invention

To provide information necessary for diagnosing atopic dermatitis

(One) From the subject  Obtaining a nucleic acid sample; And

(2) By analyzing the nucleotide sequence of the nucleic acid sample COL6A6  Single nucleotide polymorphism ( SNP ) Determining the base of the site

Containing COL6A6  Gene polymorphism With marker  Provides a method for detection.

In the present invention, the sample of the subject can be used without limitation as long as it is capable of separating nucleic acid samples such as blood, whole blood, plasma, sputum, saliva, ocular fluid, semen, cells and tissues (e.g., skin) separated from the subject. You can. Most preferably, blood can be used. The sample may be subjected to pre-treatment such as filtration, distillation, extraction, concentration, inactivation of interference components, and addition of reagents before use for detection. Methods for isolating nucleic acid samples are well known in the art.

'Detection' in the present specification means analyzing, imaging, confirming or establishing the presence or absence of a specific base of the diagnostic marker according to the present invention, specifically COL6A6 SNP.

The single base polymorphism (SNP) region of COL6A6 is as described above. Specifically, it is characterized in that it is any one or more bases selected from the group consisting of the 26th base of SEQ ID NO: 1, the 26th base of SEQ ID NO: 2 and the 26th base of SEQ ID NO: 3.

In addition, when the single base polymorphism site is any one or more bases selected from the group consisting of A in the 26th base of SEQ ID NO: 1, T in the 26th base of SEQ ID NO: 2, and T in the 26th base of SEQ ID NO: 3 Atopic dermatitis is diagnosed, or the risk or susceptibility to atopic dermatitis is high.

The step of determining the base may be used without limitation as long as it is commonly used in the art to analyze and determine the base sequence. More specifically, sequencing, exome sequencing, microarray hybridization, allele specific PCR, dynamic allele-specific hybridization, PCR extension analysis and Taqman technique can be performed by any one method selected from the group consisting of.

Accordingly, the present invention provides a method for detecting a mutation in a composition for diagnosing atopic dermatitis, comprising an agent capable of detecting a mutation in the 26th base of rs16830494, rs200963433, and rs59021909, which are the single base polymorphism (SNP) sites of the COL6A6 gene. The present inventors analyzed the protein coding region of the genomic DNA of a family member with a family history of atopic dermatitis accompanied by severe symptoms, and found that the specific base mutation of the SNP of the COL6A6 gene is closely related to atopic dermatitis. , It can be used to develop a genetic diagnostic reagent for diagnosing atopic dermatitis using this.

1 shows a family tree of a Korean family with a history of atopic dermatitis (AD). Symbols The squares represent males, the circles represent females, the black symbols represent AD patients, and the white symbols represent normal people.
2 shows genotypes for common or rare genetic mutations found in family members of the AD family. Rare variants are indicated by diagonal marks and common variants are indicated by gray marks. A gene mutation in a family has been identified as a rare and common variant (rare and common variant).
3 is a schematic diagram of a filtering process for selecting mutations in the AD household
4 is a Venn diagram showing the number of common and rare variations common to the three families.
Figure 5 shows the single nucleotide polymorphism (SNP) mutation of the protein coding region of COL6A6 identified by Sanger method in AD family.

Hereinafter, the present invention will be described in detail.

However, the following examples are only to illustrate the present invention, the content of the present invention is not limited to the following examples.

<Experiment method>

patient

This study was conducted with the approval of the Ethics Committee of Chung-Ang University Hospital. Peripheral blood samples were obtained from three family members of Koreans with AD family history. Each family consisted of 2 people with AD and 2 normal people. Each family member was diagnosed by a dermatologist. The subjects and children both had AD before 2 years of age.

Whole-exome sequencing

Genomic DNA was isolated from peripheral blood obtained from family members with AD family history using a QIAamp DNA mini kit (Qiagen Inc, Valencia, CA, USA). The amount and quality of DNA was confirmed with a Nanodrop spectrometer (Nanodrop Technologies, Wilmington, DE, USA) and a 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, MA, USA). Paired-end DNA sequencing library was constructed through shearing, end-repair, A-tailing, peak detection, PE adaptor ligation, and amplification. The prepared library (Library) was hybridized with bait sequence for 24 hours, separated, amplified using an index index barcode tag, and the quantity and quality were determined. The exome library was sequenced in a 100-bp paired-end mode of HiSeq SBS kit.

Whole - Exome Sequencing  Process and sequence alignment ( alignment )

Sequence reads in FASTQ format are created using the Burrows-Wheeler Aligner (BWA, v0.7.7) program to generate a SAM file with accurate mate pair information as 'mem' and seed value parameter '-k 45' to human assembly UCSC hg19. Mapping (Li H et al., Bioinformatics 2009, 25 (14): 1754-1760). Read group tag included sample name. The SAM file was converted into a compressed BAM file using Picard (v1.92), and the BAM file was sorted according to the chromosomal coordinate position. The Genome Analysis Toolkit (v2.3.9Lite) was used to locally rearrange BAM files at intervals that could include insertion / deletion (indel) misalignment (DePristo MA et al., Nature genetics 2011). , 43 (5): 491-498). Insertion and deletion were confirmed using Mutect (Cingolani P et al., Fly 2012, 6 (2): 80-92) and GATK Somatic Indel Detector, respectively. Single base mutation and indel are snpEff (v3.6c) (Cibulskis K et al., Nature biotechnology 2013, 31 (3): 213-219), 'synonymous','non-synonymous','missense','frameshift point mutation (frameshift point mutation) ) ',' Frameshift indel ', and so on.

Annotation

Filter 1 (Filter 1): SnpEff ( http://snpeff.sourceforge.net/index.html) is annotate the mutation such as amino acid substitution of the various genes, a program for predicting the effect of the mutation. Genetic mutations induce various effects (eg 'Non_Synonymous_coding', 'Stop gained', 'Insertion', 'Deletion', etc.).

Filter 2 (Filter 2): impact prediction analysis by SnpEff showed that the analysis of the potential impact of gene mutations should be able to quickly categorize the mutation and easily prioritize ( "High (High) ':' Splice Site Donor (Splice_Site_Acceptor), 'Splice Site Donor (Splice_Site_Donor)', 'Start_Lost', 'Frame_Shift', 'Stop_Gained';'Moderate' : 'Non_Synonymous_coding', 'Codon_Change', 'Codon_Insertion and Deletion', etc.)

Filter 3 (Filter 3): dbNSFP SIFT and the Polyphen2.

The SIFT score predicts the effect of amino acid substitutions on protein function. SIFT's predictions are based on the degree of conservation of amino acids found in alignment with closely related sequences collected through PSI-BLAST. The range is 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 higher the probability that the substitution is tolerated. The Polyphen2 HDIV score is based on HumDiv (hdiv_prob). The score is in the range of 0 to 1, if the score is between 0.957 and 1, it is 'probably damaging', between 0.453 and 0.956 is 'possibly damaging', and between 0 and 0.452 is 'good' (benign) '(higher score indicates higher damage).

Filter 4 (Filter 4): PhyloP dbNSFP of the phylogenetic differences in evolutionary rates over a branch of the (phylogenetic tree) seeks a position that is, positive or negative selection (positive or negative selection), allowing progress. The higher the PhyloP score, the more conserved it is (http://varianttools.sourceforge.net/Annotation/DbNSFP).

Filter 5 (Filter 5): PhastCons measures the strength of the tablet selection (purifying selecton) acting on the DNA sequence. The high PhastCons score (0.2) provides an important basis that the genomic region is functionally important.

Filter 6 (Filter 6): 1000 dielectric allele frequencies (the 1000 Genome allele frequency) of dbNSFP selects the mutation frequency is that low or unknown than 0.01. Filter 6 eliminates the universal variation of minor allele frequency (MAF) of more than 0.01 observed in races worldwide.

Filter 7 (Filter 7): South Korea Personal Genome Project (The Korean Personal Genome Project, KPGP ) is part of a private international genome projects. KPGP selects an 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 higher.

Sanger sequencing

PCR conditions for verifying the single base polymorphism (SNP) selected by WES by the Sanger method are as follows: 95 ° C., 10 min (1 cycle); 95 ° C, 30s; 55-58 ° C, 30s; 72 ° C., 40 s (35 cycles); 72 ℃, 1min 30s (1cycle). PCR reaction solution (total volume 50μL) contains 25μL of 2 × EF-Taq premix (SolGent, Seoul, South Korea), 2.5μL of oligonucleotide primer (10 pmol/μL), 18μL of distilled water, and 2μL containing 20ng of genomic DNA The template was included. PCR products were separated 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.

Statistical analysis

Logistic regression analysis was used to calculate the natural odds ratios (ORs) and 95% confidence intervals (CIs) for the association between the SNP and AD being analyzed. The association between COL6A6 polymorphism and AD was tested using Fisher's exact test.

< Example  1>

Whole exome sequencing  analysis

Whole-exome sequencing (WES) was performed on genomic DNA isolated from peripheral blood samples from three family members of Koreans with AD family history.

In order to analyze only the genetic factors, three families with AD family history showing severe symptoms were recruited, and in order to eliminate environmental effects as much as possible, the AD was limited to the early expression. When AD was developed before the age of two by one parent and one child, the subjects were analyzed with normal family members. As a result of analysis, all AD patients had a heterozygote genotype, and normal family members had a wild-type (WT) genotype of homozygote for all identified mutations ( FIG. 1 , Fig. 2 ).

The WES data obtained from each individual was filtered stepwise as shown in FIG . 3 in order to select mutation candidates related to AD. 176 common variants that passed filter average 4 per AD household and 48 rare variants that passed 1000 genomes of filter 5 were identified. In KPGP, an average of 44 rare mutations were detected ( Table 1 ).

Amino acid substitutions and types of mutations ('non-synonymous SNP', 'stop gained', 'insertion', 'deletion', etc.) are one-step filters (filter 1) Was decided. 'Non-like SNP' refers to a change in a single base that converts to a codon of another amino acid. In all three households, a significant number of 'unlike SNPs' were observed. In addition to the fact that the mutations found in Filter 5 are universal mutations (MAF higher than 1%), they have a well-conserved sequence among more than 100 vertebrate species and are functional genes predicted to have impaired protein function by mutations. gave.

< Example  2>

Features of genetic variation common to AD ancestry

In order to find a particularly important mutation among the selected genetic mutations, genes common to universal mutations identified through filter 5 in three households were identified ( FIGS. 2 and 4 ).

In filter 5, there were 14 types of overlapping genes, and 4 of them were those that could be classified as rare mutations until reaching filter 7 ( FIG. 2 ). The risk allele in AD patients was identified in comparison to a healthy control group ( FIG. 2 ). Among the above genes, the UNC93A gene obtained a termination codon, and other genes showed a 'unlike SNP' mutation. In particular, mutations in the COL6A6 gene were confirmed in all three households ( Table 2 to Table 4 ), and there were two COL6A6 SNPs in Household A ( Table 2 ). The genotype of 14 genes was investigated by exome analysis. Fathers and children with AD were heterozygous, and normal mothers and children had homozygous wild-type (WT) genes for all 14 genes ( FIG. 2 ).

The indexes of chromosomal location, amino acid change, and functional prediction of 14 genes with mutations common to all three families were identified. The 14 genes were functionally important, and supported by the results of SIFT (variability may be impaired or harmful) and PhyloP (preserved well among over 100 species of vertebrates). In particular, in all three households, the SIFT index of the COL6A6 gene was low, and the high PhyloP and Phastcon index was judged to be a genetic mutation impairing the function of COL6A6. In addition, the KPGP index of the three households' COL6A6 showed that the MAF would be in the range of 5 to 21% per 1,000 Koreans.

< Example  3>

AD Wow COL6A6 SNP  Variation association

COL6A6 gene, located in 3q21, known as a site of susceptibility to atopic dermatitis on the genome, having a meaningful functional prediction score with respect to atopic dermatitis, as identified in the previous example The correlation between a mutation identified by WES and atopic dermatitis was confirmed by Sanger sequencing. Primers used in Sanger seqeuncing are as described in Table 5 .

As a result, the mutation was confirmed at the same position as the WES result. In household A, the missense mutations c.5216G> A, p.Arg1739Gln (universal variation; corresponding to SNP rs16830494) and c.2716C> T, p.Arg906Cys (rare mutation; corresponding to SNP rs200963433) were found in AD patients. Healthy family members did not have these variations ( FIG. 5 ). In family B, missense mutations c.1666C> T, p.Pro556Ser (universal variation; corresponding to SNP rs59021909) existed, and in family C, missense mutations c.2716C> T, p.Arg906Cys (rare mutations) Was observed. The above results suggest that the COL6A6 SNP mutation found in AD patients in each household is closely related to the development of AD, and is highly likely to be a risk factor for developing AD.

Also, genes such as CDX1, ANKRD35, and TUFT1, which were identified in this study, were located in 5q31-33 or 1q21, which are known to be related to AD, and the COL6A6 gene, which is commonly found in three families, is also closely related to AD It was confirmed to be present at the known 3q21 position ( Table 6 ). This supports the possibility that mutation of the COL6A6 gene and impaired function of the protein may be involved in the development of AD.

As described above, the present invention can be usefully used to develop a gene diagnosis reagent and diagnostic chip of atopic dermatitis for early detection of atopic dermatitis and appropriate treatment in an early stage to effectively treat it.

<110> CHUNG ANG University industry Academic Cooperation Foundation <120> Composition and method for diagnosing atopic dermatitis using          COL6A6 SNP <130> NP16-0067 <160> 11 <170> KopatentIn 2.0 <210> 1 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Homo sapiens rs16830494 <400> 1 acatgtgagc tcattcagta tgtgcgagac cgcagtcgta agtaccctgc t 51 <210> 2 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Homo sapiens rs200963433 <400> 2 catgttcact gaagcccggg gcagccgcct gaacaagggg gtcccccaag t 51 <210> 3 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Homo sapiens rs59021909 <400> 3 catgtccaag gatagcatct tggagcctgc aaacagactg agagaagagc a 51 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> rs16830494 forward primer <400> 4 tagggtgtga aaggagcc 18 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rs16830494 reverse primer <400> 5 agcaaccaga gagggagtta 20 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> rs200963433 forward primer <400> 6 tgatgaccca gaggtgctg 19 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> rs200963433 reverse primer <400> 7 ggcaccatca atccccac 18 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> rs59021909 forward primer <400> 8 gggaatacaa acacaggcg 19 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> rs59021909 reverse primer <400> 9 gccacgattt ctcttacctt c 21 <210> 10 <211> 6792 <212> DNA <213> Artificial Sequence <220> <223> Homo sapiens COL6A6 mRNA (from start codon to stop codon; <400> 10 atgatgttgc taattttgtt cctcgtgata atttgttccc atatttctgt gaaccaagat 60 tccggccctg agtatgcaga tgttgtgttt ttggtggaca gctctgatcg cctgggatcc 120 aagtccttcc catttgtgaa aatgttcatc accaaaatga tcagcagtct ccccatagag 180 gccgacaaat accgtgtggc cctggcccag tacagtgata aacttcacag tgaattccac 240 ctgagcacct tcaaaggcag gagccccatg ctgaaccacc taaggaagaa ctttggattc 300 attggcgggt ccctgcagat aggaaaggct cttcaggagg ctcacaggac ttatttctct 360 gcacccgcaa atgggagaga caagaaacag tttcccccaa ttctagtggt cctggcttca 420 tctgagtctg aggataatgt ggaagaggca tcaaaggccc tgcggaaaga cggagtgaaa 480 atcatctctg taggggtgca gaaagcttct gaggaaaacc tgaaggccat ggccacgtct 540 cagtttcatt tcaaccttcg gacagtcaga gacctcagca tgttttccca aaacatgaca 600 cacatcatca aggatgtaat aaagtacaag gagggagcag ttgatgacat ctttgtagaa 660 gcttgccaag gcccttctat ggccgatgtt gtgttcctat tggatatgtc aatcaatgga 720 agtgaggaga actttgacta tcttaaagga ttcttggaag aaagtgtatc tgcccttgac 780 ataaaggaaa attgcatgag ggttggcctt gtggcctata gcaatgagac aaaagtgata 840 aattcactga gcatgggcat aaataagtca gaggttctcc agcatataca gaacctttct 900 ccccgaactg ggaaggccta tactggagct gccatcaaaa agctcaggaa ggaagttttt 960 agtgcacgga atggcagtcg gaagaatcag ggggtgcccc agattgccgt gctggtgacc 1020 caccgagatt cagaagacaa cgtgacaaaa gcagctgtta acctccgacg ggagggtgtg 1080 accatcttca ccctgggcat agagggcgcc agcgacaccc agttggaaaa gatagcatcc 1140 caccctgctg agcagtatgt ctccaaactg aagaccttcg ctgacctggc tgctcacaac 1200 cagacatttc tgaagaagct gcggaaccaa ataacacaca cagtctctgt cttttcagag 1260 aggactgaaa cgctcaaatc tggttgtgtg gacactgagg aagcagacat ctatctgctt 1320 atcgatggct cagggagcac ccaggccaca gatttccatg aaatgaagac gttcctgtca 1380 gaggtggtag ggatgttcaa cattgctccc cataaggtgc gggttggggc cgttcagtat 1440 gctgacagct gggacttgga atttgagatc aataaatact ccaacaagca ggatttggga 1500 aaggccattg agaatatcag gcagatgggt gggaatacaa acacaggcgc agcactgaat 1560 ttcacactga gtctgttgca aaaagcaaag aagcagcgag gaaacaaagt tccatgccac 1620 cttgttgtcc tgacaaatgg catgtccaag gatagcatct tggagcctgc aaacagactg 1680 agagaagagc acatccgagt ttatgctatc gggatcaagg aggccaacca aacacagctg 1740 agagaaattg caggagagga aaagagagtg tattacgtgc atgactttga tgcattgaaa 1800 gacataagaa accaagttgt tcaagaaatc tgtactgaag aagcttgcaa agagatgaaa 1860 gctgacatca tgtttctggt ggacagttct ggaagtatag gacctgaaaa cttcagcaaa 1920 atgaaaacat ttatgaaaaa cctggtgagc aagtctcaga ttggaccaga tcgggtgcaa 1980 attggtgtag tccagttcag cgacatcaat aaggaagagt ttcagctcaa cagattcatg 2040 tcccaaagcg acatttcaaa tgcaatagac caaatggctc acattggaca aaccaccctg 2100 actggtagtg ccctgagctt tgtgtctcag tacttcagcc ccaccaaggg cgcccggccc 2160 aacatcagaa agtttctcat cctcatcacg gatggtgaag ctcaggacat agtaaaggaa 2220 ccagcagtag tgcttcggca agaaggtgta atcatctatt ctgtgggagt gtttggctcc 2280 aatgtcaccc agcttgagga gatcagtggg aggcccgaga tggtttttta tgttgagaat 2340 tttgacattc tgcagcgcat tgaagatgat cttgtttttg gaatatgcag cccccgtgaa 2400 gaatgcaagc ggattgaagt tttagacgtt gtgtttgtca ttgatagctc tggcagtatt 2460 gactatgatg agtataatat catgaaggat tttatgattg gcttagtgaa aaaagctgat 2520 gtgggcaaga atcaggtccg gtttggggct ctgaagtatg ctgatgaccc agaggtgctg 2580 ttttatctgg atgactttgg cacaaaactg gaggtaattt cagtgctcca gaatgaccaa 2640 gccatgggtg gcagtactta tactgctgag gcactgggct tctcagacca catgttcact 2700 gaagcccggg gcagccgcct gaacaagggg gtcccccaag tcctcattgt gatcaccgat 2760 ggggaatccc atgatgctga taaactcaat gccacggcaa aggccttgcg ggacaaaggc 2820 attcttgtcc tggctgtggg gattgatggt gccaatcccg tggagctgtt agccatggca 2880 ggatcaagcg acaagtactt cttcgtggag acttttggag gtctgaaggg aatattttca 2940 gatgtgacag ccagtgtctg caactcttca aaagtagatt gtgaaattga caaagtagat 3000 cttgttttcc ttatggatgg ttcaactagc attcagccaa atgacttcaa gaaaatgaag 3060 gaatttctgg catctgttgt tcaagacttt gatgtcagcc tcaacagagt gcgaatagga 3120 gcggcccagt ttagcgatac ctatcacccg gagtttccac tgggaacttt cataggtgaa 3180 aaagagatat catttcagat tgaaaacatc aagcagatct ttggaaacac acacatcggt 3240 gctgcactca gggaggtgga acattacttc aggccagaca tgggcagcag gataaataca 3300 ggtaccccac aggtgctgct ggtccttaca gatggccagt cccaagacga ggtggcccag 3360 gccgcggaag ccctgagaca cagaggtatc gacatctact ccgtgggcat tggggatgtg 3420 gatgaccagc agctcattca gatcaccggg actgcagaga aaaaactgac agtgcacaac 3480 ttcgatgaac tgaagaaggt caataaaagg atcgttcgca acatctgtac cacagcgggt 3540 gaaagcaact gtttcgtgga tgttgtggtg ggatttgatg tctcaactca ggagaaaggg 3600 cagactttgc ttgaaggtca gccttggatg gaaacctacc ttcaagacat cttacgtgcc 3660 atcagctccc tcaatggagt aagctgtgag gtgggcacag agactcaggt cagtgtggct 3720 tttcaagtga ccaatgccat ggaaaaatat tctcccaagt ttgagatcta cagtgaaaac 3780 atactgaata gcttgaagga tataacagtt aaaggaccat ctcttctcaa tgcaaacctc 3840 ttggattctc tatgggatac atttcagaat aaatcagctg ctcgaggaaa ggtggtcctt 3900 ttattttcag atggattgga tgatgatgtt gagaaacttg aacaaaaatc tgatgaactt 3960 agaaaagaag gcctgaatgc cctcataact gttgctctgg atggacctgc tgattcaagt 4020 gacttggctg atcttcccta tattgaattt gggaaaggat ttgagtacag gacacagctc 4080 tctattggca tgagagaact tggaagccgg ctgtcaaagc agctggtcaa tgttgctgaa 4140 aggacatgct gctgtttgtt ctgcaagtgc attggaggag atggcacaat gggagatcct 4200 ggaccaccag ggaaaagggg acctccaggt tttaaaggca gtgaaggcta cctgggagag 4260 gagggaatcg ctggagaaag aggagcccct ggaccagtgg gagagcaagg tactaaggga 4320 tgctatggca ccaaaggtcc taagggaaac agaggactaa atggacagga gggagaagtt 4380 ggggaaaatg gaattgacgg attaaacgga gaacagggtg ataatggtct tcctggaaga 4440 aaaggagaaa agggagatga gggatctcag ggaagcccag ggaagagagg gactcctggt 4500 gaccgtggag caaagggcct gcgaggggat cccggagctc ctggagttga cagtagcata 4560 gaaggaccca caggcttgaa aggagaacgt ggaagacaag gcagaagagg ctggccaggc 4620 ccccccggga caccaggctc cagaagaaag acagcagctc atggcagaag gggacataca 4680 ggcccacagg gaacagcagg catcccagga ccagatggac ttgaaggctc cctgggactt 4740 aagggccctc agggcccaag aggagaggct ggtgtgaaag gagaaaaagg aggtgtggga 4800 agtaaaggtc cccaggggcc tccaggaccc ggaggagagg cagggaatca aggccgtttg 4860 ggaagccaag gaaataaagg agaacctgga gatctgggag aaaaaggagc tgttggcttt 4920 cctggtcctc gtggcttgca gggcaatgat ggcagtccag gttatggtag tgtcggacgc 4980 aagggagcaa agggacaaga aggattccct ggagaaagtg gacctaaggg tgagattggg 5040 gaccctggtg gtccaggaga gactgggctg aagggagcta gaggcaaaat gatatctgct 5100 gggcttccag gagagatggg atcccctggg gaaccaggac ctcctggacg taagggtgtg 5160 aaaggagcca aaggcttggc ttcattttct acatgtgagc tcattcagta tgtgcgagac 5220 cgcagtcctg gcaggcatgg aaaaccggaa tgcccagtgc acccaaccga gttggtgttt 5280 gccctggacc actcccggga tgtcactgag caggaatttg agcggatgaa ggagatgatg 5340 gctttcctgg tgagagacat taaggtccgg gagaacagct gccccgtggg agcgcacatc 5400 gccatcctct cctataactc ccacgccagg caccttgtgc gcttctcaga cgcctacaag 5460 aagagtcaac ttctcagaga aattgaaact attccttatg agagatcctc tgccagcagg 5520 gagattggca gagcaatgcg gtttatttcc aggaatgtct tcaagcggac gcttccgggg 5580 gcacacacga gaaaaatcgc cacatttttc agcagcggtc agtccgcgga tgcccactcc 5640 atcaccacgg ctgccatgga gttcggcgcg cttgaaatca ttcccgtggt gatcactttc 5700 agcaacgtgc cctcggtcag gcgcgcattt gcgattgacg acactggcac atttcaagta 5760 atagtggttc cctccggggc cgactacata ccagcattag agagactcca gcggtgcact 5820 ttctgctatg atgtgtgcaa gccagatgct tcttgtgacc aagccagacc accccctgtg 5880 cagtcttaca tggatgctgc tttccttctg gatgcctccc ggaacatggg aagtgctgaa 5940 tttgaagaca taagagcctt ccttggagca ctattagatc actttgaaat caccccagag 6000 ccggagactt ctgtcactgg agaccgggtg gccctattga gccatgctcc ccccgacttc 6060 ctacccaaca ctcagaagag tccagttaga gctgagttca atcttaccac ctacagaagt 6120 aagcgcctca tgaagaggca tgtgcacgag tcagttaaac aactaaatgg agatgctttt 6180 attggtcatg ccttacagtg gactctggac aatgtatttt taagtacacc caatctgaga 6240 agaaacaaag tcatatttgt gatatctgct ggggaaacca gccacttaga tggggaaatc 6300 ttaaagaagg aatccttgcg agccaaatgt cagggatatg ccttatttgt gttttccctt 6360 ggccctattt gggatgacaa ggaactggag gatctcgcca gccacccttt ggatcaccac 6420 ctggtccagc ttggccgaat tcataaacct gaccacagtt atggtgtgaa gtttgtgaag 6480 tcctttataa actcaatcag gcgtgcaatc aacaaatatc caccaataaa cttaaaaata 6540 aagtgcaaca gacttaactc tatagatcca aagcagcccc cacgaccatt ccgaagcttt 6600 gttcctggac cacttaaagc taccctcaaa gaagatgtat tacagaaggc aaaattcttt 6660 caagataaaa aatatctttc aagagtagca agaagtggca gagatgatgc tattcaaaat 6720 tttatgagaa gcacctccca tacctttaag aatggaagga tgatagaaag tgctcccaaa 6780 caacatgatt aa 6792 <210> 11 <211> 2263 <212> PRT <213> Artificial Sequence <220> <223> Homo sapiens COL6A6 protein (NP_001096078.1) <400> 11 Met Met Leu Leu Ile Leu Phe Leu Val Ile Ile Cys Ser His Ile Ser   1 5 10 15 Val Asn Gln Asp Ser Gly Pro Glu Tyr Ala Asp Val Val Phe Leu Val              20 25 30 Asp Ser Ser Asp Arg Leu Gly Ser Lys Ser Phe Pro Phe Val Lys Met          35 40 45 Phe Ile Thr Lys Met Ile Ser Ser Leu Pro Ile Glu Ala Asp Lys Tyr      50 55 60 Arg Val Ala Leu Ala Gln Tyr Ser Asp Lys Leu His Ser Glu Phe His  65 70 75 80 Leu Ser Thr Phe Lys Gly Arg Ser Pro Met Leu Asn His Leu Arg Lys                  85 90 95 Asn Phe Gly Phe Ile Gly Gly Ser Leu Gln Ile Gly Lys Ala Leu Gln             100 105 110 Glu Ala His Arg Thr Tyr Phe Ser Ala Pro Ala Asn Gly Arg Asp Lys         115 120 125 Lys Gln Phe Pro Pro Ile Leu Val Val Leu Ala Ser Ser Glu Ser Glu     130 135 140 Asp Asn Val Glu Glu Ala Ser Lys Ala Leu Arg Lys Asp Gly Val Lys 145 150 155 160 Ile Ile Ser Val Gly Val Gln Lys Ala Ser Glu Glu Asn Leu Lys Ala                 165 170 175 Met Ala Thr Ser Gln Phe His Phe Asn Leu Arg Thr Val Arg Asp Leu             180 185 190 Ser Met Phe Ser Gln Asn Met Thr His Ile Ile Lys Asp Val Ile Lys         195 200 205 Tyr Lys Glu Gly Ala Val Asp Asp Ile Phe Val Glu Ala Cys Gln Gly     210 215 220 Pro Ser Met Ala Asp Val Val Phe Leu Leu Asp Met Ser Ile Asn Gly 225 230 235 240 Ser Glu Glu Asn Phe Asp Tyr Leu Lys Gly Phe Leu Glu Glu Ser Val                 245 250 255 Ser Ala Leu Asp Ile Lys Glu Asn Cys Met Arg Val Gly Leu Val Ala             260 265 270 Tyr Ser Asn Glu Thr Lys Val Ile Asn Ser Leu Ser Met Gly Ile Asn         275 280 285 Lys Ser Glu Val Leu Gln His Ile Gln Asn Leu Ser Pro Arg Thr Gly     290 295 300 Lys Ala Tyr Thr Gly Ala Ala Ile Lys Lys Leu Arg Lys Glu Val Phe 305 310 315 320 Ser Ala Arg Asn Gly Ser Arg Lys Asn Gln Gly Val Pro Gln Ile Ala                 325 330 335 Val Leu Val Thr His Arg Asp Ser Glu Asp Asn Val Thr Lys Ala Ala             340 345 350 Val Asn Leu Arg Arg Glu Gly Val Thr Ile Phe Thr Leu Gly Ile Glu         355 360 365 Gly Ala Ser Asp Thr Gln Leu Glu Lys Ile Ala Ser His Pro Ala Glu     370 375 380 Gln Tyr Val Ser Lys Leu Lys Thr Phe Ala Asp Leu Ala Ala His Asn 385 390 395 400 Gln Thr Phe Leu Lys Lys Leu Arg Asn Gln Ile Thr His Thr Val Ser                 405 410 415 Val Phe Ser Glu Arg Thr Glu Thr Leu Lys Ser Gly Cys Val Asp Thr             420 425 430 Glu Glu Ala Asp Ile Tyr Leu Leu Ile Asp Gly Ser Gly Ser Thr Gln         435 440 445 Ala Thr Asp Phe His Glu Met Lys Thr Phe Leu Ser Glu Val Val Gly     450 455 460 Met Phe Asn Ile Ala Pro His Lys Val Arg Val Gly Ala Val Gln Tyr 465 470 475 480 Ala Asp Ser Trp Asp Leu Glu Phe Glu Ile Asn Lys Tyr Ser Asn Lys                 485 490 495 Gln Asp Leu Gly Lys Ala Ile Glu Asn Ile Arg Gln Met Gly Gly Asn             500 505 510 Thr Asn Thr Gly Ala Ala Leu Asn Phe Thr Leu Ser Leu Leu Gln Lys         515 520 525 Ala Lys Lys Gln Arg Gly Asn Lys Val Pro Cys His Leu Val Val Leu     530 535 540 Thr Asn Gly Met Ser Lys Asp Ser Ile Leu Glu Pro Ala Asn Arg Leu 545 550 555 560 Arg Glu Glu His Ile Arg Val Tyr Ala Ile Gly Ile Lys Glu Ala Asn                 565 570 575 Gln Thr Gln Leu Arg Glu Ile Ala Gly Glu Glu Lys Arg Val Tyr Tyr             580 585 590 Val His Asp Phe Asp Ala Leu Lys Asp Ile Arg Asn Gln Val Val Gln         595 600 605 Glu Ile Cys Thr Glu Glu Ala Cys Lys Glu Met Lys Ala Asp Ile Met     610 615 620 Phe Leu Val Asp Ser Ser Gly Ser Ile Gly Pro Glu Asn Phe Ser Lys 625 630 635 640 Met Lys Thr Phe Met Lys Asn Leu Val Ser Lys Ser Gln Ile Gly Pro                 645 650 655 Asp Arg Val Gln Ile Gly Val Val Gln Phe Ser Asp Ile Asn Lys Glu             660 665 670 Glu Phe Gln Leu Asn Arg Phe Met Ser Gln Ser Asp Ile Ser Asn Ala         675 680 685 Ile Asp Gln Met Ala His Ile Gly Gln Thr Thr Leu Thr Gly Ser Ala     690 695 700 Leu Ser Phe Val Ser Gln Tyr Phe Ser Pro Thr Lys Gly Ala Arg Pro 705 710 715 720 Asn Ile Arg Lys Phe Leu Ile Leu Ile Thr Asp Gly Glu Ala Gln Asp                 725 730 735 Ile Val Lys Glu Pro Ala Val Val Leu Arg Gln Glu Gly Val Ile Ile             740 745 750 Tyr Ser Val Gly Val Phe Gly Ser Asn Val Thr Gln Leu Glu Glu Ile         755 760 765 Ser Gly Arg Pro Glu Met Val Phe Tyr Val Glu Asn Phe Asp Ile Leu     770 775 780 Gln Arg Ile Glu Asp Asp Leu Val Phe Gly Ile Cys Ser Pro Arg Glu 785 790 795 800 Glu Cys Lys Arg Ile Glu Val Leu Asp Val Val Phe Val Ile Asp Ser                 805 810 815 Ser Gly Ser Ile Asp Tyr Asp Glu Tyr Asn Ile Met Lys Asp Phe Met             820 825 830 Ile Gly Leu Val Lys Lys Ala Asp Val Gly Lys Asn Gln Val Arg Phe         835 840 845 Gly Ala Leu Lys Tyr Ala Asp Asp Pro Glu Val Leu Phe Tyr Leu Asp     850 855 860 Asp Phe Gly Thr Lys Leu Glu Val Ile Ser Val Leu Gln Asn Asp Gln 865 870 875 880 Ala Met Gly Gly Ser Thr Tyr Thr Ala Glu Ala Leu Gly Phe Ser Asp                 885 890 895 His Met Phe Thr Glu Ala Arg Gly Ser Arg Leu Asn Lys Gly Val Pro             900 905 910 Gln Val Leu Ile Val Ile Thr Asp Gly Glu Ser His Asp Ala Asp Lys         915 920 925 Leu Asn Ala Thr Ala Lys Ala Leu Arg Asp Lys Gly Ile Leu Val Leu     930 935 940 Ala Val Gly Ile Asp Gly Ala Asn Pro Val Glu Leu Leu Ala Met Ala 945 950 955 960 Gly Ser Ser Asp Lys Tyr Phe Phe Val Glu Thr Phe Gly Gly Leu Lys                 965 970 975 Gly Ile Phe Ser Asp Val Thr Ala Ser Val Cys Asn Ser Ser Lys Val             980 985 990 Asp Cys Glu Ile Asp Lys Val Asp Leu Val Phe Leu Met Asp Gly Ser         995 1000 1005 Thr Ser Ile Gln Pro Asn Asp Phe Lys Lys Met Lys Glu Phe Leu Ala    1010 1015 1020 Ser Val Val Gln Asp Phe Asp Val Ser Leu Asn Arg Val Arg Ile Gly 1025 1030 1035 1040 Ala Ala Gln Phe Ser Asp Thr Tyr His Pro Glu Phe Pro Leu Gly Thr                1045 1050 1055 Phe Ile Gly Glu Lys Glu Ile Ser Phe Gln Ile Glu Asn Ile Lys Gln            1060 1065 1070 Ile Phe Gly Asn Thr His Ile Gly Ala Ala Leu Arg Glu Val Glu His        1075 1080 1085 Tyr Phe Arg Pro Asp Met Gly Ser Arg Ile Asn Thr Gly Thr Pro Gln    1090 1095 1100 Val Leu Leu Val Leu Thr Asp Gly Gln Ser Gln Asp Glu Val Ala Gln 1105 1110 1115 1120 Ala Ala Glu Ala Leu Arg His Arg Gly Ile Asp Ile Tyr Ser Val Gly                1125 1130 1135 Ile Gly Asp Val Asp Asp Gln Gln Leu Ile Gln Ile Thr Gly Thr Ala            1140 1145 1150 Glu Lys Lys Leu Thr Val His Asn Phe Asp Glu Leu Lys Lys Val Asn        1155 1160 1165 Lys Arg Ile Val Arg Asn Ile Cys Thr Thr Ala Gly Glu Ser Asn Cys    1170 1175 1180 Phe Val Asp Val Val Val Gly Phe Asp Val Ser Thr Gln Glu Lys Gly 1185 1190 1195 1200 Gln Thr Leu Leu Glu Gly Gln Pro Trp Met Glu Thr Tyr Leu Gln Asp                1205 1210 1215 Ile Leu Arg Ala Ile Ser Ser Leu Asn Gly Val Ser Cys Glu Val Gly            1220 1225 1230 Thr Glu Thr Gln Val Ser Val Ala Phe Gln Val Thr Asn Ala Met Glu        1235 1240 1245 Lys Tyr Ser Pro Lys Phe Glu Ile Tyr Ser Glu Asn Ile Leu Asn Ser    1250 1255 1260 Leu Lys Asp Ile Thr Val Lys Gly Pro Ser Leu Leu Asn Ala Asn Leu 1265 1270 1275 1280 Leu Asp Ser Leu Trp Asp Thr Phe Gln Asn Lys Ser Ala Ala Arg Gly                1285 1290 1295 Lys Val Val Leu Leu Phe Ser Asp Gly Leu Asp Asp Asp Val Glu Lys            1300 1305 1310 Leu Glu Gln Lys Ser Asp Glu Leu Arg Lys Glu Gly Leu Asn Ala Leu        1315 1320 1325 Ile Thr Val Ala Leu Asp Gly Pro Ala Asp Ser Ser Asp Leu Ala Asp    1330 1335 1340 Leu Pro Tyr Ile Glu Phe Gly Lys Gly Phe Glu Tyr Arg Thr Gln Leu 1345 1350 1355 1360 Ser Ile Gly Met Arg Glu Leu Gly Ser Arg Leu Ser Lys Gln Leu Val                1365 1370 1375 Asn Val Ala Glu Arg Thr Cys Cys Cys Leu Phe Cys Lys Cys Ile Gly            1380 1385 1390 Gly Asp Gly Thr Met Gly Asp Pro Gly Pro Pro Gly Lys Arg Gly Pro        1395 1400 1405 Pro Gly Phe Lys Gly Ser Glu Gly Tyr Leu Gly Glu Glu Gly Ile Ala    1410 1415 1420 Gly Glu Arg Gly Ala Pro Gly Pro Val Gly Glu Gln Gly Thr Lys Gly 1425 1430 1435 1440 Cys Tyr Gly Thr Lys Gly Pro Lys Gly Asn Arg Gly Leu Asn Gly Gln                1445 1450 1455 Glu Gly Glu Val Gly Glu Asn Gly Ile Asp Gly Leu Asn Gly Glu Gln            1460 1465 1470 Gly Asp Asn Gly Leu Pro Gly Arg Lys Gly Glu Lys Gly Asp Glu Gly        1475 1480 1485 Ser Gln Gly Ser Pro Gly Lys Arg Gly Thr Pro Gly Asp Arg Gly Ala    1490 1495 1500 Lys Gly Leu Arg Gly Asp Pro Gly Ala Pro Gly Val Asp Ser Ser Ile 1505 1510 1515 1520 Glu Gly Pro Thr Gly Leu Lys Gly Glu Arg Gly Arg Gln Gly Arg Arg                1525 1530 1535 Gly Trp Pro Gly Pro Pro Gly Thr Pro Gly Ser Arg Arg Lys Thr Ala            1540 1545 1550 Ala His Gly Arg Arg Gly His Thr Gly Pro Gln Gly Thr Ala Gly Ile        1555 1560 1565 Pro Gly Pro Asp Gly Leu Glu Gly Ser Leu Gly Leu Lys Gly Pro Gln    1570 1575 1580 Gly Pro Arg Gly Glu Ala Gly Val Lys Gly Glu Lys Gly Gly Val Gly 1585 1590 1595 1600 Ser Lys Gly Pro Gln Gly Pro Pro Gly Pro Gly Gly Glu Ala Gly Asn                1605 1610 1615 Gln Gly Arg Leu Gly Ser Gln Gly Asn Lys Gly Glu Pro Gly Asp Leu            1620 1625 1630 Gly Glu Lys Gly Ala Val Gly Phe Pro Gly Pro Arg Gly Leu Gln Gly        1635 1640 1645 Asn Asp Gly Ser Pro Gly Tyr Gly Ser Val Gly Arg Lys Gly Ala Lys    1650 1655 1660 Gly Gln Glu Gly Phe Pro Gly Glu Ser Gly Pro Lys Gly Glu Ile Gly 1665 1670 1675 1680 Asp Pro Gly Gly Pro Gly Glu Thr Gly Leu Lys Gly Ala Arg Gly Lys                1685 1690 1695 Met Ile Ser Ala Gly Leu Pro Gly Glu Met Gly Ser Pro Gly Glu Pro            1700 1705 1710 Gly Pro Pro Gly Arg Lys Gly Val Lys Gly Ala Lys Gly Leu Ala Ser        1715 1720 1725 Phe Ser Thr Cys Glu Leu Ile Gln Tyr Val Arg Asp Arg Ser Pro Gly    1730 1735 1740 Arg His Gly Lys Pro Glu Cys Pro Val His Pro Thr Glu Leu Val Phe 1745 1750 1755 1760 Ala Leu Asp His Ser Arg Asp Val Thr Glu Gln Glu Phe Glu Arg Met                1765 1770 1775 Lys Glu Met Met Ala Phe Leu Val Arg Asp Ile Lys Val Arg Glu Asn            1780 1785 1790 Ser Cys Pro Val Gly Ala His Ile Ala Ile Leu Ser Tyr Asn Ser His        1795 1800 1805 Ala Arg His Leu Val Arg Phe Ser Asp Ala Tyr Lys Lys Ser Gln Leu    1810 1815 1820 Leu Arg Glu Ile Glu Thr Ile Pro Tyr Glu Arg Ser Ser Ala Ser Arg 1825 1830 1835 1840 Glu Ile Gly Arg Ala Met Arg Phe Ile Ser Arg Asn Val Phe Lys Arg                1845 1850 1855 Thr Leu Pro Gly Ala His Thr Arg Lys Ile Ala Thr Phe Phe Ser Ser            1860 1865 1870 Gly Gln Ser Ala Asp Ala His Ser Ile Thr Thr Ala Ala Met Glu Phe        1875 1880 1885 Gly Ala Leu Glu Ile Ile Pro Val Val Ile Thr Phe Ser Asn Val Pro    1890 1895 1900 Ser Val Arg Arg Ala Phe Ala Ile Asp Asp Thr Gly Thr Phe Gln Val 1905 1910 1915 1920 Ile Val Val Pro Ser Gly Ala Asp Tyr Ile Pro Ala Leu Glu Arg Leu                1925 1930 1935 Gln Arg Cys Thr Phe Cys Tyr Asp Val Cys Lys Pro Asp Ala Ser Cys            1940 1945 1950 Asp Gln Ala Arg Pro Pro Pro Val Gln Ser Tyr Met Asp Ala Ala Phe        1955 1960 1965 Leu Leu Asp Ala Ser Arg Asn Met Gly Ser Ala Glu Phe Glu Asp Ile    1970 1975 1980 Arg Ala Phe Leu Gly Ala Leu Leu Asp His Phe Glu Ile Thr Pro Glu 1985 1990 1995 2000 Pro Glu Thr Ser Val Thr Gly Asp Arg Val Ala Leu Leu Ser His Ala                2005 2010 2015 Pro Pro Asp Phe Leu Pro Asn Thr Gln Lys Ser Pro Val Arg Ala Glu            2020 2025 2030 Phe Asn Leu Thr Thr Tyr Arg Ser Lys Arg Leu Met Lys Arg His Val        2035 2040 2045 His Glu Ser Val Lys Gln Leu Asn Gly Asp Ala Phe Ile Gly His Ala    2050 2055 2060 Leu Gln Trp Thr Leu Asp Asn Val Phe Leu Ser Thr Pro Asn Leu Arg 2065 2070 2075 2080 Arg Asn Lys Val Ile Phe Val Ile Ser Ala Gly Glu Thr Ser His Leu                2085 2090 2095 Asp Gly Glu Ile Leu Lys Lys Glu Ser Leu Arg Ala Lys Cys Gln Gly            2100 2105 2110 Tyr Ala Leu Phe Val Phe Ser Leu Gly Pro Ile Trp Asp Asp Lys Glu        2115 2120 2125 Leu Glu Asp Leu Ala Ser His Pro Leu Asp His His Leu Val Gln Leu    2130 2135 2140 Gly Arg Ile His Lys Pro Asp His Ser Tyr Gly Val Lys Phe Val Lys 2145 2150 2155 2160 Ser Phe Ile Asn Ser Ile Arg Arg Ala Ile Asn Lys Tyr Pro Pro Ile                2165 2170 2175 Asn Leu Lys Ile Lys Cys Asn Arg Leu Asn Ser Ile Asp Pro Lys Gln            2180 2185 2190 Pro Pro Arg Pro Phe Arg Ser Phe Val Pro Gly Pro Leu Lys Ala Thr        2195 2200 2205 Leu Lys Glu Asp Val Leu Gln Lys Ala Lys Phe Phe Gln Asp Lys Lys    2210 2215 2220 Tyr Leu Ser Arg Val Ala Arg Ser Gly Arg Asp Asp Ala Ile Gln Asn 2225 2230 2235 2240 Phe Met Arg Ser Thr Ser His Thr Phe Lys Asn Gly Arg Met Ile Glu                2245 2250 2255 Ser Ala Pro Lys Gln His Asp            2260

Claims (10)

A composition for diagnosing atopic dermatitis in Koreans comprising one or more monobasic polymorph detection agents selected from the group consisting of the GenBank SNP database rs16830494, rs200963433, and rs59021909, which are the single base polymorphism (SNP) sites of the COL6A6 gene.
The composition of claim 1, wherein the detection agent is a probe capable of detecting any one or more bases selected from the group consisting of rs16830494, rs200963433, and rs59021909.
The composition of claim 1, wherein the detection agent is a primer pair capable of amplifying any one or more bases selected from the group consisting of rs16830494, rs200963433 and rs59021909.
The primer pair according to claim 3, wherein the primer pair is a pair of base sequences represented by SEQ ID NO: 4 and SEQ ID NO: 5, a pair of base sequences represented by SEQ ID NO: 6 and SEQ ID NO: 7, and represented by SEQ ID NO: 8 and SEQ ID NO: 9. The composition characterized in that it is any one primer pair selected from the group consisting of pairs of base sequences.
The composition of claim 1, wherein the atopic dermatitis is hereditary atopic dermatitis.
The composition of claim 1, wherein the atopic dermatitis is an early-onset atopic dermatitis.
To provide information necessary for diagnosing atopic dermatitis in Koreans
Determining the base of a single nucleotide polymorphism (SNP) site of any one or more COL6A6 genes selected from the group consisting of rs16830494, rs200963433 and rs59021909 by analyzing the nucleotide sequence of the nucleic acid sample obtained from the subject
Method for detecting the polymorphism of the COL6A6 gene comprising a.
delete The method of claim 7, wherein the single base polymorphism site is determined as atopic dermatitis when the base of rs16830494 is A, the base of rs200963433 is T, and the base of rs59021909 is any one or more bases in the group consisting of T.
The method of claim 7, wherein the step of determining the base comprises sequencing, exome sequencing, microarray hybridization, allele specific PCR, dynamic allele hybridization. Method characterized by being performed by any one method selected from the group consisting of a technique (dynamic allele-specific hybridization), PCR extension analysis and Taqman technique.

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