WO2015190655A1 - Method for detecting filaggrin gene mutation for diagnosing atopic dermatitis - Google Patents

Abstract

The present invention relates to a probe complementarily binding to a filaggrin gene mutation nucleic acid sequence, and a method for detecting a filaggrin gene mutation using the same. Specifically, by preparing a PNA probe complementarily binding to a filaggrin gene and employing fluorescence melting curve analysis (FMCA) using a difference in binding strength between a normal type and a mutation type to provide the convenience of being able to determine a filaggrin gene mutation simply, promptly, and accurately, aetiological factors of atopic dermatitis patients can be diagnosed, and especially, the likelihood of atopic dermatitis during infancy immediately after birth can be promptly diagnosed, thereby decreasing the disease rate.

Description

Detection of Filaggrin Gene Mutations for Diagnosis of Atopic Dermatitis

The present invention relates to a method and kit for detecting a filaggrin gene mutation for diagnosing atopic dermatitis.

In recent years, more than 1 million patients have been treated for atopic dermatitis, and children under 9 years of age are close to half of all patients. Is reaching. Therefore, the development of a simple and accurate method for diagnosing atopic dermatitis applicable to infants and young children is required.

The skin is located at the outermost part of the body anatomically, blocking the direct inflow of pathogenic microorganisms, viruses, and chemicals in the air to protect the body from the external environment and to prevent excessive leakage of the body's moisture. 'Is doing. Filaggrin protein is expressed in the uppermost and epithelial layers, including the stratum corneum in the skin, and is one of the major proteins indispensable for the formation of the "barrier function" unique to the skin described above.

In recent years, atopic dermatitis has been largely divided into extrinsic and intrinsic (Tokura Y (2010) Extrinsic and intrinsic types of atopic). dermatitis, J. Dermatol. Sci. 58, 1-7). In the case of intrinsic atopy, the etiological factors are mainly inside the human body, which impairs the immune function, resulting in abnormal immune and inflammatory reactions on the skin. In the case of exogenous atopy, skin barrier function is caused by genetic inclination or environmental factors. This damage occurs when external pathogenic microorganisms, viruses and chemicals are directly introduced into the body, resulting in immune and inflammatory reactions of the skin. As such, there are various etiological predispositions to a single disease, and according to the etiological findings, there is an urgent need to establish a treatment method for each patient, that is, a need for different treatment methods.

On the other hand, recently, European researchers have first identified a correlation between atopic dermatitis and mutations in the filaggrin gene (Palmer CN et al. (2006) Common loss-of-function variants of the epidermal barrier protein filaggrin a). Major predisposing factor of atopic dermatits, Nat. Genet. 38, 441-6.), particularly in Europe, has detected mutations in the filaggrin gene in more than half of all atopic patients. Subsequently, over 25% of atopic patients in Japan were found to have mutations in the filaggrin gene (Osawa R et al. (2010) Japanese-specific Filaggrin gene mutations in Japanese patients suffering from Atopic Eczema and Ashthma, J. Invest) Dermatol. 130, 2834-6.), Mutations in the filaggrin gene have been detected in patients with atopic dermatitis in Asia, including China and Korea (Osawa R et al. (2011) Filaggrin gene defects and the risk of developing allergic disorders, Allergology International 60, 1-9.).

Among the findings of the study of mutations in the filaggrin gene, it is remarkable that the positions of mutations found by race and region differ significantly from one another (Osawa R et al. (2011) Filaggrin gene defects and the risk of developing allergic disorders, Allergology International 60, 1-9.). The results of the previous studies show that the location of mutations varies considerably between races and regions, with the exception of the R501X and E2422X mutations commonly found in Europeans and Asians, and 3321delA commonly found in East Asian Koreans and Chinese Japanese. It turns out that there is.

In Korea, however, even though the annual average number of patients is more than 1 million, there is no clear statistical data on the Filagrin gene mutation. Therefore, it is necessary to develop a simple and rapid method for detecting the Filagrin gene mutation.

PNA, which stands for peptide nucleic acids, is one of the substances that recognizes and binds to DNA sequences of genes, such as LNA (Locked Nucleic Acid) and MNA (Mopholino Nucleic Acid), and can be synthesized artificially. Consists of. The main reason for using PNA to detect mutations in genes is their high binding affinity and selectivity with DNA. That is, the binding force between PNA and DNA is much better than that between DNA (DNA-DNA), so even if only one base is mismatched between the PNA probe and the DNA sequence complementarily binding thereto (10-15 ° C) There is a big difference in the melting temperature (Telting Temperature = Tm) value. By using the difference in the Tm value (ie, the difference in binding capacity), it is possible to easily detect the change in the base (sequence) of the SNP and the mutation, and the development of a detection kit using the same is also easy. Other advantages include high stability against nucleases, which do not degrade with existing restriction enzymes. Thermal and chemical properties and stability make it easy to store.

In addition, PNA probes are very simple to design, unlike other well-known probes (TaqMan probes, Molecular beancon probes, and Scorpion probes), and typically contain 11-18 mer bases containing SNPs or mutation sites. Construct using sequences. In particular, it is possible to design a probe with a specific melting temperature (Tm) value desired. 1) Adjust the melting temperature (Tm) value according to the length of the PNA probe, or 2) Change the probe even with the same length of PNA probe. You can adjust the (Tm) value. PNA has better binding ability than DNA, so the basic melting temperature (Tm) value is high, so it can be designed to be shorter than DNA, and even near neighboring SNPs and mutations are very easy to detect.

Accordingly, the present inventors have made efforts to develop a method for diagnosing atopic dermatitis and a diagnostic kit. As a result, the present inventors have developed a PNA probe that selectively binds to a filaggrin gene wild type or a mutation and has a specific melting temperature (Tm) value, and measures the melting temperature thereof. The present invention was completed by revealing that atopic dermatitis can be diagnosed by simply and quickly detecting a filaggrin gene mutation.

(Prior literature)

(Non-Patent Document 1) Palmer CN et al. (2006) Common loss-of-function variants of the epidermal barrier protein filaggrin a major predisposing factor of atopic dermatits, Nat. Genet. 38, 441-6.

(Non-Patent Document 2) Osawa R et al. (2010) Japanese-specific Filaggrin gene mutations in Japanese patients suffering from Atopic Eczema and Ashthma, J. Invest. Dermatol. 130, 2834-6.

(Non-Patent Document 3) Osawa R et al. (2011) Filaggrin gene defects and the risk of developing allergic disorders, Allergology International 60, 1-9.

An object of the present invention was to develop a PNA probe that complementarily binds to a filaggrin gene wild type or mutant nucleic acid sequence, and detects the pilaggrin gene mutation by measuring the difference in melting temperature of the wild type hybridization product and the mutant hybridization product thereof. To provide a method for diagnosing atopic dermatitis and a diagnostic kit.

In order to achieve the above object,

The present invention

Amplifying the filaggrin gene region included in the specimen to obtain a PCR product;

Hybridizing with the PCR product using a PNA probe for detecting base mutations having a reporter that complementarily binds to the filaggrin gene region to obtain a hybridized product;

Varying the temperature of the hybridization product and measuring fluorescence in real time to obtain a melting curve; And

Determining the mutation or normal form of the filaggrin gene included in the sample by measuring the melting temperature difference or the melting curve peak value with the wild-type gene hybridization product caused by mismatch by the filaggrin gene mutation in the melting curve. It provides a filaggrin gene mutation detection method comprising a.

The present invention also provides a kit for diagnosing atopic dermatitis, comprising a primer for amplifying a nucleotide sequence including part or all of a filaggrin gene from a sample sample, and a PNA probe for detecting a pilaggrin gene mutation.

The PNA probe complementary to the filaggrin gene mutant nucleic acid sequence of the present invention and the method for detecting the filaggrin gene mutation using the same provide a convenient, quick and accurate method for discriminating the filaggrin base mutation. To facilitate the diagnosis of dermatitis patients.

1 is a diagram showing a PCR reaction for detecting a mutation of the filaggrin gene and a PCR condition for FMCA analysis of the PNA probe.

Figure 2 is a diagram showing the PNA fusion curve of mutant dogs and normal individuals for the filaggrin gene, the solid line represents the normal form, the dotted line represents the heterozygote mutation, the thick solid line represents the homozygote mutation.

Hereinafter, the present invention will be described in detail.

The present invention

To provide you with the information you need to diagnose atopic dermatitis,

Amplifying the filaggrin gene region included in the specimen to obtain a PCR product;

Hybridizing with the PCR product using a PNA probe for detecting base mutations having a reporter that complementarily binds to the filaggrin gene region to obtain a hybridized product;

Varying the temperature of the hybridization product and measuring fluorescence in real time to obtain a melting curve; And

Detecting the pilagrin gene mutation, characterized in that for determining the mutation or normal form of the pilagrin gene included in the sample by measuring the difference in the melting curve with the subtypes caused by mismatch by the mutation of the hybridization product Provide a method.

The determining step,

The melting curve peak value of the hybridization product was compared with the melting curve value of the hybridization product of the wild-type filaggrin gene and the PNA probe, and the homozygous filaggrin gene mutation, the peak low temperature, and the same temperature when the peak was observed only at low temperature. If observed in heterozygous mutant, if the peak is observed only at the same temperature may be determined as a wild type, in detail the fluorescence melting curve to analyze the difference in binding temperature between the PCR product and PNA probe as the difference in the melting temperature (Tm) value Fluorescence Melting Curve Analysis (FMCA) is used.

The sample may be epithelial cells in the oral cavity, blood, saliva, plasma or a solution containing the same, or a compound, and the like, and may be any bio-derived material known to be capable of amplifying gene sequences in the art.

The PCR product may be oligonucleotide, DNA, RNA, and includes all the mixtures including the same, preferably, the size of the PCR product is less than 1000bp.

The filaggrin gene of NCBI RefSeq NM_002016 (SEQ ID NO: 17) The human propyllagrin gene represented by the nucleotide sequence, or a sequence having the same activity or the same gene position on the chromosome, one or several bases are added, deleted, or substituted.

The filaggrin gene is at least 80% homology to the base sequence of NCBI RefSeq NM_002016 (SEQ ID NO: 17), more specifically at least 90% homology, most specifically 95%, 96%, 97%, 98%, 99% or It consists of a nucleotide sequence having a homology of 99.5% or more.

The PNA probe is preferably designed so that the melting temperature (Tm) at the time of hybridization with the mutant gene is 5 ° C. or more than that of the normal case.

The PNA probe may be for detecting pilaggrin gene mutations, and filaggrin gene mutations are described in Sandilands et al. (2007) Comprehensive analysis of the gene encoding filaggrin uncovers prevalent and rare mutations in ichthyosis vulgaris and atopic eczema, Nature K4022X, K4021X, K4671X, 3321delA, S2554X, S2889X, S3296X, R501X, 2282del4, 3673delC, 3702delG, R1474X, 5360delG, 6867delAG, E2422X, 72447X, based on Genetics 39, 650-654. , S3247X, 11029delCA, 11033del4, Q3683X, 441delA, 1249insG, S1695X, Q1701X, E1795X, Q2417X, 7945delA, or R4307X, but are not limited thereto, and include all genes encoding homologues, variants, derivatives, and the like. And any filaggrin gene mutation that can cause dermatitis.

The pillar green (filaggrin) gene mutation K4022X, K4021X, K4671X, 3321delA, S2554X, S2889X, S3296X, R501X, 2282del4, 3673delC, 3702delG, R1474X, 5360delG, 6867delAG, E2422X, 7267delCA, R2447X, S3247X, 11029delCA, 11033del4, Q3683X, 441delA, 1249insG, S1695X, Q1701X, E1795X, Q2417X, 7945delA, or R4307X have at least 80% homology, more specifically at least 90% homology, most specifically 95%, 96%, 97%, It consists of a nucleotide sequence having at least 98%, 99% or 99.5% homology.

The PNA probe may be substituted with an oligonucleotide probe, DNA probe, RNA probe, Taqman probe, LNA probe, MNA probe having the same nucleotide sequence.

The PNA probe may include any one of SEQ ID NO: 11 to SEQ ID NO: 16, but is not limited thereto, and one or more bases may be added, as long as it can bind complementarily to the filaggrin mutant sequence. Deleted, including all substituted nucleotide sequences.

In one embodiment, the PNA probe was prepared by a manufacturing method according to Korean Patent No. 464261.

The reporter (detector) may be a conjugate labeled with a chromophore, a fluorescent substance, a radioisotope or a colloid, and preferably a secondary antibody capable of complementarily binding to the PNA probe. For example, the chromase may be peroxidase, alkaline phosphatase or acid phosphatase (eg horseradish peroxidase); In the case of fluorescent materials, colloid gold, fluorescein carboxylic acid (FCA), fluorescein isothiocyanate (FITC), Rhodamine-B-isothiocyanate (RITC), fluorescein thiourea (FTH), 7 -Acetoxycoumarin-3-yl, fluorescein-5-yl, fluorescein-6-yl, 2 ', 7'-dichlorofluorescin-5-yl, 2', 7'-dichlorofluorescein -6-yl, dihydrotetramethyllosamine-4-yl, tetramethyldamin-5-yl, tetramethyldamin-6-yl, 4,4-difluoro-5,7-dimethyl-4- Bora-3a, 4a-diaza-s-indacene-3-ethyl or 4,4-difluoro-5,7-diphenyl-4-bora-3a, 4a-diaza-s-indacene-3 It is possible to use ethyl and the like, but is not limited thereto.

The PNA probe may be labeled with a reporter and a quencher, and such quencher may be dabcyl. Preferably, the PNA probe is labeled terminally with quencher, and most preferably, the PNA probe can be labeled C-terminally with Davyl. It is also commonly linked to the N-ε-amino group of the C-terminal lysine amino acid via methods known in the art.

The PNA probe is formed on a support such as glass slide, silica, semiconductor, plastic, gold, silver, magnetic material, nylon, polymer compound of poly (dimethylsiloxane, PDMS), cellulose or nitrocellulose, glass, etc. Immobilization is possible. The shape of the support is not particularly limited, and is not only a thin plate that can be held by hand, for example, a glass slide, but also a tube or a bead of 0.1 mm or less that can be mixed and transported in a liquid. Can be. The surface of the support can be functionalized with functional groups such as aldehyde groups, carboxyl groups, epoxy groups, isothiocyanate groups, N-hydroxysuccinimidyl groups, activated ester groups, in particular epoxy groups. After the PNA probe is immobilized on the support, functional groups such as residual amine and epoxy group may be stabilized through a blocking step.

The PNA probe can be immobilized in a plurality of, for example four or eight, independent regions on the support to detect a plurality of, for example four or eight, samples simultaneously.

The PNA probe may include a plurality of probes having different nucleotide sequences coupled with different reporters to simultaneously detect a plurality of filaggrin gene mutations, and the PNA probe independently distinguishes between the filaggrin gene wild type and the mutation. There may be multiple PNA probes to

The detection method may be for diagnosing atopic dermatitis, preferably for diagnosing exogenous atopic dermatitis.

The present invention also provides a kit for detecting a pilaggrin gene mutation comprising a PNA probe that complementarily binds to a pilaggrin gene mutant nucleic acid sequence.

The kit includes a means for PCR amplifying a filaggrin gene in a specimen containing a primer, DNA polymerase, or RNA polymerase, dNTPs, and a PCR buffer for amplifying the filaggrin gene sequence, and after the PCR amplification, the filaggrin Means for detecting whether the amplified filaggrin gene sequence and the hybridization of the probe using a PNA probe specific for the gene mutation sequence.

The primer may be any one selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 10, but is not limited thereto, and includes all sequences capable of complementarily binding to the filaggrin gene sequence.

The PNA probe is preferably designed so that the melting temperature (Tm) at the time of hybridization with the mutant gene is 5 ° C. or more than that of the normal case.

The PNA probe may be substituted with an oligonucleotide probe, DNA probe, RNA probe, Taqman probe, LNA probe, MNA probe having the same nucleotide sequence.

The PNA probe may be a probe including any one of SEQ ID NO: 11 to SEQ ID NO: 16, but is not limited thereto, and includes all sequences capable of complementarily binding to a filaggrin gene mutant sequence.

In one embodiment, the PNA probe is labeled with a reporter and a quencher, which may be dabcyl. Preferably, the PNA is labeled terminally with quencher, and most preferably, the PNA is labeled C-terminally with Davyl. It is also commonly linked to the N-ε-amino group of the C-terminal lysine amino acid via methods known in the art.

In one embodiment, the PNA probe may be associated with a reporter (detector). The reporter (detector) may be a conjugate labeled with a chromophore, a fluorescent substance, a radioisotope or a colloid, and preferably a secondary antibody capable of complementarily binding to the PNA probe. For example, the chromase may be peroxidase, alkaline phosphatase or acid phosphatase (eg horseradish peroxidase); In the case of fluorescent materials, colloid gold, fluorescein carboxylic acid (FCA), fluorescein isothiocyanate (FITC), Rhodamine-B-isothiocyanate (RITC), fluorescein thiourea (FTH), 7 -Acetoxycoumarin-3-yl, fluorescein-5-yl, fluorescein-6-yl, 2 ', 7'-dichlorofluorescin-5-yl, 2', 7'-dichlorofluorescein -6-yl, dihydrotetramethyllosamine-4-yl, tetramethyldamin-5-yl, tetramethyldamin-6-yl, 4,4-difluoro-5,7-dimethyl-4- Bora-3a, 4a-diaza-s-indacene-3-ethyl or 4,4-difluoro-5,7-diphenyl-4-bora-3a, 4a-diaza-s-indacene-3 It is possible to use ethyl and the like, but is not limited thereto.

The PNA probe may be for detecting pilaggrin gene mutations, and filaggrin gene mutations are described in Sandilands et al. (2007) Comprehensive analysis of the gene encoding filaggrin uncovers prevalent and rare mutations in ichthyosis vulgaris and atopic eczema, Nature K4022X, K4021X, K4671X, 3321delA, S2554X, S2889X, S3296X, R501X, 2282del4, 3673delC, 3702delG, R1474X, 5360delG, 6867delAG, E2422X, 72447X, based on Genetics 39, 650-654. , S3247X, 11029delCA, 11033del4, Q3683X, 441delA, 1249insG, S1695X, Q1701X, E1795X, Q2417X, 7945delA, or R4307X, but are not limited thereto, and include all genes encoding homologues, variants, derivatives, and the like. And any filaggrin gene mutation that can cause dermatitis.

The pillar green (filaggrin) gene mutation K4022X, K4021X, K4671X, 3321delA, S2554X, S2889X, S3296X, R501X, 2282del4, 3673delC, 3702delG, R1474X, 5360delG, 6867delAG, E2422X, 7267delCA, R2447X, S3247X, 11029delCA, 11033del4, Q3683X, 441delA, 1249insG, S1695X, Q1701X, E1795X, Q2417X, 7945delA, or R4307X have at least 80% homology, more specifically at least 90% homology, most specifically 95%, 96%, 97%, It consists of a nucleotide sequence having at least 98%, 99% or 99.5% homology.

The kit may be for diagnosing atopic dermatitis, preferably for diagnosing exogenous atopic dermatitis.

The kit is a method for measuring the antigen-antibody response of the antigen (or sequence) -antibody complex or a method for measuring the amount of the detector after treating the antigen to the antigen-antibody complex, thereby filaggrin Genetic mutations can be diagnosed. Examples of the antigen-antibody reaction include enzyme immunoassay (ELISA), immunoprecipitation, fluorescence immunoassay, enzyme substrate coloration, antigen-antibody aggregation, and the like. It may be through chemiluminescence, absorbance, reflection or transmission.

As a method of detecting the amount of the detector, a high throughput screening (HTS) system may be used, and a method of measuring a color reaction by treating a substrate inducing color development of a chromophore bound to the detector. , A fluorescence method performed by detecting a fluorescence by attaching a fluorescent substance to a detector, or a radiation method performed by detecting a radiation by attaching a radioisotope to a detector, and an SPR measuring a plasmon resonance change of a surface in real time without a label of the detector ( Surface plasmon resonance (SPRI) method or surface plasmon resonance imaging (SPRI) method to verify by imaging the SPR system, Fluorescence Melting Curve Analysis (Fluorescence Melting Curve Analysis, FMCA) is preferably used, but is not limited thereto.

The kit may further include a wash solution for removing the remaining substances after the antigen-antibody binding reaction and the binding reaction of the detector. The wash solution preferably comprises phosphate buffer, NaCl and Tween 20, and a buffer solution (PBST) consisting of 0.02 M phosphate buffer, 0.13 M NaCl, and 0.05% Tween 20 is more preferred. Preferably, but not limited to. After washing the antigen-antibody-binding reaction, the washing solution reacts the detector with the antigen-antibody conjugate, and then washes 3 to 6 times by adding an appropriate amount to the fixture. The reaction terminating solution may preferably be sulfuric acid solution (H 2 SO 4), but is not limited thereto.

The kit may further comprise a positive control comprising a filaggrin gene mutation or a negative control comprising a filaggrin wild type gene.

The filaggrin gene mutation diagnostic kit includes a reverse transcription polymerase chain reaction (RT-PCR) kit, a DNA chip kit, an enzyme-linked immnosorbent assay (ELISA) kit, a sandwich ELISA kit, a protein chip kit, and a rapid kit. Or MRM (Multiple reaction monitoring) kits, and the like, but is not limited to this, if the method of complementary binding to the nucleotide sequence or protein can be made through any known method known to those skilled in the art.

In a specific embodiment of the present invention, in order to detect a filaggrin gene mutation, a sample amplified by a filaggrin gene region including a mutated portion using PCR was prepared.

In addition, in a specific embodiment of the present invention, a PNA probe having a length of about 10-20mers was prepared, and a probe having a desired melting temperature value could be manufactured through a change in length or sequence.

In a specific embodiment of the present invention, after hybridization (hybridization, hybridization) of the PNA probe attached to the fluorescent label and the specimen, the fluorescence melting curve analysis (Fluorescence Melting Curve analysis) to analyze the difference in the binding force as the difference in the melting temperature value Analysis, FMCA) was used, it was confirmed that the difference in melting temperature between the normal gene and the mutant gene (Fig. 2).

As used herein, the term "complementary" means not only that it is in a completely complementary relationship with the target nucleotide sequence, but also includes having a complementary relationship that can be hybridized with the target nucleotide sequence. do.

As used herein, the term "oligonucleotide" refers to a nucleic acid or a nucleotide sequence including both RNA and DNA, and includes all cDNA, genomic DNA, and synthetic DNA. In addition, the RNA includes all mRNA, rRNA, and synthetic RNA. In this specification, oligonucleotides, nucleic acids, and bases are used interchangeably.

Hereinafter, the present invention will be described in detail by way of examples.

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

Example 1 Preparation of Specimen Samples for Filaggrin Gene Base Mutation Detection

In order to use the PNA probe for the detection of SNPs or mutations in the gene, a PCR portion of the gene region containing the SNPs or mutations was amplified to obtain a PCR product (sample). At this time, the size of the PCR product should be less than about 1000 bp to facilitate the detection by the PNA probe, and if the existing primers and PCR conditions for PCR amplification can be used as it is.

Specifically, chromosomal DNA is extracted from human and human subjects, and PCR reactions are performed on the gene region including five kinds of filaggrin mutations 3321delA, K4022X (other nomenclature: K4021X, K4671X), S2554X, S2889X, and S3296X. A specimen sample was prepared. PCR reactions for each mutation are described by Osawa R et al. (2010) Japanese-specific Filaggrin gene mutations in Japanese patients suffering from Atopic Eczema and Ashthma, J. Invest.Dermatol . 130, 2834-6, Osawa R et al. (2011) Filaggrin gene defects and the risk of developing allergic disorders, Allergology International 60, 1-9), and were amplified using the primers listed in Table 1 below. Thereafter, the specimens containing the respective mutation sites were sequenced to identify the known mutation sites.

Table 1 PCR primers Mutant direction Sequence (5 → 3) 3321delA F GCTGATAATGTGATTCTGTCTG (SEQ ID NO: 1) R GACCCCGATGATTGTTCCTGT (SEQ ID NO: 2) K4022X F CTAGTACCGCTAAGGAACATGG (SEQ ID NO: 3) R TGGCTCCTTCGATATTTCTGA (SEQ ID NO: 4) S2554X F CCACACGTGGCCGGTCAGCA (SEQ ID NO: 5) R CTGGCTAAAACTGGATCCCCA (SEQ ID NO: 6) S2889X F GATGGACGGGGCCCAGCACTA (SEQ ID NO: 7) R CTTGGTGGCTCTGCTGATGGGA (SEQ ID NO: 8) S3296X F TCTGTTCAGGAGCAGTCAAGG (SEQ ID NO: 9) R GCTTCATGGTGATGCGACCA (SEQ ID NO: 10)

references

1) Nomura T et al. (2007) Unique mutations in the filaggrin gene in Japanese patients with ichthyosis vulgaris and atopic dermatitis, J. Allergy Clin. Immun. 119, 434-440.

2) Kim EJ et al. (2011) Genetic Polymorphism of FLG in Korean Ichthyosis Vulgaris Patients, Ann. Dermatol. 23, 170-176.

Example 2 Preparation of PNA Probe for Filaggrin Gene Mutation Detection

We have constructed a PNA probe for use in detecting filaggrin gene mutations.

Specifically, PNA probes were designed using PNA Probe Designer (Appliedbiosystems, USA). In order to easily detect the presence and absence of mutations in the specimen, the design was made by considering the melting temperature (Tm) of the PNA probe to be more than 5 ° C. different from the normal case. In addition, it was designed to avoid unnecessary secondary structure of the probe for more effective binding with the target nucleic acid, all PNA probes (FAM, HEX, Texas Red-labeled, Dabcyl) used in the present invention HPLC purification in Panagene (Panagene, Korea) It was synthesized by the method, and the purity of all synthesized probes was confirmed by mass spectrometry. The sequence of the PNA probe synthesized by the above method is shown in Table 2 below.

TABLE 2 PNA Probe Oligomer Sequences for Filaggrin Gene Mutation Identification Probe Sequence (5 '→ 3') K4022Xm (SEQ ID NO: 11) Dabcyl-GATCTTTACCAAAC-OK (FAM) 3321delA (SEQ ID NO: 12) Dabcyl-GACCTTCCCCCTGACC-OK (HEX) S2554X (SEQ ID NO: 13) Dabcyl-CCCCTCTGATTGTCC-OK (FAM) S2889Xnew (SEQ ID NO: 14) Dabcyl-ACTGGATCCCTGG-OK (HEX) S2889Xmut (SEQ ID NO: 15) Dabcyl-ACACTTCAACCCTG-OK (FAM) S3296X (SEQ ID NO: 16) Dabcyl-GCAAGATCAAGTCCA-OK (Texas Red)

* O means linker and K means lysine. In parentheses are fluorescent materials.

Example 3 Analysis of FMCA Dissolution Curves According to Filaggrin Gene Variation

The present inventors attached a fluorescent marker to the PNA probe to facilitate the analysis of the results. The hybridization method used a hybridization method to determine the difference in binding force between the PCR product (sample) and the PNA probe. Fluorescence Melting Curve Analysis (FMCA) was used. All of the commonly used quantitative PCR (real-time PCR) is equipped with the function to analyze the results as described above, if only the PNA probes made for the purpose is prepared, high resolution melting (HRM) and Detection of mutations is possible without the purchase of additional additional programs or equipment.

Specifically, the PCR reaction for melting curve analysis using a PNA probe was performed using a CFX96 ™ Real-Time System (BIO-RAD, USA), and all experimental conditions produced a single strand specimen. In order to perform asymmetric PCR (asymmetric PCR). 1X EyeBio Real-Time FMCA ™ Buffer (SeaBio, Korea), forward primer for 0.05 μM sample generation and reverse primer for 0.5 μM sample generation, one 0.5 μl PNA probe or S2889X Case 2 (S2889Xnew and S2889Xmut are used simultaneously), 1 μl of human chromosomal DNA (or diluted sample PCR products) were added to a total volume of 20 μl, followed by real-time PCR. That is, one detection PNA probe was used for four types of mutations 3321delA, K4022X (other nomenclature: K4021X, K4671X), S2554X, and S3296X, and in the case of S2889X, two PNA probes were used to independently distinguish between normal and mutations. S2889Xnew and S2889Xmut) were used simultaneously. In the real-time PCR process, the sample was denatured at 95 ° C. for 10 minutes, followed by 35 cycles of reaction for 45 seconds at 95 ° C., 30 seconds at 60 ° C., and 45 seconds at 74 ° C., and fluorescence was measured in real time. . Melting curve analysis was performed after 3 minutes of denaturation at 95 ° C, hybridized for 1 minute at 72 ° C, 1 minute at 60 ° C, and 1 minute at 50 ° C, and then eluted by increasing the temperature from 40 ° C to 80 ° C by 0.5 ° C. The melting temperature (Tm) value was obtained by measuring the fluorescence intensity of the PNA probe. Each step was held stationary for 5 seconds (FIG. 1).

 As a result, in the melting curve analysis after performing the asymmetric PCR, it was confirmed that the exact melting temperature (Tm) difference occurs between the normal type and the mismatched mutant type showing complete hybridization with the PNA probe (FIG. 2).

Thus, for heterozygote mutations, one of the homologous chromosomes corresponds to the mutant and the other is normal, so two peak curves are shown, as shown in the dotted fusion curves of the K4022X, 3321delA, and S2889X mutations. The left peak coming from the lower Tm value is the mutant type, and the right peak (the higher Tm value) overlapping with the normal peak of the solid line corresponds to the normal type.

In the case of Homozygote mutations, all pairs of homologous chromosomes are mutant, so as shown in the thick solid line melting curve (peak) of S2554X, S2889X, and S3296X (without overlapping with the normal peak of the solid line) ) All show only thick solid mutant peaks with low Tm values.

In addition, if homozygote mutations were detected in the K4022X and 3321delA mutants, only the left peak from the lower Tm value would be detected, such as S2554X and S3296X in the above figure. Type) mutations, such as K4022X and 3321delA in the figure above, the left peak (mutant) coming from the lower Tm value and the right peak (higher Tm value) overlapping the normal peak of the solid line Peaks can be detected. A good example is the S2889X, which performed both the heterozygote and homozygote mutations, and the analytical diagram shown at the bottom left of FIG. 2 detects the heterozygote mutation. And, the analysis diagram shown in the middle of the bottom of Figure 2 is an experimental example of detecting Homozygote (homozygote) mutation.

Table 3 below shows the melting temperature (Melting Temperature = Tm) of normal and mutant types obtained through PNA probe and FMCA analysis, and the number of mismatched bases.

TABLE 3 SNP count and melting temperature distribution of mutant and normal individuals for filaggrin gene K4022X 3321delA S2554X S2889X S3296X Normal type (WT) Mismatched Base Number 0 0 0 0 0 Tm 54 71 62 63 69 Mutant Inconsistent base number One 1 del One 2 One Tm 46.5 60 52.5 46.5 55

* 0 indicates a perfect match with no number of mismatched bases, 1 ~ 2 indicates the number of mismatched bases, and 1 del indicates the case where one base is deleted.

* Depending on the type of PCR equipment used and the degree of temperature correction, the Tm values in the above table may be slightly different, but the difference between the normal and mutant Tm values can be distinguished as shown in FIG. 2.

Claims (15)

To provide you with the information you need to diagnose atopic dermatitis, Amplifying the filaggrin gene mutation site included in the specimen to obtain a PCR product; Hybridizing with the PCR product using a PNA probe for detecting a base mutation in which a reporter that complementarily binds to the filaggrin gene mutation site obtains a hybridization product; Obtaining a melting curve by increasing the temperature of the hybridization product and measuring the fluorescence intensity of the PNA probe eluted in real time; The melting temperature of the hybridization product or the temperature at which the melting curve peak value appears is the melting temperature or melting curve peak value of the hybridization product between the wild-type filaggrin gene PCR product and the PNA probe at the same site as the amplified pilaggrin gene mutation site. Comparing to the temperature shown; And When the melting temperature or melting curve peak value of the hybridization product between the PCR product and the PNA probe of the sample was measured at a relatively low temperature, the homozygous pilaggrin gene mutation, the relatively low temperature, and the same temperature were measured. If heterozygous mutations, when determined only at the same temperature, comprising the step of determining the wild type. The method of claim 1, wherein the filaggrin gene mutation is selected from the group consisting of K4022X, K4021X, K4671X, 3321delA, S2554X, S2889X, or S3296X. The method of claim 1, wherein the PCR product is less than 1000bp in size. The detection method according to claim 1, wherein the temperature difference at which the melting temperature difference or the melting curve peak value appears is 5 ° C or more. The detection method according to claim 1, wherein the PNA probe is two kinds of PNA probes for independently distinguishing the filaggrin gene wild type and the mutation. The detection method according to claim 1, wherein the PNA probe is a PNA probe comprising any one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 11 to 16. The method of claim 1, wherein the reporter is a fluorescent material and a quencher. The method of claim 7, wherein the PNA probe comprises a plurality of PNA probes having different nucleotide sequences coupled with different reporters, thereby simultaneously detecting a plurality of different filaggrin gene mutations. The method of claim 1, wherein the detection method is a fluorescence melting curve analysis (Fluorescence Melting Curve Analysis, FMCA) to analyze the difference in the binding force between the PCR product and the probe in terms of the difference in the melting temperature (Tm) value. The method of claim 1, wherein the atopic dermatitis is exogenous atopic dermatitis. A and atopic dermatitis diagnostic kit comprising a forward and reverse primers for amplifying a nucleotide sequence including a part or all of the filaggrin gene from a sample, a PNA probe for detecting a filaggrin gene mutation. The kit for diagnosing atopic dermatitis according to claim 11, wherein the filaggrin gene mutation is selected from the group consisting of K4022X, K4021X, K4671X, 3321delA, S2554X, S2889X, or S3296X. 12. The kit for diagnosing atopic dermatitis according to claim 11, wherein the primer is at least one selected from the group consisting of SEQ ID NOs: 1 to 10. 12. The kit for diagnosing atopic dermatitis according to claim 11, wherein the PNA probe is at least one selected from the group consisting of SEQ ID NOs: 11 to 16. The kit for diagnosing atopic dermatitis according to claim 11, wherein the PNA probe includes a plurality of PNA probes having different nucleotide sequences coupled with different reporters to simultaneously detect a plurality of different filaggrin gene mutations. .

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