WO2010013855A1

WO2010013855A1 - Biomarker for diagnosis of aspirin hypersensitivity, method for manufacturing the same, and method for diagnosis of aspirin hypersensitivity using the same

Info

Publication number: WO2010013855A1
Application number: PCT/KR2008/004484
Authority: WO
Inventors: Choon-Sik Park; Hyoung Doo Shin
Original assignee: Soonchunhyang University Industry Academy Cooperation Foundation
Priority date: 2008-07-30
Filing date: 2008-08-01
Publication date: 2010-02-04
Also published as: KR101057262B1; KR20100012986A

Abstract

The article discloses the detection of a biomarker including a single nucleotide polymorphism (SNP) gene capable of diagnosing aspirin hypersensitivity without the administration of aspirin through analysis of the correlation between aspirin-hypersensitive asthma and SNP. Furthermore, this article demonstrates a method for manufacturing the said biomarker, likewise, a method for diagnosis of aspirin hypersensitivity using the biomarker. In order to manufacture the biomarker including a SNP gene for diagnosis of aspirin hypersensitivity, genomic DNAs of aspirin- hypersensitive patients with asthma and aspirin-nonhypersensitive patients with asthma were genotyped for one or more SNP(s), multiple regression analysis was used to correlate the SNP genotyping result with aspirin hypersensitivity, and SNPs with high correlation wereselected. Using the biomarker, aspirin-hypersensitive asthma can be effectively diagnosed without the ad¬ ministration of aspirin making it very beneficial and applicable for new drug development and diagnosis and treatment of diseases.

Description

BIOMARKER FOR DIAGNOSIS OF ASPIRIN HYPERSENSITIVITY, METHOD FOR MANUFACTURING THE SAME, AND METHOD FOR DIAGNOSIS OF ASPIRIN HYPERSENSITIVITY USING THE SAME Technical Field

[1] The study discloses reports on detection of a biomarker including a single nucleotide polymorphism (SNP) gene capable of diagnosing aspirin hypersensitivity without the administration of aspirin through analysis of the correlation between aspirin- hypersensitive asthma and SNP, a method for manufacturing the said biomarker, and a method for diagnosing aspirin hypersensitivity using the biomarker. Background Art

[2] In order to diagnose for aspirin hypersensitivity, aspirin has to be administered directly either by oral intake or by inhalation of a patient suspected with hypersensitivity, after which the changes in the lung function, nasal cavity, and skin are measured and monitored for 8 hours/day within 2 days. This procedure is very excruciating and sometimes, may result to fatal side effects such as anaphylaxis. However, it is the most practical method available as of the moment due to the lack of alternative methods for safe and sure diagnosis of aspirin hypersensitivity.

[3] Aspirin hypersensitivity refers to a condition resulting in asthma, rhinitis, urticaria, or anaphylaxis after taking in aspirin or non-steroidal anti-inflammatory drugs (NSAIDs) to lower an elevated body temperature or relive pain. It is prevalent in 5-10% of the total asthmatic patients. It is also found in 10% of patients with chronic urticaria, and 0.5-1% in people who are of good condition. This syndrome is characterized by the so- called "aspirin triad", in other words, aspirin hypersensitivity, bronchial asthma, and nasal polyposis. Cases of aspirin hypersensitivity have been reported ever since aspirin was introduced as a medicine over a century ago. In 1922, it was referred to as "aspirin triad" by Widal et al. Thereafter, researches on the treatment and mechanism of aspirin-hypersensitive asthma have been actively carried out. At present, however, only a small number of related genes were identified. Disclosure of Invention

Technical Problem

[4] This study is directed towards detection of a biomarker including a single nucleotide polymorphism (SNP) gene capable of diagnosing aspirin hypersensitivity without direct administration of aspirin through analysis of the correlation between aspirin hy- persensitivity and SNP. It is also aimed at providing a method for manufacturing the said biomarker, likewise, providing a method for diagnosis of aspirin hypersensitivity using the biomarker containing an SNP gene. Technical Solution

[5] For diagnosis of aspirin hypersensitivity, a biomarker containing two or more DNA sequences selected from a group with DNA sequences of SEQ ID NO: 1 to 43 was examined in the study.

[6] A method for manufacturing the specific biomarker was also disclosed. The method includes the following procedures :(i) genotyping genomic DNAs of aspirin-hypersensitive and aspirin-nonhypersensitive patients with asthma for one or more single nucleotide polymorphism gene(s) (SNP(s)); and (ii) analyzing the correlation between the SNP genotyping result and aspirin hypersensitivity using multiple regression analysis and finally selecting SNPs with high correlation.

[7] Furthermore, the inventors also disclosed method for diagnosis of aspirin hypersensitivity, which includes (i) genotyping DNAs extracted from a subject of diagnosis for two or more SNPs as selected from a group with DNA sequences of SEQ ID NO: 1 to 43; and (ii) depending on the genotyping result, substituting a value of 0 if the selected SNP is a major allele homozygote, 1 if it is a heterozygote and 2 if it is a minor allele homozygote (refer to Equation 1), and diagnosing the subjects as aspirin-hypersensitive if the result, i.e. the estimated value of aspirin hypersensitivity diagnosis, is equal to or greater than 1 :

[8] [Equation 1]

[9] E = 0.5962 + SNP₁ x 0.11247 + SNP₂ x 0.28448 + SNP₃ x 0.21721 + SNP₄ x -

0.15349 + SNP₅ x -0.09728 + SNP₆ x -0.15391 + SNP₇ x -0.18442 + SNP₈ x -0.14884 + SNP₉ x 0.23328 + SNPi₀ x -0.12368 + SNPn x 0.1254 + SNPj₂ x 0.14993 + SNPi₃ x -0.10873 + SNPi₄ x -0.21847 + SNPi₅ x 0.1282 + SNPi₆ x 0.49166 + SNP_n x 0.12978 + SNPi₈ x -0.19498 + SNPi₉ x 0.08771 + SNP₂₀ x 0.29671 + SNP₂I x -0.10311 + SNP₂₂ x 0.10061 + SNP₂₃ x -0.17642 + SNP₂₄ x -0.19909 + SNP₂₅ x -0.13394 + SNP₂₆ x 0.12405 + SNP₂₇ x 0.18955 + SNP₂₈ x 0.20034 + SNP₂₉ x 0.18337 + SNP₃₀ x 0.12153 + SNP₃I x 0.35268 + SNP₃₂ x -0.15567 + SNP₃₃ x -0.15568 + SNP₃₄ x 0.15717 + SNP₃₅ x 0.25871 + SNP₃₆ x -0.67052 + SNP₃₇ x 0.13613 + SNP₃₈ x 0.16576 + SNP₃₉ x 0.13042 + SNP₄₀ x -0.19585 + SNP_4I x -0.15725 + SNP₄₂ x -0.26271 + SNP₄₃ x - 0.27598

[10] wherein E represents the estimated value of aspirin hypersensitivity diagnosis and

SNP_n represents the value for the genotype of the n-th SNP selected from a group consisting of DNA sequences of SEQ ID NO: 1 to 43.

Advantageous Effects [11] With the use of the biomarker as reported by this study, it is possible to diagnose aspirin hypersensitivity with high accuracy even without direct administration of aspirin and subjection to other clinical or chemical processes by means of only one operation that incorporates multiple single nucleotide polymorphism (SNP) genes. Furthermore, this procedure is not just limited to cases of asthma; it is applicable in general for diagnosis of aspirin hypersensitivity for other diseases requiring the prescription of aspirin. Brief Description of Drawings

[12] The above and other aspects, features, and advantages of the disclosed information will be more apparent from the following detailed description taken in conjunction with the accompanying figures in which:

[13] Figure 1 compares the actual aspirin hypersensitivity diagnosis result with the expected value obtained from the equation presented in Table 2 in order to confirm the accuracy of the single nucleotide polymorphism (SNP) gene set biomarker according to this disclosure; and

[14] Figure 2 shows the aspirin hypersensitivity diagnosis result for unknown sample obtained using 43 SNP gene set biomarkers according to this disclosure and the expected value obtained from the equation presented in Table 2. Best Mode for Carrying out the Invention

[15] Exemplary embodiments now will be described more fully hereinafter with reference to the accompanying figures. This disclosure may, however, be interpreted in different ways and should not be construed as limited to the demonstrations set forth therein. Rather, these explanations are provided for the completion of this study such that the scope and thought of the said domain will be properly conveyed to the readers skilled in the art. In the description, details of apparent features and techniques were omitted to avoid ambiguities within the presented embodiments.

[16] The terminologies used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of this disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, the use of the terms a, an, etc. does not denote a limitation of quantity, but rather denotes the presence of at least one of the referenced item. The use of the terms "first", "second", and the likes does not imply any particular order, but they are included to identify individual elements; does not denote any order or importance, but rather, are used to distinguish one element from another. It will be further understood that the terms "comprises" and/or "comprising", or "includes" and/or "including" when used in this specification, specify the presence of stated features, regions, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and/or groups thereof.

[17] Unless otherwise defined, all terms (technical and scientific) used herein have the same meaning as understood by the common people. These terms should be interpreted as having a meaning that is consistent with their how they are used in daily conversations and will not be interpreted in an overly formal sense unless defined herein.

[18] This study presented a biomarker including a single nucleotide polymorphism (SNP) gene set selected from a group of DNA sequences with SEQ ID NO: 1 to 43 for diagnosis of aspirin hypersensitivity. The SNP gene set may include all the SNP genes of DNA sequences with SEQ ID NO: 1 to 43.

[19] To develop the specific biomarker, genotyping of 3,760 SNPs selected from the

NCBI SNP database (DB) was carried out using samples obtained from 288 Korean patients including 84 aspirin-hypersensitive patients with asthma and 204 aspirin- nonhypersensitive patients with asthma. Genotyping was performed using the Golden gate assay (Illumina, CA, USA). Based on the genotype analysis result, 208 SNPs having a minor allele frequency (MAF) that is equal to or greater than 0.1 along with a multiple regression analysis p-value that is equal to or less than 0.05 for aspirin hypersensitivity diagnosis were selected as primary SNP gene candidates. After carrying out the procedure for the second round of selection, 43 SNP genes set with high correlation between estimated value of aspirin hypersensitivity diagnosis and actual aspirin hypersensitivity diagnosis result (p < 0.05) were selected using stepwise regression.

[20] The multiple regression analysis is an extension of simple regression analysis that is used to analyze a regression model with two or more independent variables. To ensure reliability of the statistical analysis result, SNPs with MAF that is equal to or greater than 0.1 were selected. Among those, SNPs having statistically significant correlation between the aspirin hypersensitivity diagnosis and the genotype with a p value less than 0.05 were selected.

[21] When carrying out the stepwise regression, new variables were tried out one by one using forward selection. After a new variable was added at each stage, a test was also conducted to check whether some of the existing variables can be deleted. The procedure went on until variables can no longer be added or deleted.

[22] To apply the stepwise regression, the genotype of SNP was converted into a numerical value and the aspirin hypersensitivity diagnosis was estimated. For substitution purposes in the equation for estimating the aspirin hypersensitivity diagnosis, the major allele homozygote, heterozygote, and minor allele homozygote at the SNP locus were assumed as AA, AB, and BB, respectively, and were assigned with the values 0 for AA, 1 for AB, and 2 for BB accordingly. The principle of stepwise re- gression is given in Table 1 (see Miller, A. J., Journal of the Royal Statistical Society, Series A, 147:389-425, 1984).

[23] Table 1 [Table 1] [Table ]

E = βn+SNP , x β , + SNPoX βo+ SNP, x β, SNP, x β. E: Estimated value of aspirin hypersensitivity diagnosisSNP_n: Value for n-th SNP (AA = 0, AB = 1, BB = 2)β₀: Interceptβ_n: Coefficient for n-th SNP

[24] The estimating equation for aspirin hypersensitivity diagnosis for the final 43 SNP genotypes is depicted in Table 2. [25] Table 2 [Table 2] [Table ]

E = 0.5962 + SNP₁ x 0.11247 + SNP₂ x 0.28448 + SNP₃ x 0.21721 + SNP₄ x -0.15349 + SNP₅ x -0.09728 + SNP₆ x -0.15391 + SNP₇ x -0.18442 + SNP₈ x -0.14884 + SNP₉ x 0.23328 + SNP₁₀ x -0.12368 + SNP₁₁ x 0.1254 + SNP₁₂ x 0.14993 + SNP₁₃ x - 0.10873 + SNP₁₄ x -0.21847 + SNP₁₅ x 0.1282 + SNP₁₆ x 0.49166 + SNP₁₇ x 0.12978 + SNP₁₈ x -0.19498 + SNP₁₉ x 0.08771 + SNP₂₀ x 0.29671 + SNP₂₁ x -0.10311 + SNP 2₂ x 0.10061 + SNP₂₃ x -0.17642 + SNP₂₄ x -0.19909 + SNP₂₅ x -0.13394 + SNP₂₆ x 0.12405 + SNP₂₇ x 0.18955 + SNP₂₈ x 0.20034 + SNP₂₉ x 0.18337 + SNP₃₀ x 0.12153 + SNP₃₁ x 0.35268 + SNP₃₂ x -0.15567 + SNP₃₃ x -0.15568 + SNP₃₄ x 0.15717 + SNP 3₅ x 0.25871 + SNP₃₆ x -0.67052 + SNP₃₇ x 0.13613 + SNP₃₈ x 0.16576 + SNP₃₉ x 0.13042 + SNP₄₀ x -0.19585 + SNP₄₁ x -0.15725 + SNP₄₂ x -0.26271 + SNP₄₃ x - 0.27598 (SNP_n represents the value for the genotype of the n-th SNP selected from a group consisting of DNA sequences of SEQ ID NO: 1 to 43.)

[26] In another embodiment, a polynucleotide comprising 10 or more consecutive DNA sequences including the 27th base (SNP locus) of one or more DNA sequence(s) selected from a group with DNA sequences of SEQ ID NO 1 to 43, or a polynucleotide having a sequence complementary to that of the above polynucleotide was used for diagnosis of aspirin hypersensitivity.

[27] Furthermore, a polynucleotide hybridizing with the above mentioned polynucleotides along with a kit for diagnosis of aspirin hypersensitivity comprising two or more SNP genes set selected from a group with DNA sequences of SEQ ID NO: 1 to 43 or the above polynucleotides were also presented.

[28] Finally, there was a demonstration on the method for diagnosis of aspirin hypersensitivity using a biomarker comprising two or more SNP genes set selected from a group with DNA sequences of SEQ ID NO: 1 to 43. The procedure for diagnosis comprises of; (i) genotyping DNAs extracted from a subject of diagnosis for two or more SNPs as selected from a group with DNA sequences of SEQ ID NO: 1 to 43; and (ii) diagnosing the subject as an aspirin-hypersensitive patient depending on the genotyping result. For substitution purposes in the equation estimating the aspirin hypersensitivity diagnosis, the major allele homozygote, major allele heterozygote, and minor allele homozygote at the SNP locus were assumed as AA, AB and BB, respectively, with a substitute value of 0 for AA, 1 for AB, and 2 for BB in the estimating equation found in Table 2. If the result of the procedure, i.e. the estimated value of aspirin hypersensitivity diagnosis, is equal to or greater than 1, the subject was diagnosed as an aspirin-hypersensitive patient. Mode for the Invention

[29] The examples (and experiments) will now be described in details. The following examples (and experiments) are for illustrative purposes only and are not intended to limit the scope of this study. All the literatures cited in the present description are incorporated herein as reference.

[30]

[31] Example 1 : Preparation of sample for development and verification of SNP gene biomarker for diagnosis of aspirin hypersensitivity

[32] For the development of a biomarker including a single nucleotide polymorphism

(SNP) gene, samples were obtained from a total of 288 Korean patients including 84 aspirin-hypersensitive patients with asthma and 204 aspirin-nonhypersensitive patients with asthma (Soonchunhyang University Bucheon Hospital, Chungbuk National University Hospital, Chonnam National University Hospital and Seoul National University Hospital). Consent was obtained from the Institutional Review Board (IRB) with regards to the acquisition of the samples and related clinical data. In determining the samples, certain requirements were considered for the patient group. Finally, samples from 30 patients were used for verification of the aspirin hypersensitivity diagnosis using the SNP genes set.

[33] Table 3 [Table 3] [Table ]

[34] Preparation of DNA sample [35] Blood sample was centrifuged at 8,000 rpm for 5 minutes and supernatant excluding solids was removed. After adding 0.2 M NH₄Cl (500 μL), centrifuge was performed at 1,800 rpm for 1 minute. Then, after further centrifuging at 8,000 rpm for 5 minutes, the supernatant was removed. This procedure was repeated 3-4 times until the pellet turned whitish or pinkish. After separating the buffy coat, STE [0.1 M NaCl, 10 mM Tris (pH 8.0), 1 mM EDTA (pH 8.0)] 520 μL, 10% SDS (72 μL) and protease (20 mg/mL, 30 μL) were added. The mixture was then allowed to react at 55 ⁰C for 3 hours after it was mixed properly by slightly shaking the tube. When all blood cells were lysed, 3M NaOAc (30 μL) and a mixture of phenol/chloroform/isoamyl alcohol (25:24:1, 700 μL) were added. After cautiously shaking the tube for 5 minutes, centrifuge was performed at 13,000 rpm for 10 minutes. The supernatant was carefully moved to a fresh tube and a mixture of chloroform/isoamyl alcohol (24:1, 700 μL) was added afterwards. The succeeding procedures involved repetition of the processes beginning from the mixing of the substance. However, on the second repetition of the processes, Isopropanol (400 μL) was added to the supernatant, and on the third repetition, the supernatant which was removed excluding the pellet, was added with 70% ethanol (1 mL). Afterwards, centrifuge was again performed at 13,000 rpm for 3 minutes and the supernatant was cautiously removed so as to avoid from losing the pellet. The product was allowed to be dried in the air and dissolved in TE buffer [10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0)].

[36] [37] Example 2: Selection and genotvping of candidate SNP [38] Among the SNPs listed in the NCBI SNP database (DB)

(http://www.ncbi.nlm.nih.gov/SNP/), 3,760 SNPs with allele frequency information were selected.

[39] The selected 3,760 SNPs were genotyped using the Golden gate assay (Illumina, CA, USA). The Golden gate genotyping assay (BeadXpress) is a Veracode technology for SNP genotyping. All SNPs are assembled into one oligo pool all (OPA). Oligos amplified by allele-specific primer extension are hybridized onto beads and genotyping is performed automatically from the fluorescence intensity data of each SNP read by the fluorescence scanner BeadXpress Reader, by the BeadStudio software. The Golden gate assay is optimized for genotyping of a larger-scale SNP than that can be achieved by single-base extension or Taqman method (100 or less SNPs).

[40] To describe the procedures in more detail, a DNA plate preparation reagent (5 μX) was prepared on a DNA plate and DNA sample (50 ng/μJl) was added. The DNA plate was heat-sealed and incubated for 30 minutes on a heater preheated to 90 ⁰C. The seal was cautiously removed and, after adding a precipitation reagent (5 μX), the resulting solution was sufficiently mixed until the solution turned blue. Subsequently, 2-propanol (15 μX) was added to the mixture which was then centrifuged at 3000 x g for 20 minutes. Afterwards, the supernatant was removed and the product was dried at room temperature for 15-20 minutes. After adding turbid solution (10 μX) to each well of the DNA plate, the mixture was sufficiently mixed until the blue precipitate became completely loose.

[41] Following that, an oligo reagent (10 μX) was initially prepared on each well of a genotype-specific extension plate after which an oligo hybridization reagent (30 μX) was added. The seal of the DNA plate was cautiously removed and, after transferring a DNA reaction solution (10 μX) to the genotype- specific extension plate, the mixture was sufficiently mixed until the beads were completely released. The genotype- specific extension plate was placed on a heater preheated to 70 ⁰C. Immediately thereafter, the heater was adjusted to 30 ⁰C and the temperature was allowed to decrease to 30 ⁰C.

[42] The genotype- specific extension plate was placed on a magnet plate for 2 minutes or until the beads gathered together. Excluding the beads, the supernatant (about 50 μX) was removed and an extension reaction solution (50 μX) was added. The mixture was sufficiently mixed until the beads were completely released. The genotype-specific extension plate was placed again on the magnet plate and the extension reaction solution was removed excluding the beads. This procedure was repeated once more. A general-use buffer (50 μX) was added to each well of the genotype- specific extension plate using an 8-channel pipette and the genotype- specific extension plate was placed on the magnet plate for 2 minutes until the beads gathered together. Using an 8-channel pipette, the general-use buffer was removed excluding the beads. This procedure was repeated once more. Extension enzyme (37 μX) was added to each well of the genotype- specific extension plate using an 8-channel pipette and the plate was sealed using an adhesive film. Then, the mixture was sufficiently mixed at 1,600-1,700 rpm for 1 minute until the beads were completely released. Afterwards, the genotype- specific extension plate was incubated for 15 minutes on a heater preheated to 45 ⁰C.

[43] DNA polymerase (64 μA) and uracil-DNA glycosylase (50 μA) were then added to a

PCR mixture tube. After sufficiently mixing, the PCR mixture solution (30 μA) was added to each well of a PCR plate. The genotype-specific extension plate was placed on a magnet plate for 2 minutes or until the beads gathered together. Excluding the beads, the Supernatant (about 50 μA) was removed and a general-use buffer (50 μA) was added. The genotype- specific extension plate was placed again on the magnet plate for 2 minutes until the beads gathered together. After removing supernatant, DNA lysis solution (35 μA) was added to each well of the genotype- specific extension plate. After sufficiently mixing the mixture at 1,800-1,900 rpm for 1 minute to release the beads, the plate was placed for 1 minute on a heater preheated to 95 ⁰C. Once the plate was removed from the heater, the plate was placed again on the magnet plate for 2 minutes or until the beads gathered together. Thereafter, supernatant (30 μX) was moved to a PCR plate. The PCR plate was heat-sealed and immediately moved to a gene amplification apparatus. PCR was performed as follows: 10 minutes at 37 ⁰C; 3 minutes at 95 ⁰C; 34 cycles of 35 seconds at 95 ⁰C, 35 seconds at 56 ⁰C and 2 minutes at 72 ⁰C; followed by 10 minutes at 72 ⁰C.

[44] Afterwards, the bead solution was carefully mixed and moved to a PCR plate, 20 μA, each. After mixing well, the solution, which was covered with a lid to block the light was moved to a filter plate and was allowed to polymerize at room temperature for 60 minutes. An intermediate plate for a Veracode bead plate was prepared. A filter plate adaptor was placed on an empty 96- well waste collection plate and a filter plate holding a PCR solution was added. Centrifuge was performed at 1,000 x g for 5 minutes at 25 ⁰C. After adding a general-use buffer (50 μA) to the filter plate, centrifuge was again performed at 1,000 x g for 5 minutes at 25 ⁰C. A hybridization solution (30 μA) was added to the intermediate plate and after replacing the latter and the waste collection plate, 0.1 N NaOH (30 μA) was added to the filter plate. Immediately thereafter, centrifuge again was performed at 1,000 x g for 5 minutes, at 25 0C.

[45] Moving on, in a 15 mL tube, 0.1 N NaOH (3 mL) was mixed with a hybridization solution (3 mL) to neutralize the latter. The neutralized hybridization solution (50 μA) was added to each well of the intermediate plate and the sample in the plate was mixed by pipetting for 4-5 times. The mixture (100 μA) was moved to a Veracode bead plate and was allowed to hybridize for 3 hours (850 rpm, 45 ⁰C). Washing solution (200 μA) was added to the Veracode bead plate which was then mixed using a pipette. After 2 minutes when the beads were collected at the bottom of each well, the supernatant was removed. Upon completion of the reaction, the Veracode bead plate was scanned using the BeadXpress reader. This experiment was carried out using Illumina's Golden gate genotyping kit for Veracode (VC-201-0096, VC-201-0384) and BeadXpress reader.

[46] When the scanning was completed, the genotype was determined using the

BeadStudio software. The final forward strand genotype was printed out in file format of BeadStudio software report according to the instructions included in the kit.

[47]

[48] Example 3: Development of SNP gene for aspirin hypersensitivity diagnosis and sta- tistical analysis

[49] Multiple regression analysis was performed for correlation analysis between aspirin hypersensitivity and SNP genotypes. Among the SNPs, 208 SNPs having an MAF that is equal to or greater than 0.1 along with a p-value 0.05 or less for multiple regression analysis with respect to aspirin hypersensitivity were selected as primary SNP gene candidates. SNPs having an MAF that is equal to or greater than 0.1 were selected for reliable statistical analysis since it qualify for the principle that a sufficient genetic diversity of minor alleles is required for aspirin hypersensitivity diagnosis. Among the mentioned SNPs, the ones depicting statistically significant correlation between aspirin-hypersensitive asthma and genotype, likewise with a p-value of 0.05 or less, were selected.

[50] Stepwise regression was performed for the second round of selection. To start off, the genotype of SNP was converted into a numerical value. For substitution purposes in the equation for estimating the aspirin hypersensitivity diagnosis (refer to Table 1), the major allele homozygote, heterozygote, and minor allele homozygote at the SNP locus were assumed as AA, AB, and BB, respectively, and were assigned with the values 0 for AA, 1 for AB, and 2 for BB, accordingly.

[51] As a result, 43 SNP genes set with high correlation with the actual aspirin hypersensitivity (p-value < 0.05) were selected, as seen in Fig. 1. The estimating equation for aspirin hypersensitivity diagnosis for the final 43 SNP genes is given in Table 2. Furthermore, Figure 1 compares the actual aspirin hypersensitivity diagnosis result with the expected value obtained from the equation presented in Table 2 in order to confirm the accuracy of the single nucleotide polymorphism (SNP) gene set biomarker according to this disclosure. A significant relationship among the variables is verified from the figure which means that the estimated diagnosis of aspirin hypersensitivity based on the genotype is similar to the actual diagnosis result. This highly suggests that aspirin hypersensitivity can be diagnosed using the estimating equation based on gene test and not solely on drug response.

[52] In the equation of Table 2, SNP_n represents the SNP ID listed in the NCBI SNP DB

(http://www.ncbi.nlm.nih.gov/SNP/). For example, SNPi stands for rsl327506 (see Tables 4 and 5). A numerical value is substituted for the genotype of each SNP. For example, for SNPi (rs 1327506), a value of 0 is given for CC (major allele ho- mozygote), 1 for CT (heterozygote), and 2 for TT (minor allele homozygote). Possible genotypes for each SNP are presented in the form of "major allele > minor allele" in Table 4. Minor allele frequency (MAF), heterozygosity, and Hardy- Weinberg equilibrium p-value (HWE) for 43 SNP genes selected according to an embodiment of this disclosure are given in Table 4. [53] Table 4

[Table 4] [Table ]

[54] The flanking sequence information of the selected 43 SNP genes is summarized in

Table 5. The [B] depicts the base, i.e. A, T, G or C, of the minor allele.

[55] The SNP ID denotes the sequence and location of each SNP. Those who are well acquainted on the domain will easily understand the sequence and location of the SNP from the ID. It has been made known that the sequences corresponding to the rs numbers of the SNPs listed in the NCBI DB may be modified in the future. In response to that, the scope of this disclosure encompasses such modification and thus, may be considered a subject of interest for the academe and the people knowledgeable on the domain.

[56] Table 5

[Table 5] [Table ]

[57] Example 5: Kit for diagnosis of aspirin hypersensitivity using SNP gene set biomarkers. and diagnosis using the same

[58] Using a kit for diagnosis of aspirin hypersensitivity comprising two or more SNP genes set selected from a group having DNA sequences of SEQ ID NO: 1 to 43, aspirin hypersensitivity was diagnosed as follows for patients (subjects) suspected with aspirin hypersensitivity.

[59] DNA was extracted from the blood of 30 patients suspected with aspirin hypersensitivity and genotyped using the diagnosis kit. Once the genotype was determined, the major allele homozygote, heterozygote and minor allele homozygote were assumed as AA, AB and BB, respectively, and were assigned with the values 0 for AA, 1 for AB, and 2 for BB. This was done for substitution purposes in the estimating equation in Table 2. If the estimated value of aspirin hypersensitivity diagnosis was equal to or greater than 1, the patient was diagnosed as having aspirin hypersensitivity.

[60] As evident in Figure 2, a similar tendency was observed which is apparent in the development of SNP genes in Example 3. Among the 30 patients with or without aspirin hypersensitivity, 27 patients were accurately diagnosed by the estimation.

[61]

[62] While the discussions have been fully described, it should be understood by people who are knowledgeable on the domain that various changes in form and details may be made thereto without straying away from the scope of this disclosure as defined by the appended claims.

[63] In addition, many modifications can be made to adapt a particular situation or material to the teachings of this disclosure without departing from the essential scope thereof. In accordance with that, this disclosure is not intended to be limited to the particular explanations given as the best mode for carrying out the reports regarding the study, but rather, this disclosure will include all the embodiments falling within the scope of the appended claims.

Claims

[1] A biomarker for diagnosis of aspirin hypersensitivity, comprising two or more

DNA sequences selected from a group having DNA sequences of SEQ ID NO: 1 to 43.

[2] A biomarker for diagnosis of aspirin hypersensitivity, comprising a polynucleotide with 10 or more consecutive DNA sequences including the 27th base of one or more DNA sequence(s) as selected from a group of DNA having sequences of SEQ ID NO: 1 to 43, or a polynucleotide having a sequence complementary to that of said polynucleotide.

[3] A method for manufacturing a biomarker for diagnosis of aspirin hypersensitivity, comprising:

(i) genotyping genomic DNAs of aspirin-hypersensitive patients with asthma and aspirin-nonhypersensitive patients with asthma for one or more single nucleotide polymorphism gene(s) (SNP(s)); and

(ii) analyzing correlation of the genotyping result with aspirin hypersensitivity using multiple regression analysis and subsequently selecting SNPs with high correlation.

[4] The method for manufacturing a biomarker according to claim 3, wherein said selecting comprises:

(i) based on the multiple regression analysis result, primarily selecting SNPs having a minor allele frequency (MAF) that is equal to or greater than 0.1 along with a multiple regression analysis p-value that is equal to or less than 0.05 for aspirin hypersensitivity diagnosis; and

(ii) going through second round of selection by performing stepwise regression after converting the genotypes of the primarily selected SNPs into numerical values, and selecting SNPs with a p-value between actual diagnosis result and estimated value of aspirin hypersensitivity diagnosis following a stepwise regression that is equal to or less than 0.05.

[5] A method for diagnosis of aspirin hypersensitivity, comprising:

(i) genotyping DNAs extracted from a subject of diagnosis for two or more SNPs selected from a group of DNA sequences having an SEQ ID NO. of 1 to 43; and (ii) depending on the genotyping result, in Equation 1, substituting a value of 2 if the selected SNP is a minor allele homozygote, substituting a value of 1 if it is a heterozygote and substituting a value of 0 if is a major allele homozygote, and if the result is equal to or greater than 1, diagnosing the subject as an aspirin- hypersensitive patient: [Equation 1] E = 0.5962 + SNP₁ x 0.11247 + SNP₂ x 0.28448 + SNP₃ x 0.21721 + SNP₄ x - 0.15349 + SNP₅ x -0.09728 + SNP₆ x -0.15391 + SNP₇ x -0.18442 + SNP₈ x - 0.14884 + SNP₉ x 0.23328 + SNP₁₀ x -0.12368 + SNP₁₁ x 0.1254 + SNP₁₂ x 0.14993 + SNP₁₃ x -0.10873 + SNP₁₄ x -0.21847 + SNP₁₅ x 0.1282 + SNP₁₆ x 0.49166 + SNP₁₇ x 0.12978 + SNP₁₈ x -0.19498 + SNP₁₉ x 0.08771 + SNP₂₀ x 0.29671 + SNP₂₁ x -0.10311 + SNP₂₂ x 0.10061 + SNP₂₃ x -0.17642 + SNP₂₄ x - 0.19909 + SNP₂₅ x -0.13394 + SNP₂₆ x 0.12405 + SNP₂₇ x 0.18955 + SNP₂₈ x 0.20034 + SNP₂₉ x 0.18337 + SNP₃₀ x 0.12153 + SNP₃₁ x 0.35268 + SNP₃₂ x - 0.15567 + SNP₃₃ x -0.15568 + SNP₃₄ x 0.15717 + SNP₃₅ x 0.25871 + SNP₃₆ x - 0.67052 + SNP₃₇ x 0.13613 + SNP₃₈ x 0.16576 + SNP₃₉ x 0.13042 + SNP₄₀ x - 0.19585 + SNP₄₁ x -0.15725 + SNP₄₂ x -0.26271 + SNP₄₃ x -0.27598 wherein E represents the estimated value of aspirin hypersensitivity diagnosis, and SNP_n represents the value for the genotype of the n-th SNP selected from a group consisting of DNA sequences of SEQ ID NO: 1 to 43.