Placenta-derived DNA methylation-based biomarker for predicting persistent food allergy risk and composition comprising the same

Abstract

The present invention relates to a biomarker for predicting the risk of early-onset persistent food allergy, a composition including the same, and a prediction method. By using the DNA methylation level of at least one gene selected from the group consisting of the RPS6KA2 gene and the GSCAML gene measured in a placental-derived biological sample as an indicator, the risk of food allergy that develops in infancy continuing into the school-age period can be predicted early at the time of birth.

Description

Technology Field

[0001] The present invention relates to the prediction of food allergies, and more specifically, to a biomarker for predicting the persistent risk of food allergies using DNA methylation information measured in a placenta-derived biological sample, a composition containing the same, and a prediction method. Background Technology

[0002] Food allergies primarily occur during infancy and early childhood; while some patients experience spontaneous remission before school age, others exhibit a course that persists beyond this age. This persistent food allergy is known to increase the risk of progression to other allergic diseases, such as allergic rhinitis or asthma, and the increasing global prevalence is placing a growing socio-medical burden. Therefore, there is a need for the development of technologies to predict the natural course of food allergies early and identify high-risk groups.

[0003] While it has been previously reported that food antigen-specific IgE levels are associated with the persistence of the disease, there are many cases where spontaneous remission occurs even with initially elevated IgE levels, limiting the ability to predict the long-term course using IgE as a sole indicator. Accordingly, there is a growing need for new biomarkers capable of more accurately predicting the persistence of food allergies.

[0004] Meanwhile, epigenetic changes, including DNA methylation, are closely related to the regulation of immune responses and are receiving attention as factors that can influence the onset and course of diseases.

[0005] Accordingly, the inventors classified the natural course of food allergies through birth cohort-based analysis and confirmed the correlation between DNA methylation patterns in placental tissue and the persistence of the disease, and based on this, completed the present invention. Prior art literature

[0006] Republic of Korea Registered Patent No. 10-2501298 (Published on Feb. 21, 2023) The problem to be solved

[0007] The present invention has been devised to solve the aforementioned problems and aims to provide a biomarker capable of predicting early at the time of birth the risk that a food allergy developed in infancy will persist into the school-age period.

[0008] In addition, the present invention aims to provide a predictive biomarker capable of determining the long-term persistence of a food allergy using DNA methylation information measured in a placenta-derived biological sample, a composition containing the same, and a prediction method.

[0009] Furthermore, the present invention aims to overcome the limitations of predictions relying solely on existing serum IgE levels and to provide a new epigenetic indicator capable of more accurately distinguishing the natural course of food allergies. means of solving the problem

[0010] To solve the above-mentioned problem, the present invention provides a biomarker for predicting the risk of early-onset food allergy development, characterized by an increased DNA methylation level of at least one gene selected from the group consisting of the RPS6KA2 gene and the GSCAML gene measured in a placenta-derived biological sample.

[0011] According to one embodiment of the present invention, the DNA methylation level may be characterized as being increased compared to the food allergy-free group or the early remission type group.

[0012] According to one embodiment of the present invention, the biomarker may be characterized by having increased levels of DNA methylation of both the RPS6KA2 gene and the GSCAML gene.

[0013] According to one embodiment of the present invention, the biological sample may be characterized as being selected from placental tissue, placental-derived cells, or placental-derived nucleic acids.

[0014] According to one embodiment of the present invention, the DNA methylation level may be characterized by being measured by a methylation array analysis, methylation-specific PCR, non-sulfite conversion-based analysis, or methylation sequencing method.

[0015] In addition, the present invention provides a biomarker composition for predicting the risk of early-onset food allergy development, comprising an oligonucleotide primer, a probe, or a kit containing the same for detecting the aforementioned biomarker.

[0016] In addition, the present invention provides a method for predicting the risk of developing an early-onset food allergy, comprising the steps of: measuring the DNA methylation level of at least one gene selected from the group consisting of the RPS6KA2 gene and the GSCAML gene in a placenta-derived biological sample; and determining that the risk of developing an early-onset food allergy is high when the DNA methylation level is increased.

[0017] According to one embodiment of the present invention, if the DNA methylation level is increased compared to the food allergy-free group or the early remission group, it can be characterized as being determined to have a high risk of developing early-onset persistent food allergy.

[0018] According to one embodiment of the present invention, the biological sample may be characterized as placental tissue obtained during childbirth, cells derived therefrom, or nucleic acid.

[0019] According to one embodiment of the present invention, the prediction may be characterized as being for determining the risk that a food allergy that developed in infancy will persist until after the school-age period. Effects of the invention

[0020] According to the present invention, the risk of food allergies that develop in infancy continuing into the school-age period can be predicted early by using the DNA methylation level of a specific gene in a placenta-derived biological sample as an indicator.

[0021] In addition, the present invention has the advantage of being able to predict the long-term course of a disease at a stage prior to the clear appearance of clinical symptoms by utilizing placental tissue available at birth, cells derived therefrom, or nucleic acids. Accordingly, high-risk groups can be identified early to establish customized management and prevention strategies, and this can contribute to minimizing unnecessary dietary restrictions or excessive treatment.

[0022] Furthermore, the present invention provides a technological advancement in the field of food allergy persistence risk assessment by complementing the limitations of predictions relying solely on existing serum IgE level indicators and by providing an epigenetic-based predictive indicator capable of more precisely distinguishing the natural course of food allergies.

[0023] In addition, the biomarker according to the present invention can be implemented in the form of a composition and a kit, thus having high applicability in clinical settings and excellent potential for industrial use. Brief explanation of the drawing

[0024] Figure 1 is a graph comparing the DNA methylation levels of the RPS6KA2 and GSCAML genes in the placenta according to the natural course and phenotype of food allergy. As shown in Figure 1, the methylation levels of the two genes in the early persistent food allergy group are significantly increased compared to the food allergy-free group or the early remission group. Figure 2 is a graph showing the correlation between the DNA methylation levels of RPS6KA2 and GSCAML genes in the placenta and the egg white-specific IgE concentration at 1 year of age. As shown in Figure 2, there is a significant positive correlation in which the egg white-specific IgE concentration at 1 year of age increases as the methylation level of the said genes increases. Figure 3 is a graph showing the correlation between the DNA methylation levels of RPS6KA2 and GSCAML genes in the placenta and the serum total IgE concentration at age 3. As shown in Figure 3, there is a significant positive correlation in which the serum total IgE concentration at age 3 increases as the methylation level of the said genes increases. Specific details for implementing the invention

[0025] Preferred embodiments according to the present invention will be described in detail below with reference to the attached drawings.

[0026] The advantages and features of the present invention and the method for achieving them will become clear by referring to the embodiments described in detail below together with the accompanying drawings.

[0027] However, the present invention is not limited by the embodiments disclosed below but may be implemented in various different forms, and these embodiments are provided merely to make the disclosure of the present invention complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the scope of the claims.

[0028] Unless otherwise specifically defined, terms used in this specification have the meanings commonly used in the relevant technical field.

[0029] In addition, in describing the present invention, if it is determined that related known technologies, etc., may obscure the essence of the present invention, a detailed explanation thereof will be omitted.

[0030] The present invention will be described in detail below.

[0031] The present invention relates to a biomarker for predicting the risk of early-onset persistent food allergy, and provides a biomarker for predicting the risk of early-onset persistent food allergy characterized by an increased DNA methylation level of at least one gene selected from the group consisting of the RPS6KA2 gene and the GSCAML gene measured in a placental-derived biological sample.

[0032] As described above, food allergies may exhibit different clinical progression patterns over time. In the present invention, food allergies are classified into the following three types based on the clinical course during the follow-up period: (1) no food allergy group, (2) early remission type, and (3) early persistence type.

[0033] Here, the "no food allergy group" refers to cases where no clinically significant symptoms of food allergy are observed during the follow-up period. The "early remission type" refers to cases where a food allergy develops during infancy but resolves before a certain age. The "early persistence type" refers to cases where a food allergy developed during infancy persists into the school-age period or beyond.

[0034] In the present invention, "risk of early-onset persistent food allergy development" refers to the possibility of corresponding to the early-onset persistent type among the progression patterns, and this can be evaluated in advance based on epigenetic information of a placenta-derived biological sample obtained at the time of birth.

[0035] The above RPS6KA2 gene is a gene encoding serine / threonine protein kinase, which is involved in cell signaling pathways and may be associated with the activation and differentiation processes of immune cells.

[0036] The aforementioned GSCAML gene is known to encode a protein involved in signaling processes associated with immune cells, particularly B cells and mast cells, and may be functionally associated with immunological pathways related to IgE-mediated immune responses.

[0037] In the present invention, the genes were identified as indicators in which the level of DNA methylation in placental-derived biological samples is statistically significant in relation to the progression of food allergies, particularly the early persistent course.

[0038] In other words, the methylation level of the above gene is not limited to being a simple indicator of changes in gene expression, but functions as a biomarker reflecting the epigenetic state at the time of birth, and can be used to predict the persistent risk of food allergies in the future.

[0039] The present invention can apply the two genes individually or in combination, and can improve prediction accuracy by considering the methylation information of multiple genes together.

[0040] The above placenta-derived biological sample may include placenta tissue obtained during childbirth, cells isolated from the placenta, or placenta-derived nucleic acids.

[0041] Specifically, the biological sample may be the placental tissue itself, a cell population isolated from the placental tissue, or a nucleic acid such as genomic DNA extracted from the placental tissue. Additionally, a DNA sample isolated and purified from the placental tissue may be used as a target sample for epigenetic analysis.

[0042] The present invention is not limited to the specific form of the above biological sample and includes all biological samples derived from the placenta that can measure the DNA methylation level of the gene.

[0043] In the above, "increased DNA methylation level" means that the degree of methylation at CpG sites within a specific gene region shows a relatively high value compared to the reference group.

[0044] In the present invention, the reference group may include a group with no food allergy or an early remission type, and a case showing a statistically significantly higher methylation level compared to the reference group may be judged as "increased."

[0045] The above DNA methylation level can be expressed as the relative ratio of methylation signals to non-methylation signals, the β value of CpG sites, or a corresponding quantitative indicator, and can be calculated as a normalized value depending on the analysis method.

[0046] Here, "increase" is not limited to meaning a specific threshold of an absolute value, but can be determined based on the relative difference with a comparison group. Additionally, the methylation level can be calculated using the average value of multiple CpG sites or the value of a representative CpG site.

[0047] Accordingly, "increase in DNA methylation level" in the present invention includes a state in which the degree of methylation of a specific gene measured in a placenta-derived biological sample changes in a direction that has a significant correlation with early-onset persistent food allergy.

[0048] The above DNA methylation level can be measured by various known methylation analysis methods. For example, it can be quantified using methylation array analysis, methylation-specific PCR, bisulfite conversion-based analysis methods, methylation sequencing analysis methods, or substantially equivalent methods.

[0049] According to the above analysis method, the methylation status or methylation ratio of a specific CpG site may be measured, and the analysis results may be expressed in the form of quantitative values, relative ratios, or normalized indicators. The present invention is not limited to a specific analysis platform and includes all methods capable of reliably measuring the methylation level of the gene.

[0050] In addition, the present invention provides a biomarker composition for predicting the risk of early-onset food allergy development, comprising an oligonucleotide primer, a probe, or a kit containing the same for detecting the aforementioned biomarker.

[0051] The above oligonucleotide primer or probe may be designed to amplify or detect regions containing specific CpG sites of the RPS6KA2 gene and / or GSCAML gene. The primer may be designed to enable selective amplification based on methylation status using non-sulfite-converted DNA as a template and may be applied to methylation-specific polymerase chain reaction (methylation-specific PCR).

[0052] In addition, the probe may include a fluorescent label, an enzyme label, or other detectable label to detect the methylation status of a specific CpG site, and may be applied to real-time PCR, hybridization analysis, methylation array analysis, or sequencing-based analysis methods.

[0053] The kit according to the present invention may include at least one of the primer or probe, and may additionally include a reagent for DNA extraction, a non-sulfite conversion reagent, a buffer solution for amplification reaction, an enzyme, a control sample, or reference data for interpreting analysis results.

[0054] The above kit may be provided in a form for research or clinical application and may be used to assess the risk of early-onset persistent food allergy by measuring the DNA methylation level of the gene from a placenta-derived biological sample.

[0055] The composition of the present invention is not limited to a specific analysis platform and encompasses a form including all suitable components capable of detecting the methylation status of the gene.

[0056] In addition, the present invention provides a method for predicting the risk of developing an early-onset food allergy, comprising the steps of: measuring the DNA methylation level of at least one gene selected from the group consisting of the RPS6KA2 gene and the GSCAML gene in a placenta-derived biological sample; and determining that the risk of developing an early-onset food allergy is high when the DNA methylation level is increased.

[0057] The step of measuring the DNA methylation level may include extracting nucleic acids from a placental-derived biological sample and then quantifying the methylation status of a region containing specific CpG sites of the RPS6KA2 gene and / or GSCAML gene. The measurement may be performed by methylation array analysis, methylation-specific polymerase chain reaction, non-sulfite conversion-based analysis, methylation sequencing analysis, or an equivalent known method.

[0058] The above DNA methylation level can be calculated in the form of a relative ratio of methylation signals to non-methylation signals, a β value of a CpG site, or a corresponding quantitative indicator, and can be expressed as a value that has been normalized or statistically corrected as needed.

[0059] The above determination step includes a step of determining that there is a high risk of early-onset persistent food allergy development if the measured DNA methylation level is relatively increased compared to a reference group. Here, the reference group may include a group without food allergies or an early-onset remission group, and if a significant difference is shown based on the results of statistical analysis, it may be determined that the risk is increased.

[0060] The prediction method according to the present invention is not limited to specific analysis equipment or platforms and includes all appropriate technical means capable of quantitatively evaluating the methylation level of the gene. In addition, it can be determined based on the methylation level of one of the genes, and the prediction accuracy can be improved by combining methylation information of two or more genes.

[0061] The above biological sample may be placental tissue obtained during the process of childbirth, or cells or nucleic acids derived therefrom. The placenta is a tissue that can typically be isolated and obtained immediately after birth, and has the advantage of being obtainable without additional invasive procedures.

[0062] The placental tissue can be used in its whole tissue form, and if necessary, specific parts may be excised and used for analysis. Additionally, cell populations isolated from the placental tissue or nucleic acid forms, such as genomic DNA extracted from the placental tissue, may be purified and used for methylation analysis.

[0063] Since the prediction method according to the present invention can be performed using placental-derived samples obtained at the time of birth or immediately after birth, it has the advantage of being able to assess in advance the risk of an early, persistent course of food allergy at a stage prior to the clinical manifestation of food allergy in infants.

[0064] In addition, the above method is not limited to the placental tissue itself, but includes any sample in the form of cells or nucleic acids derived from the placenta that can reliably measure the DNA methylation level of the gene.

[0065] The above prediction is intended to assess the risk that food allergies developed during infancy will not naturally resolve during subsequent growth and will persist into the school-age period and beyond.

[0066] Here, "onset in infancy" refers to cases where clinical symptoms consistent with an IgE-mediated immune response are observed after the consumption of a specific food during the early stages of life, and "persistence beyond school age" includes cases where the same or similar clinical symptoms recur after a certain age or where a condition requiring continuous avoidance of the causative food is maintained. In this specification, "infancy" generally refers to an early life stage including the period up to approximately 2 years after birth, and "school age" typically refers to the childhood period after school age, including the age range of approximately 6 to 12 years after birth. In the present invention, the above terms are not strictly limited to specific ages but refer to relative time divisions to distinguish between the onset of a food allergy and its subsequent persistence.

[0067] The prediction method according to the present invention is not merely intended to diagnose the current presence of a food allergy, but rather to distinguish whether a food allergy that developed during infancy will show an early remission course or an early persistence course in the future.

[0068] Therefore, the prediction method of the present invention can be utilized to identify high-risk groups and establish customized management strategies by evaluating the long-term course of the disease in advance.

[0069] [Example]

[0070] The present invention will be explained in more detail below through examples, but the following examples are not intended to limit the scope of the invention and should be interpreted as being for the purpose of aiding understanding of the invention.

[0071] 1. Classification of birth cohort-based food allergy progression trajectories

[0072] The progression of food allergy (FA) over time was analyzed in subjects followed from birth to school age in a Korean birth cohort. Food allergy was defined as the appearance of clinical symptoms consistent with IgE-mediated reactions following the consumption of a specific food.

[0073] As a result of classifying FA progression patterns using group-based trajectory modeling, no food allergy (no FA), early remission, and early persistent types were observed.

[0074] Among these, the early persistence type was defined as a group in which food allergies that developed in infancy persist into the school-age period.

[0075] 2. Placental Methylation Results in the Food Allergy (FA) Group

[0076] To identify the epigenetic characteristics at birth associated with the occurrence of early persistent FA, DNA methylation analysis was performed on randomly selected placental tissue samples representing each trajectory.

[0077] After extracting genomic DNA from placental tissue, full-length CpG methylation profiling was performed using the Infinium MethylationEPIC BeadChip. Preprocessing and normalization of the methylation data were performed according to the manufacturer's recommended protocol.

[0078] The methylation level of each CpG site was calculated as a β value using the methylation signal (M) and the non-methylation signal (U). The β value was calculated according to the following formula: β = M / (M + U + 100). Here, CpG sites satisfying a significance level P < 0.05 and a mean methylation difference |Δβ| > 0.2 were defined as differentially methylated sites. Comparisons of methylation levels between loci were performed using analysis of variance or nonparametric tests.

[0079] As a result, the early-onset FA group showed significant hypermethylation in the RPS6KA2 and GSCAML sites compared to the food allergy-free group and the early-onset remission group.

[0080] The above results are illustrated in Figure 1, and as shown in Figure 1, it can be confirmed that the methylation levels of RPS6KA2 and GSCAML in the early-acting group are significantly increased compared to other groups.

[0081] 3. Correlation between placental methylation and antigen-specific IgE in 1-year-olds

[0082] The correlation between the methylation levels of RPS6KA2 and GSCAML identified in the placenta and egg white-specific IgE concentrations at 1 year of age was analyzed. Correlation analysis was performed using the Pearson correlation coefficient, and P < 0.05 was considered statistically significant.

[0083] As a result, the methylation levels of the two genes showed a statistically significant positive correlation with the specific IgE concentration in 1-year-old egg whites. In other words, a tendency was confirmed in which the specific IgE concentration increased as the methylation level increased.

[0084] The above correlation results are illustrated in Fig. 2, and as shown in Fig. 2, it can be confirmed that there is a significant positive correlation between the degree of methylation of RPS6KA2 and GSCAML in the placenta and the 1-year-old egg white-specific IgE concentration.

[0085] 4. Correlation between placental methylation and 3-year-old total IgE

[0086] The correlation between the methylation levels of RPS6KA2 and GSCAML identified in the placenta and the serum total IgE concentration at age 3 was analyzed. Correlation analysis was performed using the Pearson correlation coefficient, and P < 0.05 was considered statistically significant.

[0087] As a result, the methylation levels of the two genes showed a statistically significant positive correlation with the serum total IgE concentration of 3-year-olds. In other words, a tendency was confirmed for total IgE concentration to increase as the methylation level increased.

[0088] The above correlation results are illustrated in Figure 3, and as shown in Figure 3, it can be confirmed that there is a significant positive correlation between the degree of methylation of RPS6KA2 and GSCAML in the placenta and the total serum IgE concentration of 3-year-olds.

[0089] According to the results of Examples 1 to 4 above, it was confirmed that hypermethylation of the RPS6KA2 and GSCAML genes in placental tissue is specifically associated with the progression pattern of early-onset persistent food allergy.

[0090] In addition, the methylation levels of the two genes showed a significant positive correlation with egg white-specific IgE concentration at 1 year of age and serum total IgE concentration at 3 years of age, respectively. This suggests that the placental epigenetic status at birth is closely related to the persistence of subsequent IgE-mediated immune responses.

[0091] Therefore, by measuring the methylation levels of RPS6KA2 and / or GSCAML in placental-derived biological samples, the risk of food allergies that develop in infancy persisting into school age can be predicted early.

[0092] That is, the methylation status of the two genes mentioned above is useful as a predictive biomarker for screening early-onset persistent food allergies.

[0093] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. That is, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

Claim 1 A biomarker for predicting the risk of early-onset food allergy development, characterized by an increased DNA methylation level of at least one gene selected from the group consisting of the RPS6KA2 gene and the GSCAML gene measured in a placenta-derived biological sample. Claim 2 A biomarker for predicting the risk of early-onset food allergy development according to claim 1, characterized in that the DNA methylation level is increased compared to the food allergy-free group or the early-onset remission group. Claim 3 A biomarker for predicting the risk of early-onset, sustained-release food allergy, according to claim 1, characterized in that the DNA methylation levels of both the RPS6KA2 gene and the GSCAML gene are increased. Claim 4 A biomarker for predicting the risk of early-onset food allergy development according to claim 1, wherein the biological sample is selected from placental tissue, placental-derived cells, or placental-derived nucleic acids. Claim 5 A biomarker for predicting the risk of early-onset food allergy development, wherein, in claim 1, the DNA methylation level is measured by a methylation array analysis, methylation-specific PCR, non-sulfite conversion-based analysis, or methylation sequencing method. Claim 6 A biomarker composition for predicting the risk of early-onset food allergy, comprising an oligonucleotide primer, probe, or kit containing the same for detecting the biomarker according to claim 1. Claim 7 A method for predicting the risk of early-onset food allergy development, comprising: a step of measuring the DNA methylation level of at least one gene selected from the group consisting of the RPS6KA2 gene and the GSCAML gene in a placental-derived biological sample; and a step of determining that there is a high risk of early-onset food allergy development when the DNA methylation level is increased. Claim 8 A method for predicting the risk of early-persistent food allergy development according to claim 7, characterized in that the DNA methylation level is increased compared to the food allergy-free group or the early remission group, thereby determining that there is a high risk of early-persistent food allergy development. Claim 9 A method for predicting the risk of early-onset food allergy occurrence according to claim 7, wherein the biological sample is placental tissue obtained during childbirth, cells derived therefrom, or nucleic acid. Claim 10 A method for predicting the risk of early persistence of food allergy, characterized in that, in claim 7, the prediction is intended to determine the risk that a food allergy that developed in infancy will persist until after the school-age period.

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