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WO2016094961A1 - Essai de méthylation - Google Patents

Essai de méthylation Download PDF

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Publication number
WO2016094961A1
WO2016094961A1 PCT/AU2015/050813 AU2015050813W WO2016094961A1 WO 2016094961 A1 WO2016094961 A1 WO 2016094961A1 AU 2015050813 W AU2015050813 W AU 2015050813W WO 2016094961 A1 WO2016094961 A1 WO 2016094961A1
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Prior art keywords
methylation
cpg sites
methylation status
allergy
food allergy
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PCT/AU2015/050813
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English (en)
Inventor
David MARTINO
Richard Saffery
Katie ALLEN
Mimi TANG
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Murdoch Childrens Research Institute
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Murdoch Childrens Research Institute
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Priority claimed from AU2014905153A external-priority patent/AU2014905153A0/en
Application filed by Murdoch Childrens Research Institute filed Critical Murdoch Childrens Research Institute
Publication of WO2016094961A1 publication Critical patent/WO2016094961A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • the present disclosure relates to methods and assays for detecting a food allergy or a propensity to develop a food allergy in a subject based on gene methylation status.
  • OFC oral food challenges
  • the present inventors have identified that various sites within the genome are differentially methylated in subjects with food allergy or a propensity to develop food allergy. Surprisingly, the present inventors have found that the methylation status of the identified sites is predictive of food allergy or a propensity to develop food allergy in a subject.
  • the identified sites are often referred to as "CpG sites” or "CG sites” or “CpNpG sites”. These sites are regions of DNA where a cytosine nucleotide occurs next to a guanine nucleotide in the linear sequence of bases along its length.
  • the present disclosure provides an in-vitro method to detect a food allergy in a subject.
  • the present disclosure provides an in-vitro method to detect a propensity to develop a food allergy in a subject.
  • the present inventors have also identified that the predictive power of the identified CpG sites is additive. Accordingly, the methylation status of multiple sites may be combined to produce a multivariate methylation pattern or methylation signature with improved accuracy or predictive power for diagnosing a food allergy or a propensity to develop a food allergy in a subject.
  • methylation patterns or signatures comprising a plurality of the methylation sites disclosed herein may be assessed to detect a food allergy or a propensity to develop a food allergy in a subject.
  • the present disclosure provides an in-vitro method to detect a food allergy or a propensity to develop a food allergy in a subject, the method comprising, determining the methylation status of at least 2 CpG sites in DNA obtained from the subject, the CpG sites being selected from the group shown in Table 2, and detecting a food allergy or the propensity to develop a food allergy based on a methylation status of the CpG sites.
  • the present disclosure relates to an in-vitro method to determine a subjects response to allergen immunotherapy, the method comprising, determining the methylation status of at least 2 CpG sites in DNA obtained from the subject, the CpG sites being selected from the group shown in Table 2, and determining whether the subject has responded to allergen immunotherapy based on the methylation status of the CpG sites.
  • the methylation status of at least 5 CpG sites may be determined.
  • the methylation status of at least 10 CpG sites are determined.
  • the methylation status of at least 15 CpG sites are determined.
  • the methylation status of at least 20 CpG sites are determined.
  • the methylation status of at least 50 CpG sites are determined.
  • the present inventors found that the methylation status of some CpG sites were more predictive than others for detecting a food allergy or a propensity to develop a food allergy in a subject. Accordingly, in one example, the CpG sites are selected from the group shown in Table 3. In another example, the CpG sites are selected from the group shown in Table 4. In another example, the CpG sites are selected from the group shown in Table 5.
  • the methylation status of the CpG sites selected from the group shown in Table 2 are determined, wherein the CpG sites reside in or across the genes shown in Table 2.
  • the methylation status of the CpG sites selected from the group shown in Table 2 are determined, wherein the CpG sites reside in or across genes in the mitogen activated protein (MAP) kinase pathway.
  • MAP mitogen activated protein
  • determining the methylation status involves comparing the methylation status of the CpG sites in DNA obtained from the subject relative to the methylation status of the CpG sites in DNA obtained from a control.
  • the control is a positive control and no change in the methylation status relative to the control is indicative of the presence of food allergy or a propensity to develop food allergy.
  • the control is a negative control and a change in the methylation status relative to the negative control is indicative of food allergy or a propensity to develop food allergy.
  • determining the methylation status comprises calculating the methylation ratio of each CpG site and/or gene assessed. In another example, determining the methylation status further comprises calculating the sum of the methylation ratios for each CpG site and/or gene assessed to obtain a methylation score. In another example, the indicative change in the methylation status relative to the control is an increase in methylation status and/or methylation score relative to a negative control.
  • Methylation status may be determined using an assay selected from the group consisting of bisulfite MALDI-TOF methylation, methylation sensitive PCR, methylation specific melting curve analysis (MS-MCA), high resolution melting (MS- HRM), MALDI-TOF MS, methylation specific MLPA; combination of methylated - DNA precipitation and methylation-sensitive restriction enzymes (COMPARE-MS), methylation sensitive oligonucleotide microarray, antibody immunoprecipitation, pyrosequencing, NEXT generation sequencing, DEEP sequencing.
  • an assay selected from the group consisting of bisulfite MALDI-TOF methylation, methylation sensitive PCR, methylation specific melting curve analysis (MS-MCA), high resolution melting (MS- HRM), MALDI-TOF MS, methylation specific MLPA; combination of methylated - DNA precipitation and methylation-sensitive restriction enzymes (COMPARE-MS), methylation sensitive oligonucleotide
  • the food allergy detected using the methods of the present disclosure is selected from the group consisting of milk, buckwheat, egg, tree nuts, peanut, fish, shellfish, fruit, yeast, onion, tomato, legume, lupin, garlic, oats, peppers, gluten, tartazine, sulfites, sesame, wheat, rice, corn, soy, meats, meat substitutes.
  • the food allergy can be a peanut allergy.
  • the food allergy can be an egg or egg white allergy.
  • the DNA used in the methods of the present disclosure is isolated from a biological sample selected from the group consisting of blood (including whole blood), blood mononuclear cells, blood plasma, blood serum, hemolysate, lymph, synovial fluid, spinal fluid, urine, cerebrospinal fluid, stool, sputum, mucus, amniotic fluid, lacrimal fluid, cyst fluid, sweat gland secretion, bile, milk, tears, saliva, earwax, B cells, dendritic cells, granulocytes ,innate lymphoid cells (ILCs), megakaryocytes, monocytes/macrophages, natural killer (NK) cells ,platelets, red blood cells (RBCs), T cells, thymocytes.
  • blood including whole blood
  • blood mononuclear cells blood plasma
  • blood serum hemolysate
  • lymph synovial fluid
  • spinal fluid urine
  • cerebrospinal fluid spinal fluid
  • stool sputum
  • mucus amniotic fluid
  • the methods of the present disclosure further comprise, performing an additional test for food allergy selected from the group consisting of skin prick test, slgE test, component diagnostic tests.
  • the additional test is a skin prick test.
  • a food allergy or a propensity to develop a food allergy is detected based on the methylation status of the CpG sites and the results of the additional test.
  • the methods of the present disclosure are used to resolve an inconclusive diagnostic test for allergy such as a skin prick test.
  • the methods of the present disclosure can be used to detect a food allergy or the propensity to develop a food allergy based on the methylation status of the CpG sites in a subject with a resulting skin prick test that produces a wheal size of between about 2mm and about 8mm in response to an allergen.
  • the methods of the present disclosure can be used to detect a food allergy or the propensity to develop a food allergy based on the methylation status of the CpG sites in a subject with a resulting skin prick test that produces a wheal size of between about 3mm and about 7mm in response to an allergen.
  • Wheal size indicative of an inconclusive skin prick test can vary depending on the allergen.
  • the methods of the present disclosure can be used to detect peanut allergy or the propensity to develop peanut allergy based on the methylation status of the CpG sites in a subject with a resulting skin prick test that produces a wheal size of between about 2mm and about 8mm in response to peanut allergen.
  • the methods of the present disclosure can be used to detect egg allergy or the propensity to develop egg allergy based on the methylation status of the CpG sites in a subject with a resulting skin prick test that produces a wheal size of between about 2mm and about 4mm in response to egg allergen.
  • the additional test is an sIgE test.
  • the methods of the present disclosure are used to resolve an inconclusive sIgE test.
  • the methods of the present disclosure can be used to detect peanut allergy or the propensity to develop peanut allergy based on the methylation status of the CpG sites in a subject with an sIgE test that provides peanut sIgE between about 0.30 kUA/L and about 15 kUA/L in response to peanut allergen.
  • the methods of the present disclosure can be used to detect egg allergy or the propensity to develop egg allergy based on the methylation status of the CpG sites in a subject with an sIgE test that produces egg sIgE between about 0.30 kUA/L and about 1.20kUA/L in response to egg allergen.
  • kits comprising:
  • the present disclosure relates to a microarray having probes and/or primers able to determine the methylation status of at least 2 CpG sites selected from the group shown in Table 2.
  • the present disclosure encompasses a system configured to perform the methods of the present disclosure.
  • the present disclosure relates to a method of treating allergy comprising, detecting a food allergy or the propensity to develop a food allergy using the methods of the present disclosure and administering allergen immunotherapy.
  • genomic DNA can be sent for assessment of methylation and subsequently assessed to detect a food allergy or a propensity to develop a food allergy in a test subject using a computer implemented method.
  • the present disclosure provides a computer implemented method of detecting a food allergy or a propensity to develop a food allergy in a test subject, the method operable in a computing system comprising a processor and a memory, the method comprising:
  • the receiving comprises receiving data indicating the methylation status of the CpG sites relative to the control from a user interface coupled to the computing system.
  • the receiving comprises receiving data indicating the methylation status of the CpG sites relative to the control from a remote device across a wireless communications network.
  • the user interface or remote device may be a methylation array platform.
  • outputting comprises outputting information to a user interface coupled to the computing system.
  • outputting comprises transmitting information to a remote device across a wireless communications network.
  • the present disclosure relates to a computer program product comprising a non-transitory computer readable medium with computer executable instructions, which when executed is effective to carry out the methods of the present disclosure.
  • FA food allergy
  • FS food sensitized
  • y-axis is Beta methylation value
  • e MDS analysis of validation study.
  • Top panel shows sample relationships based on all somatic probes
  • PC principal component of variation.
  • Figure 2 Sensitivity and specificity analysis of DNAm patient scores, (a) Distribution of methylation ratios for the 96 CpG signature stratified by phenotype. (b) Boxplots of total patient DNAm scores showing median and range. Statistical analysis by Man- Whitney test, (c) ROC curve analysis of DNAm scores for predicting clinical allergy between different groups (d) Performance comparisons of DNAm scores against serum IgE measures (e) Performance comparisons of DNAm scores against egg skin prick test wheal size among egg sensitized individuals (f) Performance comparisons of DNAm scores against peanut skin prick test wheal size among peanut sensitized individuals
  • Figure 3 Starburst plot of patient methylation scores for discovery (left) and replication cohorts (right). Concentric numerals denotes sample number, patient scores are shown on the vertical axis in bold. Patient methylation scores derived from CD4+ T-cells (red) were consistently lower than scores derived from total PBMCS (blue) however; differences between phenotype classes were conserved in each cohort. Solid circles are visual guides for diagnostic cutoffs determined by sensitivity analysis.
  • the CpG sites of the present disclosure encompass related sites in linkage disequilibrium. Moreover, determining the methylation status of the CpG sites of the present disclosure includes determining the methylation status of other markers in linkage disequilibrium with the particular CpG sites.
  • an assay is an investigative (analytic) procedure or method for qualitatively assessing or quantitatively measuring the presence or amount or the functional activity of a target. For example, an assay can assess methylation of various CpG sites.
  • a method or assay according to the present disclosure may be incorporated into a treatment regimen.
  • a method of treating allergy in a subject in need thereof may comprise performing an assay that embodies the methods of the present disclosure.
  • a clinician or similar may wish to perform or request performance of an assay according to the present disclosure before administering or modifying treatment to a patient.
  • a clinician may perform or request performance of an assay according to the present disclosure on a subject before electing to administer or modify therapy such as allergen immunotherapy.
  • composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.
  • the term "food allergy” is used in the context of the present disclosure to refer to allergies caused by exposure or ingestion of food allergens.
  • the methods of the present disclosure may be used to detect a food allergy or propensity to develop a food allergy to allergens such as milk, buckwheat, egg, tree nuts, peanut, fish, shellfish, fruit, yeast, onion, tomato, legume, lupin, garlic, oats, peppers, gluten, tartazine, sulfites, sesame, wheat, rice, corn, soy, meats, meat substitutes.
  • the food allergy detected using the methods of the present disclosure is a legume allergy.
  • the allergy is an peanut allergy.
  • the allergy is an egg allergy.
  • the allergy is an egg white allergy.
  • the methods of the present disclosure are used to detect a food allergy or the propensity to develop a food allergy in a subject based on methylation status.
  • methylation status is used to indicate whether a particular site or gene is methylated or not.
  • the term “methylation status” encompasses methylation status or hydroxymethylation status of "— C— phosphate— G— " (CpG) sites or "— C— phosphate— any base (N)— phosphate— G— " (CpNpG) sites and genes.
  • the term “methylation status” also encompasses methylation status of non CpG sites or non-CG methylation.
  • methylation status can be determined using assays such as bisulfite MALDI-TOF methylation, methylation sensitive PCR, methylation specific melting curve analysis (MS-MCA), high resolution melting (MS-HRM), MALDI-TOF MS, methylation specific MLPA; combination of methylated-DNA precipitation and methylation- sensitive restriction enzymes (COMPARE-MS), methylation sensitive oligonucleotide microarray, antibody immunoprecipitation, pyrosequencing, NEXT generation sequencing, DEEP sequencing.
  • assays such as bisulfite MALDI-TOF methylation, methylation sensitive PCR, methylation specific melting curve analysis (MS-MCA), high resolution melting (MS-HRM), MALDI-TOF MS, methylation specific MLPA; combination of methylated-DNA precipitation and methylation- sensitive restriction enzymes (COMPARE-MS), methylation sensitive oligonucleotide microarray, antibody immunoprecipitation, p
  • DNA obtained from a subject can be modified relative to its naturally occurring counterpart before determining methylation status.
  • genomic DNA isolated from a subject can be treated with bisulfite for a time and under conditions sufficient to convert non-methylated cytosine to uracils.
  • DNA can be digested with methylation sensitive restriction endonucleases.
  • DNA can amplified to produce cDNA. Methylation status of treated/digested DNA or cDNA can then be assessed via sequencing or via a suitable amplification based system such as those discussed above.
  • the methylation status of multiple sites will be assessed.
  • the methylation status of the CpG sites of the present disclosure can be combined to produce a multivariate methylation pattern or methylation signature indicative of a food allergy or a propensity to develop a food allergy in a subject.
  • Such a pattern or signature can be used as a comparative reference for determining whether a subject has an allergy or a propensity to develop an allergy.
  • the methylation status of at least 2 CpG sites selected from the group shown in Table 2 are determined.
  • the methylation status of at least 5 CpG sites selected from the group shown in Table 2 are determined.
  • the methylation status of at least 10 CpG sites selected from the group shown in Table 2 are determined. In another example, the methylation status of at least 20 CpG sites selected from the group shown in Table 2 are determined. In another example, the methylation status of at least 50 CpG sites selected from the group shown in Table 2 are determined. In another example, the methylation status of at least 70 CpG sites selected from the group shown in Table 2 are determined. In another example, the methylation status of at least 90 CpG sites selected from the group shown in Table 2 are determined. In another example, the methylation status of at least 96 CpG sites selected from the group shown in Table 2 are determined.
  • the methylation status of at least 100 CpG sites selected from the group shown in Table 2 are determined. In another example, the methylation status of at least 110 CpG sites selected from the group shown in Table 2 are determined. In another example, the methylation status of at least 120 CpG sites selected from the group shown in Table 2 are determined. In another example, the methylation status of at least 140 CpG sites selected from the group shown in Table 2 are determined. In another example, the methylation status of at least 160 CpG sites selected from the group shown in Table 2 are determined. In another example, the methylation status of at least 180 CpG sites selected from the group shown in Table 2 are determined.
  • the methylation status of at least 200 CpG sites selected from the group shown in Table 2 are determined. In another example, the methylation status of at least 220 CpG sites selected from the group shown in Table 2 are determined. In another example, the methylation status of at least 240 CpG sites selected from the group shown in Table 2 are determined. In another example, the methylation status of at least 260 CpG sites selected from the group shown in Table 2 are determined. In another example, the methylation status of at least 280 CpG sites selected from the group shown in Table 2 are determined. In another example, the methylation status of at least 300 CpG sites selected from the group shown in Table 2 are determined.
  • the methylation status of at least 320 CpG sites selected from the group shown in Table 2 are determined. In another example, the methylation status of at least 340 CpG sites selected from the group shown in Table 2 are determined. In another example, the methylation status of at least 360 CpG sites selected from the group shown in Table 2 are determined. In another example, the methylation status of at least 380 CpG sites selected from the group shown in Table 2 are determined. In another example, the methylation status of at least 400 CpG sites selected from the group shown in Table 2 are determined. In another example, the methylation status of at least 420 CpG sites selected from the group shown in Table 2 are determined.
  • the methylation status of at least 440 CpG sites selected from the group shown in Table 2 are determined. In another example, the methylation status of at least 460 CpG sites selected from the group shown in Table 2 are determined. In another example, the methylation status of at least 480 CpG sites selected from the group shown in Table 2 are determined. In another example, the methylation status of at least 500 CpG sites selected from the group shown in Table 2 are determined. In another example, the methylation status of at least 520 CpG sites selected from the group shown in Table 2 are determined. In another example, the methylation status of at least 540 CpG sites selected from the group shown in Table 2 are determined.
  • the methylation status of at least 560 CpG sites selected from the group shown in Table 2 are determined. In another example, the methylation status of at least 580 CpG sites selected from the group shown in Table 2 are determined. In another example, the methylation status of at least 600 CpG sites selected from the group shown in Table 2 are determined. In another example, the methylation status of at least 620 CpG sites selected from the group shown in Table 2 are determined. In another example, the methylation status of at least 640 CpG sites selected from the group shown in Table 2 are determined. In another example, the methylation status of at least 649 CpG sites selected from the group shown in Table 2 are determined.
  • the methylation status of at least 2 CpG sites selected from the group shown in Table 3 are determined. In another example, the methylation status of at least 5 CpG sites selected from the group shown in Table 3 are determined. In another example, the methylation status of at least 10 CpG sites selected from the group shown in Table 3 are determined. In another example, the methylation status of at least 15 CpG sites selected from the group shown in Table 3 are determined. In another example, the methylation status of at least 20 CpG sites selected from the group shown in Table 3 are determined. In another example, the methylation status of at least 25 CpG sites selected from the group shown in Table 3 are determined.
  • the methylation status of at least 30 CpG sites selected from the group shown in Table 3 are determined. In another example, the methylation status of at least 35 CpG sites selected from the group shown in Table 3 are determined. In another example, the methylation status of at least 40 CpG sites selected from the group shown in Table 3 are determined. In another example, the methylation status of at least 45 CpG sites selected from the group shown in Table 3 are determined. In another example, the methylation status of at least 50 CpG sites selected from the group shown in Table 3 are determined. In another example, the methylation status of at least 55 CpG sites selected from the group shown in Table 3 are determined.
  • the methylation status of at least 60 CpG sites selected from the group shown in Table 3 are determined. In another example, the methylation status of at least 65 CpG sites selected from the group shown in Table 3 are determined. In another example, the methylation status of at least 70 CpG sites selected from the group shown in Table 3 are determined. In another example, the methylation status of at least 75 CpG sites selected from the group shown in Table 3 are determined. In another example, the methylation status of at least 80 CpG sites selected from the group shown in Table 3 are determined. In another example, the methylation status of at least 81 CpG sites selected from the group shown in Table 3 are determined.
  • the methylation status of at least 82 CpG sites selected from the group shown in Table 3 are determined. In another example, the methylation status of at least 83 CpG sites selected from the group shown in Table 3 are determined. In another example, the methylation status of at least 84 CpG sites selected from the group shown in Table 3 are determined. In another example, the methylation status of at least 85 CpG sites selected from the group shown in Table 3 are determined. In another example, the methylation status of at least 86 CpG sites selected from the group shown in Table 3 are determined. In another example, the methylation status of at least 87 CpG sites selected from the group shown in Table 3 are determined.
  • the methylation status of at least 88 CpG sites selected from the group shown in Table 3 are determined. In another example, the methylation status of at least 89 CpG sites selected from the group shown in Table 3 are determined. In another example, the methylation status of at least 90 CpG sites selected from the group shown in Table 3 are determined. In another example, the methylation status of at least 91 CpG sites selected from the group shown in Table 3 are determined. In another example, the methylation status of at least 92 CpG sites selected from the group shown in Table 3 are determined. In another example, the methylation status of at least 93 CpG sites selected from the group shown in Table 3 are determined.
  • the methylation status of at least 94 CpG sites selected from the group shown in Table 3 are determined. In another example, the methylation status of at least 95 CpG sites selected from the group shown in Table 3 are determined. In another example, the methylation status of at least 96 CpG sites selected from the group shown in Table 3 are determined.
  • the methylation status of at least 2 CpG sites selected from the group shown in Table 4 are determined. In another example, the methylation status of at least 3 CpG sites selected from the group shown in Table 4 are determined. In another example, the methylation status of at least 4 CpG sites selected from the group shown in Table 4 are determined. In another example, the methylation status of at least 4 CpG sites selected from the group shown in Table 4 are determined. In another example, the methylation status of at least 5 CpG sites selected from the group shown in Table 4 are determined. In another example, the methylation status of at least 6 CpG sites selected from the group shown in Table 4 are determined.
  • the methylation status of at least 7 CpG sites selected from the group shown in Table 4 are determined. In another example, the methylation status of at least 8 CpG sites selected from the group shown in Table 4 are determined. In another example, the methylation status of at least 9 CpG sites selected from the group shown in Table 3 are determined. In another example, the methylation status of 10 CpG sites selected from the group shown in Table 4 are determined.
  • the methylation status of at least 2 CpG sites selected from the group shown in Table 5 are determined. In another, example the methylation status of at least 5 CpG sites selected from the group shown in Table 5 are determined. In another example, the methylation status of at least 10 CpG sites selected from the group shown in Table 5 are determined. In another example, the methylation status of at least 15 CpG sites selected from the group shown in Table 5 are determined. In another example, the methylation status of at least 20 CpG sites selected from the group shown in Table 5 are determined. In another example, the methylation status of at least 25 CpG sites selected from the group shown in Table 5 are determined.
  • the methylation status of at least 30 CpG sites selected from the group shown in Table 5 are determined. In another example, the methylation status of at least 35 CpG sites selected from the group shown in Table 5 are determined. In another example, the methylation status of at least 40 CpG sites selected from the group shown in Table 5 are determined. In another example, the methylation status of at least 45 CpG sites selected from the group shown in Table 5 are determined. In another example, the methylation status of 50 CpG sites selected from the group shown in Table 5 are determined.
  • CpG sites can reside within or overlapping genes and regulatory regions thereof.
  • CpG sites may reside upstream of genes important for mounting an allergic response to an allergen.
  • the methods of the present disclosure encompass assessing methylation sites in coding and non-coding regions such as introns, in or across intron/exon boundaries, in or across splicing regions of the gene transcripts.
  • the methods of the present disclosure can encompass assessing methylation status of genes. Exemplary genes that may be assessed using the methods of the present disclosure are provided in Tables 2, 3, 5 and 6.
  • the methods of the present disclosure encompass assessing the methylation status of one or more genes selected from the group shown in Table 2. For example, the methylation status of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at
  • All selected CpG sites of the present disclosure need not be completely methylated to indicate allergy or a propensity to develop allergy.
  • predictive CpG methylation status can range from about 10 % to about 90 % methylation of CpG sites in a particular gene or regulatory region thereof.
  • predictive CpG methylation status can range from about 20 % to about 80 % methylation of CpG sites in a particular gene or regulatory region thereof.
  • predictive CpG methylation status can range from about 25 % to about 75 % methylation of CpG sites in a particular gene or regulatory region thereof.
  • predictive CpG methylation status can range from about 30 % to about 70 % methylation of CpG sites in a particular gene or regulatory region thereof.
  • predictive CpG methylation status is at least about 10 % methylation, at least about 15 % methylation, at least about 20 % methylation, at least about 25 % methylation, at least about 30 % methylation, at least about 35 % methylation, at least about 40 % methylation, at least about 45 % methylation, at least about 55 % methylation, at least about 60 % methylation, at least about 65 % methylation, at least about 70 % methylation, at least about 75 % methylation, at least about 80 % methylation, at least about 85 % methylation, at least about 90 % methylation, at least about 95 % methylation of CpG sites in a particular gene or regulatory region thereof.
  • predictive CpG methylation status is 100% methyl
  • determining the methylation status comprises calculating the ratio between methylated and un-methylated alleles for each CpG site and/or gene assessed.
  • the ratio based on the methylated and un-methylated status can be represented as:
  • methylation status for each allele is determined using an Infinium HumanMethylation450 BeadChip as exemplified below.
  • the ratio based on the methylated and un-methylated intensity can be represented as:
  • the process of determining the methylation ratio can be performed for each CpG assessed and the resulting ratios can be added together to provide a methylation score.
  • a methylation score indicative of a food allergy or the propensity to develop a food allergy will largely depend on the number of CpG sites assessed. For example, when the 96 CpG sites shown in Table 3 are assessed, a methylation score of at least about 30 is indicative of a food allergy or the propensity to develop a food allergy.
  • a methylation score of at least about 31, at least about 32, at least about 33, at least about 34, at least about 35, at least about 36, at least about 37, at least about 38, at least about 39, at least about 40, at least about 41, at least about 42, at least about 43, at least about 44, at least about 45, at least about 46, at least about 46.5, at least about 47, at least about 48, at least about 49, at least about 50, at least about 51, at least about 52, at least about 53, at least about 54, at least about 55, at least about 56, at least about 57, at least about 58, at least about 59, at least about 60 is indicative of a food allergy or the propensity to develop a food allergy.
  • a methylation status indicative of a food allergy or the propensity to develop a food allergy can be identified by assessing the CpG sites of the present disclosure relative to a control.
  • the control sample may be a biological sample obtained from a subject either positive (positive control) or negative (negative control) for allergy. As one of skill in the art would appreciate, the control sample is dictated by the test or experimental sample in that it must provide the necessary comparison for detecting allergy.
  • the control sample could be negative for the allergy being tested, a negative control.
  • the control sample may be from a healthy mammal that has no symptoms of the allergy (e.g. does not mount an allergic response to the food allergen).
  • the negative control is not allergy free but rather has an alternative allergy to the allergy being detected.
  • the negative control does not have a food allergy.
  • a change in the methylation status in the test sample relative to the control is indicative of the presence of the allergy or a propensity to develop the allergy.
  • an increase in methylation status or score relative to the negative control is indicative of allergy or propensity to develop a food allergy.
  • control sample is positive for the allergy being tested.
  • control sample provides a comparative or baseline level of methylation that indicates the presence of a food allergy or the propensity to develop a food allergy.
  • a test sample having comparative or increased levels of methylation relative to the positive control indicates that the subject has the allergy or a propensity to develop the allergy.
  • the positive control is a statistically validated standard.
  • control sample may consist of a plurality of samples.
  • a plurality of control samples are used to establish a robust baseline or threshold level of methylation that can be compared with a test sample to detect a food allergy of the propensity to develop a food allergy.
  • the control sample may include samples positive for various different allergies.
  • the control may comprise samples obtained from individuals known to have peanut, egg, milk or shellfish allergies.
  • the control samples can be used to provide a control panel that can be compared against a sample from a subject to detect the presence or absence of a series of allergies or a propensity to develop a series of allergies.
  • Various methods can be used to determine a change in the methylation status in the test sample relative to the control. For example, a change may be evident from a side by side comparison of methylation status between a test sample and a control(s).
  • methylation status of test samples and controls can be compared statistically to identify a statistically significant difference in methylation status.
  • sample In performing the methods of the present disclosure, a sample from a subject is required. It is considered that terms such as “sample” and “specimen” are terms that can, in context, be used interchangeably in the present disclosure.
  • the sample used in the present disclosure can be a biological sample from a human. Any biological material can be used as the above-mentioned sample so long as it can be collected from the subject and DNA can be isolated and analysed according to the methods of the present disclosure.
  • the sample may be selected from the group consisting of blood (including whole blood), blood plasma, blood serum, hemolysate, lymph, synovial fluid, spinal fluid, urine, cerebrospinal fluid, stool, sputum, mucus, amniotic fluid, lacrimal fluid, cyst fluid, sweat gland secretion, bile, milk, tears, saliva, earwax.
  • a sample such as a blood sample may be treated to remove particular cells using various methods such as such centrifugation, affinity chromatography (e.g. immunoabsorbent means), immuno selection and filtration.
  • the sample can comprise a specific cell type or mixture of cell types isolated directly from the subject or purified from a sample obtained from the subject (e.g. purifying T-cells from whole blood).
  • the biological sample is peripheral blood mononuclear cells (pBMC).
  • the sample may be selected from the group consisting of B cells, dendritic cells, granulocytes, innate lymphoid cells (ILCs), megakaryocytes, monocytes/macrophages, natural killer (NK) cells ,platelets, red blood cells (RBCs), T cells, thymocytes.
  • pBMC can be purified from whole blood using various known Ficoll based centrifugation methods (e.g. Ficoll-Hypaque density gradient centrifugation).
  • Other cells such as T-cells can also be purified by selecting for the appropriate phenotype using techniques such as immunomagnetic cell sorting (e.g.
  • T-cells can be purified using a two step selection process that firstly removes CD8+ cells and then selects CD4+ cells. Cell population purity can be confirmed by assessing the appropriate markers such as CD19-FITC, CD3-PE, CD8-PerCP, CDl lc-PE Cy7, CD4-APC and CD14-APC Cy7 using commercially available antibodies (e.g. BD Biosciences).
  • markers such as CD19-FITC, CD3-PE, CD8-PerCP, CDl lc-PE Cy7, CD4-APC and CD14-APC Cy7 using commercially available antibodies (e.g. BD Biosciences).
  • DNA is extracted from the sample for methylation analysis.
  • the DNA is genomic DNA.
  • genomic DNA Various methods of isolating DNA, in particular genomic DNA are known to those of skill in the art. In general, known methods involve disruption and lysis of the starting material followed by the removal of proteins and other contaminants and finally recovery of the DNA. For example, techniques involving alcohol precipitation; organic phenol/chloroform extraction and salting out have been used for many years to extract and isolate DNA.
  • DNA isolation is exemplified below (e.g. Qiagen All-prep kit). However, there are various other commercially available kits for genomic DNA extraction (Life technologies; Sigma). Purity and concentration of DNA can be assessed by various methods, for example, spectrophotometry.
  • a "reflexive test” or “reflex test” can refer to a subsequent test (e.g., a second test) that is undertaken based upon the results obtained in a previous test (e.g., a first test).
  • a subsequent test e.g., a second test
  • a previous test e.g., a first test.
  • positive skin prick or IgE-based (e.g. total IgE, specific-IgE, component IgE) testing can lead to a desire to perform a further, more conclusive test to determine whether a subject has a food allergy or a propensity to develop the allergy.
  • the desire to perform an additional test is driven by previous finding that the majority of individuals that test positive for skin prick or IgE tests do not have a food allergy to the tested allergen.
  • the methods of the present disclosure can be used as a reflex test from a positive or inconclusive skin prick or slgE test.
  • the results of the claimed method may also direct the performance of one or more additional tests. For example, determining methylation status according to the present disclosure and detecting a food allergy in a subject can lead to a desire to perform a further "gold standard" food challenge test to confirm that a subject has a food allergy.
  • the methods of the present disclosure can be used as a reflex test to an oral food challenge.
  • a diagnostic determination regarding a food allergy or the propensity to develop and allergy can be made by determining methylation status according to the present disclosure.
  • the diagnostic determination may or may not be conclusive with respect to the definitive diagnosis upon which a treating physician will determine a course of treatment or intervention.
  • the methods of the present disclosure can be used in combination with other methods or "additional test(s)" of clinical assessment known in the art (e.g. IgE-targeted tests) in providing an evaluation of the presence of a food allergy or an increased risk of developing an allergy.
  • the methods of the present disclosure may also be performed as an adjunctive test.
  • an "adjunctive test” provides information that adds to or assists in the interpretation of the results of other tests, and/or provides information useful for confirming or resolving an inconclusive test.
  • a skin patch test, slgE test or similar may reveal that a subject has a food allergy or propensity to develop an allergy. Such an assessment is generally inconclusive and requires confirmation.
  • the methylation status can also be determined using the methods of the present disclosure as an adjunct to the skin prick, slgE or similar IgE-based tests.
  • a wheal size less than about 2 mm is indicative that the subject is not allergic to the particular allergen and no further testing is required.
  • a wheal size of greater than about 2 mm suggests that the subject may be clinically sensitised to a particular allergen.
  • a skin prick test is generally inconclusive with a wheal size greater than between about 2 mm (all food allergies) and less than about 4mm (egg allergy) or 8 mm (peanut allergy).
  • a large number of subjects with a wheal size greater than about 2 to 3mm and less than about 7 to 8mm may be diagnosed as clinically sensitised to an allergen and subsequently prescribed an unnecessary avoidance program or additional testing such as an oral food challenge.
  • the methods of the present disclosure may be used to reduce implementation of unnecessary avoidance programs or additional testing such as oral food challenge.
  • the methods of the present disclosure may be used to resolve an inconclusive skin prick test or slgE test, or to resolve inconclusive diagnoses based on a combination of the two.
  • the present disclosure encompasses a method of detecting a food allergy or a propensity to develop a food allergy in a subject, wherein a skin prick test performed on the subject produces a wheal size of between about 2mm and about 8mm.
  • a skin prick test performed on the subject produces a wheal size of between about 3mm and about 7mm, between about 4mm and about 6mm.
  • the subjects skin prick test produces a wheal size of about 2mm, about 3mm, about 4mm, about 5mm, about 6mm, about 7mm, about 8mm, about 9mm, about 10 mm.
  • the present disclosure encompasses a method of detecting a food allergy or a propensity to develop a food allergy in a subject, wherein a slgE test performed on the subject provides peanut slgE between about 0.30 kUA/L and about 15 kUA/L in response to peanut allergen or between about 0.30 kUA/L and about 1.20 kUA/L in response to egg allergen.
  • the present disclosure encompasses a method of detecting a food allergy or a propensity to develop a food allergy in a subject, wherein a slgE test performed on the subject provides peanut slgE between about 0.35 kUA/L and about 14.9 kUA/L in response to peanut allergen or between about 0.35 kUA/L and about 1.17 kUA/L in response to egg allergen.
  • adjunctive testing methylation status can be determined at or about the same time as the additional test (e.g. skin prick or slgE tests). However, these steps may be performed separately.
  • each test may be performed on separate samples obtained from the same subject.
  • a subject may provide two samples with one being sent for assessment using the methods of the present disclosure and the other for assessment using a slgE test.
  • obtaining additional samples for further testing can be difficult and can delay further testing that is desirable.
  • additional samples need to be processed prior to testing, it is desirable to minimize the costs associated with processing (e.g., reagents, and the like). Accordingly, when performing the methods of the present disclosure in combination with an additional test each test may be performed on the same sample obtained from the patient.
  • the "subject" can be any organism which can have an allergy.
  • the subject is a mammal.
  • the mammal may be a companion animal such as a dog or cat, or a livestock animal such as a horse or cow.
  • the subject is a human.
  • the human subject can be a child.
  • Terms such as "subject", “patient” or “individual” are terms that can, in context, be used interchangeably in the present disclosure.
  • the methods of the present disclosure can be used for routine screening of subjects.
  • the methods of the present disclosure can be used to detect a food allergy or the propensity to develop a food allergy in a subject with symptoms that may be indicative of an allergy.
  • the present disclosure would be applicable to a subject presenting to the clinic with symptoms such as tingling or itching in the mouth, hives, itching or eczema, swelling of the lips, face, tongue and throat or other parts of the body, wheezing, nasal congestion or trouble breathing, abdominal pain, diarrhoea, nausea or vomiting, dizziness, light headedness or fainting.
  • a subject In all cases, a subject must first be exposed to an allergen to develop antibodies, which then react to further exposures. Such subjects are referred to as being “sensitised” due to the presence of Immunoglobulin E (IgE) antibodies.
  • IgE antibodies specific to food allergens are detectable and such subjects are referred to as being food “sensitised”.
  • a food "sensitised” subject has a positive skin prick test for a food allergen and a negative oral food challenge outcome for that same allergen.
  • the methods of the present disclosure may be used to detect whether a "sensitised” subject, such as a food sensitised subject has a food allergy or the propensity to develop an allergy.
  • the immune system of a "sensitised" subject can react to an allergen by way of an immune response characterised by the excessive activation of specific white blood cells by allergen binding to cognate IgE present on the surface of these cells.
  • Subjects with an immune system that reacts to an allergen are known as "allergic" to the allergen.
  • an "allergic" subject has a positive skin prick test for an allergen and a positive oral food challenge outcome for the allergen.
  • the methods of the present disclosure may also be used to detect whether a subject is allergic to a particular food. Accordingly, the methods of the present disclosure may be used to detect whether a subject, "allergic" or "sensitized", has a food allergy or the propensity to develop an allergy.
  • the methods of the present disclosure may be used to determine whether a subject does not have a food allergy based on the methylation status of the CpG sites of the present disclosure.
  • the disclosed methods can be used to identify subjects that are not "allergic" for an allergen.
  • the disclosed methods can be used to identify subjects that are not food "reactive” or food “sensitised”.
  • the methylation status of the CpG sites of the present disclosure would indicate the lack of an allergy.
  • the methods of the present disclosure can be incorporated into methods of treating food allergy. If a food allergy or a propensity to develop a food allergy is detected in a subject using the methods of the present disclosure the subject can be directed or prescribed an appropriate treatment for the allergy. For example, allergy detected using the methods of the present disclosure may be treated with a pharmacological agent.
  • Suitable exemplary therapies include, administration of an antihistamine or allergen immunotherapy or IgE neutralization using anti-IgE monoclonal antibody or vaccination with an allergen peptide.
  • WO 2009/094717 Another exemplary therapy is described in WO 2009/094717 and relates to the administration of a probiotic and an appropriate food allergen to induce tolerance to the food allergen.
  • the probiotic may be from a species such as Lactobacillus, Bifidobacterium, Escherichia, Saccharomyces, Streptococcus or Bacillus or a combination thereof.
  • the appropriate allergen will depend on the allergy being treated. For example, treatment of a peanut allergy will require administration of a peanut allergen.
  • allergy detected using the methods of the present disclosure may be treated by behavioural intervention such as establishing an avoidance program for the subject.
  • the methods of the present disclosure can be used in a pre- screening or prognostic manner to assess whether a subject has an allergy, and if warranted, a further definitive diagnosis can be conducted.
  • a "food challenge” test could be used to obtain a definitive diagnosis of an allergy.
  • the methods of the present disclosure are used to determine the likelihood that a subject will develop a food allergy (prognostic).
  • the methods of the present disclosure can provide measures of relative risk that a subject has a food allergy or a propensity to develop an allergy.
  • the methods of the present disclosure can indicate or determine the therapeutic effectiveness of a drug or therapy (theranostic).
  • the methods of the present disclosure can be used to determine a subjects response to allergen immunotherapy.
  • Allergen immunotherapy also known as desensitisation ⁇ aims to desensitise patients to a particular allergen by administering increasing doses of allergen(s) to accustom a patients body to particular substances.
  • a reduction in methylation of the CpG sites of the present disclosure is indicative of a positive response to allergen immunotherapy.
  • a patient may provide a sample before allergen immunotherapy is initiated and provide additional samples over time as treatment progresses.
  • the initial sample can be used as a baseline and a decrease in methylation indicates that the patient is responding to immunotherapy.
  • a sample can be obtained from patients subject to allergen immunotherapy and compared with a control sample. Such assessments can be repeated at various time points as treatment progresses and/or escalates to detect whether the subject is responding to therapy.
  • the methods of the present disclosure may be implemented by a system.
  • the system is a computer system comprising one or a plurality of processors which may operate together (referred to for convenience as "processor") connected to a memory.
  • the memory may be a non-transitory computer readable medium, such as a hard drive, a solid state disk or CD-ROM.
  • Software that is executable instructions or program code, such as program code grouped into code modules, may be stored on the memory, and may, when executed by the processor, cause the computer system to perform functions such as determining that a task is to be performed to assist a user to determine the methylation status of CpG sites in DNA obtained from the subject, the CpG sites being selected from the present disclosure (e.g Tables 2 to 5); receiving data indicating the methylation status of CpG sites in DNA obtained from the subject; processing the data to detect a food allergy or the propensity to develop a food allergy based on a methylation status of the CpG sites; outputting the presence of the allergy or a propensity to develop the allergy in a subject.
  • the memory comprises program code which when executed by the processor causes the system to determine the methylation status of CpG sites in DNA obtained from the subject, the CpG sites being selected from the present disclosure or receive data indicating the methylation status of the CpG sites in the subject; process the data to detect a food allergy or the propensity to develop a food allergy based on the methylation status of the CpG sites; report the presence of the allergy or a propensity to develop the allergy in a subject.
  • the system may be coupled to a user interface to enable the system to receive information from a user and/or to output or display information.
  • the user interface may comprise a graphical user interface, a voice user interface or a touchscreen.
  • the system may be configured to communicate with at least one remote device or server across a communications network such as a wireless communications network.
  • a communications network such as a wireless communications network.
  • the system may be configured to receive information from the device or server across the communications network and to transmit information to the same or a different device or server across the communications network.
  • the system may be isolated from direct user interaction.
  • performing the methods of the present disclosure to detect a food allergy or a propensity to develop a food allergy in a subject by determining the methylation status of CpG sites in DNA obtained from the subject, enables establishment of a diagnostic or prognostic rule based on the methylation status.
  • the diagnostic or prognostic rule can be based on the methylation status relative to a control.
  • the diagnostic or prognostic rule is based on the application of a statistical and machine learning algorithm.
  • a statistical and machine learning algorithm uses relationships between methylation profiles and disease status observed in training data (with known disease status) to infer relationships which are then used to predict the status of patients with unknown status.
  • An algorithm is employed which provides an index of probability that a patient has a food allergy or propensity to develop an allergy. The algorithm performs a multivariate or univariate analysis function.
  • the present disclosure relates to a knowledge base of training data comprising methylation status at a particular CpG site(s) or in or across a gene(s) in DNA obtained from a subject with a food allergy to generate an algorithm which, upon input of a second knowledge base of data comprising corresponding levels of methylation from a patient with an unknown allergy status, provides a probability that predicts the nature of unknown allergy status or response to treatment.
  • training data can include knowledge of the methylation status in individuals with confirmed allergy status, i.e. allergic status and non-allergic status.
  • the present disclosure relates to a method of allowing a user to determine the status, prognosis and/or treatment response of a subject with an allergy, the method including (a) receiving data indicating the methylation status of the CpG sites in the subject; b) processing the data to determine the methylation status of the CpG sites; and c) outputting the status, prognosis and/or treatment response of a subject.
  • the methylation status provides a correlation to the presence, state, classification, remission or progression of the allergy.
  • the sensitivity achieved by the methods of the present disclosure is at least about 50%, at least about 60%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%.
  • the specificity achieved by the methods of the present disclosure is at least about 50%, at least about 60%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%.
  • the present disclosure relates to a kit comprising PCR primer pairs specifically configured to amplify the CpG sites outlined in the present disclosure (see for example Tables 2, 3, 6 and 7) for use in the methods of the present disclosure.
  • the kit can comprise:
  • kit components may be packaged in or with a suitable solvent or in lyophilised form.
  • kit components may optionally be packaged in a suitable container with written instructions for performing the method of the present disclosure, such as treating genomic DNA isolated from a subject for a time and under conditions sufficient to convert non-methylated cytosine to uracils; digesting and amplifying treated DNA using PCR using primer pairs specifically configured to amplify the CpG sites outlined in the present disclosure (see for example Tables 2, 3, 6 and 7).
  • FA Food Allergic
  • FS Food Sensitized
  • NA Nonallergic control
  • SPT skin prick test
  • OFC oral food challenge.
  • Non-allergic (NA) controls had a negative SPT with a negative oral food challenge outcome.
  • Food sensitized (FS) individuals had a positive skin prick test (either egg or peanut), and a negative OFC outcome (either egg or peanut).
  • Food allergic (FA) individuals had a positive skin prick test (either egg or peanut) and an unequivocal allergic reaction during OFC (either egg or peanut).
  • probes most variably methylated between subgroups of interest were identified using ANOVA.
  • a classifier model trained to predict food challenge outcome was then built from these most variably methylated probes.
  • a nearest shrunken centroid classifier was trained to predict the food challenge outcome group (FA or FS) to which each patient belonged.
  • the 649 predictive CpG were then annotated according to their genomic location to identify overlapping protein coding genes and performed an enrichment test for the gene list.
  • the list was substantially enriched for immunological signatures derived from numerous cell types, including genes differentially expressed between plasma cells and memory B-lymphocytes (VEGFA, TG, TIMP2, PLA2G16, VKORC1) (FDR q-value 7.09 E-04), effector memory and central memory cells (DTX1, MAP3K1, SLC24A6, PKMYT1) (FDR q-value 8.70E-04), Tregs and conventional T-cells (CAMKKl, TRPM1, SCL45A2, ARF4) (FDR q-value 8.70E-04) and genes induced by dendritic cells upon stimulation.
  • NFYC;NFYC;NFYC;NFYC;N cg24700993 0.0187 -0.0171 523.4 chrl 41222901 FYQMIR30C1 cg21382923 0.0187 -0.0171 547.2 chrl 21055163 + SH2D5;SH2D5 cg06657560 0.0183 -0.0168 540.4 chrl2 121841372 + RNF34;RNF34 cg06634552 0.0182 -0.0167 553.4 chr6 13873667 +
  • the 649 probes were then ranked according to their absolute between group difference for the main outcomes of interest (FA v FS).
  • a cutoff for a minimum 5% difference in methylation between groups was applied, a cutoff for which the sample size had reliable detection power, and also within the measurable range of EpiTYPER Mass Spectrometry.
  • the cutoff reduced the list to 96 probes, which overlapped with 72 genes (Table 6).
  • MAP3K1 mitogen-activated protein kinase kinase kinase 1
  • SLC23A2 solute carrier family 23 (nucleobase transporters), member 2
  • HLA-DMA major histocompatibility complex class II, DM alpha
  • MRPS33 mitochondrial ribosomal protein S33
  • SIN3A SIN3 homolog A, transcription regulator (yeast)
  • ZAK sterile alpha motif and leucine zipper containing kinase AZK
  • GNB 1 guanine nucleotide binding protein (G protein), beta polypeptide 1
  • FARP1 FERM RhoGEF (ARHGEF) and pleckstrin domain protein 1 (chondrocyte-derived) ATP 1 OA ATPase, class V, type 10A
  • MAPK8IP1 mitogen-activated protein kinase 8 interacting protein 1
  • LTB lymphotoxin beta (TNF superfamily, member 3)
  • RASGRP2 RAS guanyl releasing protein 2 (calcium and DAG-regulated)
  • APH1B anterior pharynx defective 1 homolog B (C. elegans)
  • CTBP2 C-terminal binding protein 2
  • CACYBP similar to calcyclin binding protein
  • HLA-DQB1 major histocompatibility complex, class II, DQ beta 1
  • MAPK8IP2 mitogen-activated protein kinase 8 interacting protein 2
  • Multidimensional scaling analysis was applied to assess sample relationships based on the predictive methylation signature. MDS analysis of all probes or the top 1000 most variable probes did not discriminate FA from NA groups in this data set, suggesting the principal components of variation are driven by unknown effects.
  • This difference in methylation profiles due to cell type meant that the diagnostics cutoffs determined for total PBMC required recalibration based on total CD4+ T-cells in order to perform predictions.
  • performing the ROC curve analysis on T-cell-derived patient methylation scores produced an area under the curve of 0.8368 (P ⁇ 0.001, CI 0.7139 to 0.9598).
  • a sensitivity analysis suggested a patient score of > 45.52 resulted in a test sensitivity of 87.5% (CI 67.64% to 97.34%) and a specificity of 70.83% (CI 48.91% to 87.38%) with a likelihood ratio of 3.0.
  • the difference in diagnostic cutoffs between the PBMC-derived and T-cell derived patient scores are illustrated in Figure 3.
  • the criterion for a positive food challenge result was at least one of the following signs present during OFC: three or more concurrent noncontact hives (urticarial lesion) lasting for more than 5 min, perioral or periorbital angioedema, vomiting (excluding immediate post-ingestion gag/vomits) or evidence of anaphylaxis as defined by the Australian Society of Clinical Allergy and Immunology (evidence of circulatory or respiratory compromise) within 2 h of the last dose of the OFC (Koplin, J. J. et al. (2012) J Allergy Clin Immunol, 129, pg 1145-1147).
  • PBMC Peripheral blood mononuclear cells
  • Raw .iDAT files were preprocessed using the Minfi package (Aryee, M. J. et al. (2014) Bioinformatics doi: 10.1093/bioinformatics/btu049) from the bioconductor project (http://www.bioconductor.org) in the R statistical environment (http://cran.r- project.org/, version 3.0.2). Quality assessment of control probes on the array indicated high quality data with excellent performance of control probes in all samples.
  • the Minfi package was used for array preprocessing using the stratified quantile normalization method. Technical bias attributable to different probe chemistries between Type I and Type II probes were adjusted in this procedure.
  • Posterior probabilities were estimated for each prediction call to estimate confidence in the prediction.
  • the classifier model was built on a training set consisting of 80% of the sample population and performance was validated in the reserved 20% that was previously unseen by the model. To determine the optimal number of CpGs in the prediction model, 10-fold cross-validations were performed on the training samples and classification errors were estimated for a range of CpGs included in the model. The minimum number of CpGs that produce no misclassification errors was determined empirically from the training data. The false discovery rate was estimated using the q- value method of Storey et al (Storey & Tibshirani (2003) PNAS, 100, pg 9440-9445). The model was validated on the unseen 20% of samples and performance was recorded.
  • Food allergy was defined by clear immediate symptoms (1-2 hours) on exposure to egg, milk or peanut (including anaphylaxis, angioedema or urticaria) and confirmed IgE-mediated sensitivity by virtue of a positive skin prick test > 3mm above negative control at 12-months of age.
  • Matched DNA methylation data derived from total CD4+ T-cells was available at two time-points, birth and 12-months and more detailed experimental procedures can be found at (Martino, D. et al. (2014) Epigenetics, 9).

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Abstract

La présente invention concerne des procédés et des essais permettant de détecter une allergie alimentaire ou une propension à développer une allergie alimentaire chez un sujet sur la base de l'état de méthylation de gènes.
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CN109880889B (zh) * 2019-04-08 2022-12-06 厦门市妇幼保健院(厦门市优生优育服务中心、厦门大学附属妇女儿童医院、厦门市林巧稚妇女儿童医院) 华法林药物靶点基因特定位点甲基化水平定量检测的引物及其方法
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