GM Food Allergy Biomarkers

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  • 1. Development of Biomarkers for the Detection of Potential Allergenicity of Novel Food Mainul Husain Department of Animal & Poultry Science University of Guelph Ontario, Canada
  • 2. What is a Novel Food?
    • A substance, including a microorganism, that does not have a history of safe use as a food
    • A food that has been prepared, preserved or packaged by a process that has not been previously applied to that food, and causes the food to undergo a major change
    • A food that is derived from a plant, animal or microorganism that has been genetically modified
  • 3. Concerns With Novel Food
    • Development of novel food or novel food processing technique may sometimes cause an unwanted change in the food
    • Genetically Modified (GM) foods around the world have very high health safety concern, because of its altered genetic nature
    • Novel foods including any GM foods could be potentially allergic to human
  • 4. Why Allergenicity of GM Food Is So Important?
    • Global GM food market now is about $6b
    • Canada produces about 10% of the world’s transgenic crops
    • About 1-2% of adults are affected by food allergy
    • About 5-8% of children of 3 years and under are affected by food allergy
    • Canadian Food Inspection Agency reported a total of 4744 food recall and 54% of these are related with food allergen
  • 5. Rationale
    • At present, there is no suitable and efficient techniques to determine the allergenicity of GM food
    • So, there is an urgent need to develop some technique, which can efficiently able to detect allergenicity of GM food
    • This research would help to find some transcriptome based bio-marker, which would help to detect of GM food allergy
  • 6. Objectives
    • To determine the gene expression profiles in Balb/c mice in response to egg ovomucoid, a known food allergen
    • To determine the gene expression profiles in C3H/HeJ mice in response to peanut protein, a strong food allergen
    • To find common or overlapping genes from objective 1 and objective 2, which could be considered as potential bio-markers
    • To do the data mining and pathway analysis on the identified genes to understand their biological role
    • To validate the potential bio-marker genes, which were commonly expressed in all of the 2 experimental groups by real time RT-PCR
  • 7. Overall Project Design Validation of Candidate Bio-Marker Genes By Real Time RT- PCR Data Analysis Mapping Genes To Functional Pathways Allergen Treated Experimental Mice Control Mice Total RNA Extracted From Spleens Total RNA Extracted From Spleens Pooled Total RNA Pooled Total RNA SELECTION OF GENES AS ALLERGY BIO-MARKERS Affymetrix Microarray Classification of Genes Using Gene Ontology
  • 8. Balb/c Mice Challenged With Egg Ovomucoid Protein Total 30 Female BALB/c Mice In Control and Treated Groups 15 Control Mice Challenged with Amino Acid Solution (1mg/100  l+10  g CT) 15 Experimental Mice Challenged with Ovomucoid (1mg/100  l+10  g CT) Group 1 3 Mice Group 2 3 Mice Group 3 3 Mice Group 4 3 Mice Group 5 3 Mice Group 1 3 Mice Group 2 3 Mice Group 3 3 Mice Group 4 3 Mice Group 5 3 Mice Pooled Total RNA Pooled Total RNA Pooled Total RNA Pooled Total RNA Pooled Total RNA Pooled Total RNA
  • 9. Results
    • There are about 1649 genes significantly differentially expressed in response to ovomucoid challenge in Balb/c mice
      • About 737 genes up-regulated
      • About 912 genes down-regulated
  • 10. Significant Genes with High Correlation with Allergy
    • The clustering was done based on co-expression of genes, in response to the treatment of egg ovomucoid protein
    • Co-Expressed Up-regulated Genes:
      • Ptgs2
      • Il6
      • Igh4
      • Adn
      • Car3
      • Errfi1 (Mig6)
      • CD47
    • No such pattern is seen in case of down regulated genes
  • 11. Biological Classification of The Significant Genes Ontology ID Gene Ontology Terms # of Genes P-Value FDR 6457 Protein Folding 39 <0.0001 <0.0001 6396 RNA Processing 52 <0.0001 <0.0001 44238 Primary Metabolism 502 <0.0001 <0.0001 44237 Cellular Metabolism 523 <0.0001 <0.0001 43283 Biopolymer Metabolism 235 <0.0001 <0.0001 43170 Macromolecule Metabolism 349 <0.0001 <0.0001 8152 Metabolism 547 <0.0001 <0.0001 19538 Protein Metabolism 248 <0.0001 0.0021 6139 Nucleobase, Nucleoside, Nucleotide and Nucleic Acid Metabolism 256 <0.0001 0.0028 6512 Ubiquitin Cycle 52 0.0001 0.0044 43285 Biopolymer Catabolism 32 0.0002 0.0068 9057 Macromolecule Catabolism 37 0.0002 0.0086 50875 Cellular Physiological Process 696 0.0003 0.0086 30163 Protein Catabolism 26 0.0003 0.0087 19941 Modification-Dependent Protein Catabolism 20 0.0003 0.0092 6984 ER-Nuclear Signaling Pathway 5 0.0003 0.0096 6986 Response to Unfolded Protein 12 0.0011 0.0241 7015 Actin Filament Organization 8 0.0033 0.0571 7205 Protein Kinase C Activation 4 0.0061 0.0963 6325 Establishment and/or Maintenance of Chromatin Architecture 25 0.0073 0.1489
  • 12. Biological Network of Genes
  • 13. KEGG Pathways Affected In Response to Ovomucoid Challenge Pathways Number of Genes Identified MAPK SIGNALING PATHWAY 27 OXIDATIVE PHOSPHORYLATION 24 FOCAL ADHESION 24 CYTOKINE-CYTOKINE RECEPTOR INTERACTION 24 REGULATION OF ACTIN CYTOSKELETON 20 WNT SIGNALING PATHWAY 19 CELL CYCLE 18 PURINE METABOLISM 16 INSULIN SIGNALING PATHWAY 15 PENTOSE AND GLUCURONATE INTERCONVERSIONS 13 PORPHYRIN AND CHLOROPHYLL METABOLISM 13 STARCH AND SUCROSE METABOLISM 13 METABOLISM OF XENOBIOTICS BY CYTOCHROME P450 13 T CELL RECEPTOR SIGNALING PATHWAY 13 JAK-STAT SIGNALING PATHWAY 13 HEMATOPOIETIC CELL LINEAGE 12 AXON GUIDANCE 12 ANDROGEN AND ESTROGEN METABOLISM 11 TGF-BETA SIGNALING PATHWAY 11 RIBOSOME 11 ADHERENS JUNCTION 10 PHOSPHATIDYLINOSITOL SIGNALING SYSTEM 10
  • 14. Validation of Genes
    • Validation of 5 representative genes by real time RT-PCR, which were significantly differentially expressed in response to ovomucoid challenge in Balb/c mice
    Accession No. Gene Name Gene Symbol Real Time RT-PCR Results Microarray Results Fold Change P Value Fold Change P Value NM_007393 Actin, beta Actb House Keeping Gene House Keeping Gene NM_013459 Adipsin Adn 7.13 0.001 1.71 0.031 NM_007606 Carbonic Anhydrase 3 Car3 4.57 0.001 7.56 0.017 BG143662 Immunoglobulin Heavy Chain 4 (serum IgG1) Igh4 3.66 0.015 3.38 0.001 NM_031168 Interleukin 6 IL6 4.12 0.011 1.78 0.019 M94967 Prostaglandin-Endoperoxide Synthase 2 Ptgs2 21.51 0.001 2.28 0.045
  • 15. 15 Control Mice Treated with Amino Acid Solution (1mg/100  l + 10  g CT) C3H/HeJ Mice Challenged With Peanut Protein Total 30 C3H/HeJ Mice In Both Control and Treated Groups 15 Experimental Mice Challenged with Peanut Protein (1mg/100  l + 10  g CT) Group 1 3 Mice Group 2 3 Mice Group 3 3 Mice Group 4 3 Mice Group 5 3 Mice Group 1 3 Mice Group 2 3 Mice Group 3 3 Mice Group 4 3 Mice Group 5 3 Mice Total RNA Total RNA Total RNA Total RNA Total RNA Total RNA Total RNA Total RNA Total RNA Total RNA RNA Pool (5 random samples) RNA Pool (5 random samples) RNA Pool (5 random samples) RNA Pool (4 High Score Samples) RNA Pool (4 Low Score Samples) RNA Pool (4 High Score Samples) RNA Pool (3 Low Score Samples)
  • 16.
    • There are about 1535 genes significantly differentially expressed in response to peanut challenge in C3H/HeJ mice
      • About 622 genes up-regulated
      • About 913 genes down-regulated
    Results
  • 17. Biological Classification of The Significant Genes Ontology ID Gene Ontology Terms # of Genes P-Value FDR 44238 Primary Metabolism 467 <0.0001 <0.0001 44237 Cellular Metabolism 475 <0.0001 <0.0001 43283 Biopolymer Metabolism 204 <0.0001 <0.0001 8152 Metabolism 504 <0.0001 <0.0001 7582 Physiological Process 705 0.0001 0.0023 43170 Macromolecule Metabolism 296 <0.0001 0.0025 50875 Cellular Physiological Process 650 0.0001 0.0029 6323 DNA Packaging 29 0.0002 0.0069 6325 Establishment and/or Maintenance of Chromatin Architecture 28 0.0003 0.0076 6259 DNA Metabolism 54 0.0006 0.0395 6139 Nucleobase, Nucleoside, Nucleotide And Nucleic Acid Metabolism 227 0.0007 0.0405 7001 Chromosome Organization and Biogenesis 29 0.0014 0.0700 16568 Chromatin Modification 20 0.0012 0.0705 6461 Protein Complex Assembly 18 0.0022 0.1158 8652 Amino Acid Biosynthesis 8 0.0037 0.1167 51244 Regulation of Cellular Physiological Process 215 0.0035 0.1176 51128 Regulation of Cell Organization and Biogenesis 10 0.0026 0.1186 6470 Protein Amino Acid Dephosphorylation 17 0.0025 0.1193 15031 Protein Transport 60 0.0027 0.1193 19538 Protein Metabolism 211 0.0034 0.1194
  • 18. Directed Acyclic Graph (DAG) of Enriched Immune Response Category Some other significantly enriched sub-categories are: (1) Innate immune response, (2) Positive regulation of inflammatory response, (3) T-cell proliferation, (4) B-cell proliferation, (5) Positive regulation of interferon-beta biosynthesis.
  • 19. KEGG Pathways Affected In Response to Peanut Challenge PATHWAYS NUMBER OF GENES IDENTIFIED MAPK SIGNALING PATHWAY 31 REGULATION OF ACTIN CYTOSKELETON 29 FOCAL ADHESION 27 ANTIGEN PROCESSING AND PRESENTATION 21 PURINE METABOLISM 18 CELL ADHESION MOLECULES 17 TIGHT JUNCTION 16 NATURAL KILLER CELL MEDIATED CYTOTOXICITY 16 T CELL RECEPTOR SIGNALING PATHWAY 15 AXON GUIDANCE 15 WNT SIGNALING PATHWAY 14 LEUKOCYTE TRANSENDOTHELIAL MIGRATION 13 B CELL RECEPTOR SIGNALING PATHWAY 12 PYRIMIDINE METABOLISM 12 APOPTOSIS 12 JAK-STAT SIGNALING PATHWAY 12 CALCIUM SIGNALING PATHWAY 12 CYTOKINE-CYTOKINE RECEPTOR INTERACTION 12 LONG-TERM POTENTIATION 11
  • 20. Common Genes from Experiment 1 and Experiment 2
  • 21. Validation of Potential Candidate Bio-Marker Genes with Real Time RT-PCR
    • Initially 9 genes were considered for validation with Real Time RT-PCR
    • These genes were significantly differentially expresses and were common to both Balb/c-ovomucoid treated group and C3H/HeJ-peanut treated group
    Gene Symbol Gene Name Ovomucoid-Balb/c Peanut-C3H/HeJ Real Time RT-PCR Microarray Real Time RT-PCR Microarray Up Down P Up Down P Up Down P Up Down P Actb Actin, beta House Keeping Gene House Keeping Gene Adn Adipsin 7.1   0.00 1.7   0.03   -1.5 0.44   -2.3 0.04 Car3 Carbonic Anhydrase 3 4.6   0.00 7.6   0.02   -1.85 0.23   -2.7 0.02 Cyp7b1 Cytochrome P450, Family 7, Subfamily B, Polypeptide 1 1.5   0.15 4.9   0.00 1.43   0.17 2.6   0.01 Fcamr Fc Receptor, IgA, IgM, High Affinity 1.1   0.88   -2.0 0.04 1.4   0.41 2   0.02 Hspa5 Heat Shock 70kD Protein 5 1.1   0.65   -2.0 0.03 1.2   0.70   -1.6 0.05 Ms4a1 Membrane-Spanning 4-Domains, Subfamily A, Member 1 1.3   0.27   -1.8 0.04 1.2   0.50   -1.5 0.02 Ppil3 Peptidylprolyl Isomerase (Cyclophilin)-Like 3 1.3   0.40 4.7   0.03 1.3   0.42 3.5   0.01 Serpinh1 Serine (Or Cysteine) Peptidase Inhibitor, Clade H, Member 1 7.5   0.20   -1.8 0.02 14.3   0.00   -1.6 0.00 Tlr4 Toll-Like Receptor 4 1.2   0.76 2.4   0.01 1.2   0.50 4.2   0.02
  • 22. Validation of More Common Genes By Real Time RT-PCR
    • Five more common genes were considered for validation with Real Time RT-PCR
    • These genes were highly (>1.5 fold) differentially expresses
    Gene Symbol Gene Name Ovomucoid-Balb/c Peanut-C3H/HeJ Real Time RT-PCR Results Microarray Results Real Time RT-PCR Results Microarray Results Up Down P Up Down P Up Down P Up Down P Actb Actin, beta House Keeping Gene House Keeping Gene Ela3b Elastase 3b, Pancreatic 100 0.11 10.9 0.23 77 0.16 31.7 0.17 Prss2 Protease, Serine, 2 48 0.12 5.9 0.24 67 0.1 15.5 0.18 Pnliprp1 Pancreatic Lipase Related Protein 1 38 0.11 7.1 0.29 31 0.2 16.3 0.22 Cel Carboxyl Ester Lipase 69 0.11 5.5 0.28 292 0.13 17.8 0.16 Clps Colipase, Pancreatic 66 0.15 10.5 0.32 55 0.15 35.8 0.13
  • 23. Common Biological Classes of Genes from Ovomucoid-Balb/c and Peanut-C3H/HeJ Group Ontology ID Gene Ontology Terms Ovomucoid Challenged Balb/c Mice Peanut Challenged C3H/HeJ Mice Common Genes No. of Genes P-Value FDR No. of Genes P-Value FDR 8152 Metabolism 547 <0.0001 <0.0001 504 <0.0001 <0.0001 45 44237 Cellular Metabolism 523 <0.0001 <0.0001 475 <0.0001 <0.0001 42 44238 Primary Metabolism 502 <0.0001 <0.0001 467 <0.0001 <0.0001 42 43283 Biopolymer Metabolism 235 <0.0001 <0.0001 204 <0.0001 <0.0001 21 43170 Macromolecule Metabolism 349 <0.0001 <0.0001 296 <0.0001 0.0025 30 19538 Protein Metabolism 248 <0.0001 0.0021 211 0.0034 0.1194 20 6139 Nucleobase, Nucleoside, Nucleotide and Nucleic Acid Metabolism 256 <0.0001 0.0028 227 0.0007 0.0405 18 50875 Cellular Physiological Process 696 0.0003 0.0086 650 0.0001 0.0029 58 6996 Organelle Organization and Biogenesis 85 0.0024 0.0504 79 0.0031 0.1291 7 16043 Cell Organization and Biogenesis 147 0.0023 0.0511 135 0.0045 0.1419 13 7582 Physiological Process 743 0.0029 0.0552 705 0.0001 0.0023 63 6259 DNA Metabolism 52 0.0082 0.1487 54 0.0006 0.0395 5 6325 Establishment And/Or Maintenance of Chromatin Architecture 25 0.0073 0.1489 28 0.0003 0.0076 2 43412 Biopolymer Modification 127 0.0077 0.1491 120 0.0049 0.147 10
  • 24. Common Biological Pathways from Ovomucoid-Balb/c and Peanut-C3H/HeJ Group Pathways Ovomucoid Challenged Balb/c Mice Peanut Challenged C3H/HeJ Mice Common Genes No. of Genes No. of Genes MAPK Signaling Pathway 27 31 2 Regulation of Actin Cytoskeleton 20 29 1 Antigen Processing and Presentation 8 21 1 Purine Metabolism 16 18 1 Cell Adhesion Molecules 9 17 Tight Junction 9 16 2 Natural Killer Cell Mediated Cytotoxicity 9 16 1 T Cell Receptor Signaling Pathway 13 15 1 WNT Signaling Pathway 19 14 1 Leukocyte Transendothelial Migration 6 13 1 Jak-Stat Signaling Pathway 13 12 2 Cytokine-Cytokine Receptor Interaction 24 12 1 Pyrimidine Metabolism 8 12 1 Calcium Signaling Pathway 9 12 B Cell Receptor Signaling Pathway 6 12
  • 25. Conclusion
    • Balb/c and C3H/HeJ mice are good animal models for the study of food allergy
    • There are good number of genes identified in these two different strains of mice in response to the treatment with two different allergens
    • These common genes may be good candidates to be food allergy bio-markers