gm food allergy biomarkers
TRANSCRIPT
Development of Biomarkers for Development of Biomarkers for the Detection of Potential the Detection of Potential
Allergenicity of Novel FoodAllergenicity of Novel Food
Mainul Husain
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
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
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
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
Objectives
1. To determine the gene expression profiles in Balb/c mice in response to egg ovomucoid, a known food allergen
2. To determine the gene expression profiles in C3H/HeJ mice in response to peanut protein, a strong food allergen
3. To find common or overlapping genes from objective 1 and objective 2, which could be considered as potential bio-markers
4. To do the data mining and pathway analysis on the identified genes to understand their biological role
5. To validate the potential bio-marker genes, which were commonly expressed in all of the 2 experimental groups by real time RT-PCR
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 SELECTION OF GENES AS ALLERGY BIO-MARKERSBIO-MARKERS
Affymetrix Microarray
Classification of Genes Using Gene Ontology
Total 30 Female BALB/c Mice In Control and Treated Groups
15 Control Mice Challenged with Amino Acid Solution (1mg/100µl+10µg CT)
15 Experimental Mice Challenged with Ovomucoid (1mg/100µl+10µg CT)
Group 13 Mice
Group 23 Mice
Group 33 Mice
Group 43 Mice
Group 53 Mice
Group 13 Mice
Group 23 Mice
Group 33 Mice
Group 43 Mice
Group 53 Mice
Pooled Total RNA
Pooled Total RNA
Pooled Total RNA
Pooled Total RNA
Pooled Total RNA
Pooled Total RNA
Balb/c Mice Challenged With Egg Ovomucoid Protein
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
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
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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
Biological Network of Genes Biological Network of Genes
KEGG Pathways Affected In Response to Ovomucoid ChallengePathways 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
Validation of Genes
Accession No. Gene NameGene 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
BG143662Immunoglobulin 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
M94967Prostaglandin-Endoperoxide Synthase 2
Ptgs2 21.51 0.001 2.28 0.045
Validation of 5 representative genes by real time RT-PCR, which were significantly differentially expressed in response to ovomucoid challenge in Balb/c mice
Total 30 C3H/HeJ Mice In Both Control and Treated Groups
15 Experimental Mice Challenged with Peanut Protein (1mg/100µl + 10µg CT)
Group 13 Mice
Group 23 Mice
Group 33 Mice
Group 43 Mice
Group 53 Mice
Group 13 Mice
Group 23 Mice
Group 33 Mice
Group 43 Mice
Group 53 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)
15 Control Mice Treated with Amino Acid Solution (1mg/100µl + 10µg CT)
C3H/HeJ Mice Challenged With Peanut Protein
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
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
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.
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
Common Genes from Experiment 1 and Experiment 2
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
Cyp7b1Cytochrome P450, Family 7, Subfamily B, Polypeptide 1
1.5 0.15 4.9 0.00 1.43 0.17 2.6 0.01
FcamrFc 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
Ms4a1Membrane-Spanning 4-Domains, Subfamily A, Member 1
1.3 0.27 -1.8 0.04 1.2 0.50 -1.5 0.02
Ppil3Peptidylprolyl Isomerase (Cyclophilin)-Like 3
1.3 0.40 4.7 0.03 1.3 0.42 3.5 0.01
Serpinh1Serine (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
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 ResultsReal Time RT-PCR
ResultsMicroarray 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
Pnliprp1Pancreatic 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
Common Biological Classes of Genes from Ovomucoid-Balb/c and Peanut-C3H/HeJ Group
Ontology ID
Gene Ontology TermsOvomucoid Challenged Balb/c Mice Peanut Challenged C3H/HeJ Mice
Common GenesNo. 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
6139Nucleobase, 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
6325Establishment 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
Common Biological Pathways from Ovomucoid-Balb/c and Peanut-C3H/HeJ Group
PathwaysOvomucoid Challenged Balb/c Mice Peanut Challenged C3H/HeJ Mice Common
GenesNo. 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
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