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

M1

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M2

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M3

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M6

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M7

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M8

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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