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The Genetics of
Eosinophilic Esophagitis
Philip E. Putnam, MD, FAAP
Professor of Pediatrics
Division of Gastroenterology, Hepatology, and Nutrition
Cincinnati Children’s Hospital Medical Center
University of Cincinnati College of Medicine
Cincinnati OH USA
Disclosures
• There are no conflicts of interest related to the
content of this presentation.
• Speakers Bureau
• AbbottNutrition
• Nutricia
• Thanks to Leah Kottyan, PhD for guidance and
slides!
Eosinophilic Esophagitis (EoE)
• Eosinophil-predominant inflammation
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Chronic
Immune-mediated, antigen-driven
Not associated with inflammation elsewhere in the GI tract
Not responsive to acid suppression by Proton Pump Inhibition (PPI)
Liacouras C, et al, 2011, JACI
EoE
• Non-specific symptoms
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Dysphagia
Abdominal or chest pain
Vomiting
Feeding disorder in infants and toddlers
• Onset of symptoms at any age
• Misdiagnosed as gastroesophageal reflux disease in
infants
• Vomiting with introduction of solids after 6 months
• Later: onset of dysphagia in preteens through adulthood
EoE as a manifestation of food “allergy”
• Complete clinical and histologic remission in response to antigen
elimination diet
• Recurrent inflammation upon re-exposure to provocative antigens
• Non-IgE mediated response
• ~2/3 patients have other atopic disorders
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•
•
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Asthma
Environmental allergy
Atopic Dermatitis
Food allergy (immediate hypersensitivity)
Simon D, et al., Allergy, 2016
Photos by Philip Putnam
Untreated/persistent EoE
• Lamina propria fibrosis
• Loss of compliance in the esophageal wall
• Ineffective bolus transit to stomach
• Dysphagia
• Food sticking/going down slow
• Food stuck
EoE—It’s not just eosinophils
• Basal cell layer hyperplasia
• Rete peg elongation
• Eosinophils, lymphocytes, mast cells in the infiltrate
• Dilated intercellular spaces
• Exudate—eosinophilic abscesses
• Lamina propria fibrosis
• Superficial layering of eosinophils
Stratified Squamous Epithelium
• Normal
• Lining of mouth, pharynx, esophagus
• Ends at the Squamocolumnar junction at the diaphragm
(esophagogastric junction)
• Barrier function—tight junctions between cells
• Controls permeability and antigen entry
• Dilated intercellular spaces in esophagitis
Sherrill, J et al., Mucosal Immunol. 2014 May ; 7(3): 718–729
Why do some people develop EoE and
others don’t?
Genetic
factors
Susceptibility
Heritability
Epithelial defects
Immune response
Healing/repair
Sequelae (fibrosis)
Environmental
factors
Immunological
pathways
EoE --Epidemiologic factoids that
make you go Hmmm….
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Prevalence ~56/100000
Male predominance (~80%)
Affected fathers and sons
Geographic/Ethnic variability
• 90% European descent
• Multiple affected family members
• Recurrence risk ratios: 10 to 64-fold increase
• Brothers >males>fathers compared to female
• Association/comorbidity with other genetic conditions
Sherrill J and Rothenberg. Gastroenterol Clin N Am 43 (2014) 269–280
Dellon E, et al. Clin Gastroenterol Hepatol. 2014 Apr;12(4):589-96
EoE Genetic Studies—Varied Approaches
• Epidemiology-Twin Study
• Genome Wide Association Study
• Epigenetic studies
• Specific gene-protein investigation
Nuclear family and twin study
• Retrospective cross-sectional study
• Nuclear-Family cohort
• 914 probands and 2192 first-degree relatives.
• Twin cohort
• 63 proband families
• No differences between the Nuclear-Family and Twin cohorts in
sex, race, ethnicity, or age.
• Both cohorts had
• 73% to 74% males
• 87% to 94% whites
• 94% non-Hispanics
• Questionnaire discerning environmental factors
Alexander et al. JACI 2014
Nuclear family relative risks
Frequency in pop. 0.055%
Alexander E, et al. JACI 2014
RRR = Recurrence Risk Ratio
Epidemiological study to assess disease
concordance in affected twins and their families
41%
22%
2.4%
5.5/10,000
Alexander et al. JACI 2014
Environmental factors assessed
• Prenatal vitamins
• Gestational age
• Breast-feeding
• Birth weight, length, and order.
• Greater difference in twin birth weight (p = .01)
• Birth seasons
• Fall (p = .02)
• Food and medication (penicillin) allergy
Alexander et al. JACI 2014
Family study summary
• Increased risk in males > females
• No suggestion of simple Mendelian inheritance
• Lower than expected concordance in monozygotic twins
• BUT: increased risk within families and dizygotic twins
• Suggests a substantial environmental contribution to
recurrence risk
• Early life exposures prime genetically susceptible
individuals
Alexander et al. JACI 2014
The Human Genome
• DNA plus mtDNA
• 23 chromosomes in pairs
• Chromosomes are non-uniformly divided into
regions
•
<2% “Coding”—genes that are transcribed into
RNA and translated into proteins = exons
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•
“Exome” is the sum total of the exons within the
genome—about 20,000 genes
“Non-coding” DNA is the rest of the genome
The Human Genome
• DNA plus mtDNA
• 46 chromosomes
• (23 pairs, one from each parent)
DNA “SEQUENCE”
• 6 billion base-pairs in the human genome
DNA sequence
• The order of the base pairs is the “DNA sequence”
and contains the “code” that when translated
results in the ability to make different proteins.
• About 1.8% of the genome is the ‘coding’ part of
the genome
• ~20-25000 genes
Genetic variation
• >99% of the human genome is
identical across the population
•
Alleles are 1 or more alternative forms of a gene
differing in nucleotide sequence
•
Allele frequency: the proportion of a particular allele
in a population
Genetic Variation
• Alleles can be due to
• SNP—single nucleotide polymorphism
• Change in a single base pair
• ~10 million SNPs in the human genome
• Insertion/deletion (InDels):
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Small: 1-50 nucleotides net change
Large: 50- 1000 nucleotides net change
• Copy number variations: greater than 1 kb
net change
•
A-B-C-D might have sections A-B-C-C-D (a duplication of
"C") or A-B-D (a deletion of "C").
•
Common within the genome and not generally associated with
disease
SNP
• Can fall within
• coding sequence
• non-coding regions
• intergenic regions (regions between genes).
• SNPs within a coding sequence
• Synonymous SNPs—base change does not change the
amino acid sequence in the protein
• Nonsynonymous SNPs change the amino acid sequence of
protein.
• Missense mutation changes the amino acid
• Nonsense—creates a stop codon.
• SNPs that are not in protein-coding regions may affect
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gene splicing
transcription factor binding
messenger RNA degradation,
sequence of non-coding RNA
GWAS
• Genome Wide Association Study
• Determines the likelihood of an association between a
SNP and a disease
• Known SNP ‘library’ on a chip (~1.5million SNPs)
• The frequency of each SNP is determined in subjects
with particular phenotype vs. controls.
• Statistical analysis only
• Cannot ASSUME causality
GWAS Result
• Manhattan plot
• The negative log of the p value is plotted on y axis
• SNPs, by chromosome,on x axis
• P value represents the statistical significance of the difference in
allele frequency for each SNP in two populations (case vs. control)
• Threshold of p <5 x 10-8
• If met, highly likely that the frequency of a SNP is different between the
two groups
Dueling GWAS replicate EoE risk Loci
CCHMC
Nature Genetics
July 2014
CHOP
Nature Communications
November 2014
Replicated Genetic Loci:
2p23 – CAPN14
5q22 – TSLP/WDR36
11q13 - EMSY/LRRC32
GWAS: EoE population vs. control
736 subjects with EoE
9,246 controls
1,468,075 genetic variants with minor allele frequency > 1% in subjects
Kottyan and Davis et al. Nature Genetics 2014
NOW WHAT?
• If GWAS identifies a SNP with high likelihood of association
• Further study is required to determine the genetics and biology of
the gene and its protein
• What does the protein do in normal?
• Is the protein relevant and participate in disease causation or
susceptibility?
• What factors modify it’s normal function?
• Is the SNP is a Coding or Non-coding region?
• Protein sequence affected?
• What factors influence the expression of the gene?
• Environmental factors, inflammatory mediators, etc
• Epigenetics
2p23 encodes Calpain-14
• Member of the classical calpain family
• Regulatory proteases- involved in
activating and degrading
• Structural proteins
• Signaling molecules
• Transcription factors
• Barrier proteins
• Inflammatory mediators
• Calcium-dependent activity
• Intracellular localization
• Calpain-1 and Calpain-2 are fairly ubiquitous
Jeffrey Rymer
CAPN14: Specifically expressed in the
esophagus as a function of disease activity
Esophagus
Pharyngeal mucosa
Tonsil epithelium
Tongue squamous cells
Head and neck epithelial cells
Nasopharyngeal epithelial cells
Plasma cells
Memory b cells
Oral mucosa
Lymph nodes
T cells
Skin
Bone marrow
Blood
Neutrophils
Pbmc
Airway epithelial cells
Colonic mucosa
Spleen
Colon
Lung
Sinus mucosa
Stomach
***
4
***
2
1
tiv
iv
ac
ct
(i n
(a
Eo
E
E
Eo
20
15
10
5
0
CAPN14 expression
e)
L
e)
0
N
Relative mRNA expression
3
Kottyan and Davis et al. Nature Genetics 2014
Calpain-14 has important regulatory activity
• Overexpression of
calpain-14 results in
accentuated defects
in barrier function
Decreased transepithelial resistance (2.1 fold)
Increased FITC-dextran flux (2.6 fold)
• Knockdown of
calpain-14 leads to
defects in repair of
epithelial cell
morphology after
exposure to IL-13
Davis et al. JCI Insights. In press.
Increased dilated space (5.5 fold)
Disorganized basal cell layering (1.5 fold)
Other Loci?
• TSLP (5q22) has also been associated EoE, but also
with other atopic disorders in promoting Th2 immune
responses
• Present in esophageal epithelium
• Lacks the tissue specificity of CAPN-14
• Susceptibility to atopy
mol-biol4masters.masters.grkraj.org
Epigenetics--Background
• Factors that influence the expression of genes without changing
the DNA sequence
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•
•
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•
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DNA methylation
Histone modification
Inflammatory mediators
Environmental factors
miRNA
RNA transcripts
• Cell/tissue differentiation depends on epigenetic factors
• All cells have the same DNA
• Differentially expressed to account for tissue specific anatomy and
function
• Cells are able to react to ‘environmental’ conditions by
changing the expression of appropriate genes
Epigenetics-- Background
The process of cell/tissue differentiation requires
activation and silencing of appropriate genes
• Epigenetic factors allow the tissue to change to meet
new challenges
•
• Repair of injury (acid, other chemical)
• Inflammatory response in esophageal disease
• Viral (herpes)
• Candida
• IBD—Crohn’s, Behcet’s
• EoE--“Allergy”—abnormal immune response to dietary
protein exposure
Epigenetic Studies in EoE
• Gene expression in tissue
• DNA
RNA
Protein
• Assess the number of copies of transcribed RNA
• Single gene vs. many
• Whole-genome-wide transcript expression profile
• RNA extraction from esophageal biopsy
• Relative RNA levels of many individual genes compared to
normal tissue
• Guides further study of which genes may be ‘involved’
Blanchard C, et al JCI 2006
Demonstrating the EoE Transcriptome
• Snapshot
• Quantitative assessment of the RNA present in the tissue at a
single point in time.
• Oligonucleotide-based DNA microarray chips.
• Affymetrix U133 Plus 2.0 GeneChip
• 54,681 transcripts
• Assesses the number of copies RNA for each
• RESULT:
• EoE: 574 genes are differentially expressed
• 344 genes over-expressed
• 230 under-expressed
Blanchard C, et al JCI 2006
Heat Map, EoE transcriptome, Blanchard C et al. 2006 JCI
Patient:
1 2
3 4
NL
5 6
7 8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
“Reflux”
Esophagitis
Eosinophilic Esophagitis
Esophageal Epithelium “Transcriptome”
• Activation or down-regulation of particular nuclear
genes occurs in a distinct pattern in EoE as
compared to normal and GERD
• EoE Transcriptome “Signature”
• Genes most highly overexpressed
• Eotaxin-3
• Periostin
Blanchard C, et al. JCI 2006
What is the role of a particular gene in the complex
interplay between genetics and immunology, and what
modifies its expression?
Esophageal Epithelial Transcriptome for Diagnosis?
• Human subjects
• 96 genes
• selected to create a smaller panel of genes whose pattern of
activation/down-regulation is highly suggestive of EoE
• EDP--Eosinophil Diagnostic Panel
• Esophageal transcriptome from endoscopic biopsy
• EoE diagnosis with high sensitivity and specificity (92–100%,
96–100%, respectively), based on dual computational
algorithms
• Commercially available through Miraca Life Sciences, Irving Texas
• EoGenius
Wen T, et al., Gastroenterology 2013
Major EDP Gene Categories
Cell adhesion
Epithelial
related
Inflammation
Remodeling
Eosinophil/m
ast cell
Chemokine/c
ytokine
CDH26
FLG
TNFAIP6
POSTN
CLC
CCL26
DSG1
UPK1A
ALOX15
KRT23
CCR3
CXCL1
CLDN10
SPINK7
ARG1
COL8A2
TPSB2/AB1
IL4
CTNNAL1
CRISP3
MMP12
CTSC
CPA3
IL5
CHL1
MUC4
IGJ
ACTG2
CMA1
IL13
Wen T, et al., Gastroenterology 2013
Wen T, et al., Gastroenterology 2013
RNA transcriptome
in esophageal
epithelium, by
phenotype
PPI-REE: PPI responsive
Esophageal eosinophilia
Rothenberg ME. Gastro 2015
EoE Transcriptome Summary
• Genetic evidence for key aspects of disease process
• eotaxin-3 in eosinophil accumulation and activation;
• periostin in facilitating eosinophil recruitment and tissue
remodeling
• the critical role of mast cells and T cells
• the local cytokine milieu
• impaired local barrier function
Wen T, et al. Gastro 2103
Rothenberg ME, Gastro 2015
EoE
• Multifaceted disorder of epithelial homeostasis
• Genetic basis for impaired barrier function
• Desmoglein, CAPN14
• Genetic susceptibility to non-IgE mediated
hypersensitivity (Thymic Stromal Lymphopoetin, etc)
• Eosinophilic inflammation after dietary antigen exposure
• Eosinophils as effectors along with other immune cells (mast
cells and lymphocytes)
• Early life environmental triggers
Rothenberg, Gastro 2015