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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 • • • • 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 • • • • 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 • • • • 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…. • • • • 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 • • “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): • • 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 • • • • 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 • • • • • • 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