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Genetics & Epigenetics in IPF Katerina M. Antoniou As. Professor in Thoracic Medicine Head of the Molecular & Cellular Pneumonology Lab ERS ILD Group Chair Medical School, University of Crete UNIVERSITY OF CRETE SCHOOL OF MEDICINE What is a molecular “signature”? •It is a set of specific changes in a molecular profile, which are characteristic for a particular pathological condition. • Molecular expression profiling of human diseases have identified “signatures” associated with diagnosis, staging, progression, prognosis and response to treatment. Common Genetic Variants in IPF associated with IPF risk or progression • Variants in several genes related to inflammation and immune response, including : • • • • • • transforming growth factor beta-1 (TGFB1) interleukin-1 receptor alpha (IL1RN) interleukin 8 (IL8) toll-like receptor 3 (TLR3) HLA DRB1*150 Cell cycle progression related genes CDKN1A and TP53 ERJ 2015 Surfactant protein C mutations • heterozygous missense SFTPC mutation (L188Q): mutated protein precursor that accumulates in the endoplasmic reticulum and causes endoplasmic reticulum stress in pts. • This protein accumulation activates the unfolded protein response, a cascade of events that, although designed to protect the cell, could lead to alveolar epithelial cell apoptosis in cases of longterm or severe activation. Thomas AQ, Lane K, Phillips J 3rd, et al. AJRCCM 2002 In 2011: MUC5B gene • A common polymorphism in the promoter of the MUC5B gene has been associated with both sporadic and familial IPF. • Associated with a 20-fold increased risk of IPF in subjects that were homozygous for the polymorphism and a 7-fold increased risk in heterozygous subjects. • At least one copy of the promoter polymorphism was present in 34-38% of IPF subjects compared with 9% of healthy controls. Seibold et al. NEJM 2011 Zhang Y, et al. NEJM 2011 The polymorphism was shown to lead to markedly increased MUC5B expression in the lung Control IPF MUC5B distribution in the cytoplasm of the secretory columnar cells of the bronchi and larger proximal bronchioles in a specimen of lung tissue. Dense accumulation of MUC5B: In areas of microscopical honeycombing and involved patchy staining of the metaplastic epithelia lining the honeycomb cysts (Panel B). Accumulation was also observed in the mucous plugs within the cysts (Panel C). Hypothesis • It is not known whether this polymorphism is associated with ILD in the general population. • The relationship between ILA and the genotype at the rs35705950 locus for a modification of effect according to age and smoking was evaluated. Hunninghake GM, et al. NEJM, June 2013 52% 41% 7% Participants with ILA were older, increased smoking history % more respiratory symptoms Main results • ILA were found in 7% of the sample. • 50% of the participants who had ILA had reduced lung volumes • In participants with ILA: • the odds of having each copy of the minor rs35705950 allele were increased by a factor of 2.8, with a frequency of 10.5% for the minor allele in the population. • About one quarter of the participants with ILA had CT abnormalities that were diagnostic of pulmonary fibrosis. • in these participants the odds of having each copy of the minor rs35705950 allele were increased by a factor of 6.3. • The INSPIRE cohort was used to model the association of the MUC5B genotype • with survival, accounting for the effect MMP7 blood concentration • and other demographic and clinical covariates. Peljto Anna, et al. MUC5B Promoter SNP is Associated with Improved Survival Probability of Survival [INSPIRE; N=438; 73 deaths] P<0.001 Hazard Ratio (95% CI) MUC5B GT 0.46 (.30-.70) MUC5B TT 0.21 (.09-.49) (age, gender, FVC, DLCO, and MMP7) Time to Death (days) Peljto. JAMA 2013; 309:2232 Results • The observed association of MUC5B with survival was independent of age, sex, FVC, DLCO, MMP-7, and treatment status. • MUC5B promoter polymorphism explains a portion of the variation in survival among IPF participants beyond that explained by MMP-7 levels. The MUC5B risk variant is observed in ~19% of unaffected individuals, and approximately one-third of individuals with IIP do not have any identifiable genetic risk factors for this disease, suggesting that other genetic variants contribute to disease risk alone or in combination with the MUC5B variant. UNIVERSITY OF CRETE SCHOOL OF MEDICINE Results • • • • Confirmed association with: TERT at 5p15 MUC5B at 11p15 3q26 region near TERC • Seven newly associated loci : • FAM13A (4q22), DSP (6p24), OBFC1 (10q24), ATP11A (13q34), DPP9 (19p13) • chromosomal regions 7q22 and 15q14-15. Second Genome Association Study • 5 loci achieved genome-wide significance, including four SNPs on chromosome 11p15 and one on 17q21. • Among the 11p15 SNPs were MUC5B rs35705950 and three SNPs within the Toll-interacting protein (TOLLIP) locus. • Linkage disequilibrium was reported to be low with rs35705950, suggesting TOLLIP may represent an independent risk locus. • Similar to MUC5B rs35705950, IPF cases with the TOLLIP risk allele (the major allele) had decreased mortality compared to minor allele carriers. Noth I, Zhang Y, Ma SF, et al. Lancet Respir Med 2013 Dysregulated lung mucins initiate or exacerbate lung fibrosis through: • Altered mucosal defense; • Interference with alveolar repair • Direct cell toxicity stimulating a fibroproliferative response initiated by unfolded intracellular MUC5B. • Agents that reduce MUC5B transcriptional activity in vitro should be tested for activity in vivo! New concepts • Identification of the putative environmental factors should be a priority for research. • Chronic occult infections must be high on the list of suspects. • Genetic studies can provide to the understanding of complex diseases. NEJM 2013 Spagnolo P, et al. Lancet Respir 2014 The hallmarks of aging are present in IPF lung Lopez-Otin, Cell 2013 1. 2. 3. 4. 5. 6. 7. 8. 9. Altered intercellular communication Genomic instability Telomere attrition Epigenetic alterations Loss of proteostasis Deregulation of nutrient sensing Mitochondrial dysfunction Cellular senescence Stem cell exhaustion Thannickal, Biogerontology 2013; Selman, Pardo, AJRCCM 2014 UNIVERSITY OF CRETE SCHOOL OF MEDICINE Spagnolo P, et al. Lancet Respir 2014 UNIVERSITY OF CRETE SCHOOL OF MEDICINE UNIVERSITY OF CRETE SCHOOL OF MEDICINE Eur Respir J 2015 UNIVERSITY OF CRETE SCHOOL OF MEDICINE What is Epigenetics? • Epigenetics: “on top of” Genetics • Modifications to nucleotides or histones that do not change the sequence but can alter gene expression • Epigenetic regulation has emerged as a key adaptive mechanism by which environmental or other stresses can induce lasting changes in the gene expression repertoire and thus in the phenotype of a cell or an organism The Idiopathic pulmonary fibrosis transcriptome is influenced by both environmental and genetic factors Antoniou KM, et al. AJRCCM 2008 Tzouvelekis A, Kaminski N. Biochem Cell Biol 2015 Expert Opin Drug Discov 2014 1est level 2nd level 3rd level UNIVERSITY OF CRETE SCHOOL OF MEDICINE Nucleosomes are the basic building block of Chromatin Ubiquitilation H2A H3 U H2Β H4 Rodenhiser& Mann CMAJ 2006 174:341 Epi-miRNAs: microRNAs and epigenetics • Investigations revealed that certain miRNAs (epimiRNAs) themselves counteract CpG methylation. • Regulate the components of epigenetic machinery, creating a tightly controlled feedback mechanism. • Histone modification is another epigenetic mechanism that can affect miRNA expression as shown in breast cancer cells UNIVERSITY OF CRETE SCHOOL OF MEDICINE MicroRNAs • MicroRNAs are members of non-coding RNAs that range in size from 18-24 nucleotides. • miRNAs regulate a large variety of biological functions by controlling gene expression. Apoptosis Inflammation Proliferation Development Angiogenesis Differentiation Stem cell maintenance Motility Metabolism • So far, about 2,000 miRNAs have been discovered in humans. • Each miRNA expressed in a cell may target about 100 to 200 mRNAs that it downregulates. • It appears that about 60% of human protein coding genes are regulated by miRNAs. UNIVERSITY OF CRETE SCHOOL OF MEDICINE Expression of DNA Methyltransferases in IPF Sanders YY, et al. Am J Respir Crit Care Med, 186, 525–535, 2012 Impaired DNA methylation studies in IPF CASZ1 Tzouvelekis A, Kaminski N. Biochem Cell Biol 2015 Aberrant histone modifications studies in IPF sirtuin Tzouvelekis A, Kaminski N. Biochem Cell Biol 2015 Noncoding RNA regulation IPF studies Tzouvelekis A, Kaminski N. Biochem Cell Biol 2015 TGF-b mediated EMT and fibroblast activation-proliferation is reflected in the miRNAs affected in IPF miR-21 miR-154 cluster Targets tumor suppressors/promotes EMT Targets Inhibitors of the WNT pathway fibroblast proliferation Let-7d Proinflammatory Activates WNT/ Targets SMADS TGF-b Targets HMG2 allowing TWIST and SNAI EMT miR-31 Targets Integrin-a,Rhoa Inhibis fibroblast proliferation migration miR-29 Targets ECM associated types of Collagen Laminins and Integrins miR-155 miR-23b cluster miR-200 ZEB1, ZEB2 Promotes EMT Targets SMADS Regulating TGFb Induced genes by Tsitoura E. mirRNA expression results • The expression of most miRNAs tested in BALF cells from IPF patients was down-regulated relative to controls - excluding mir-210 • miR-29a and miR-185 were significantly down-regulated in IPF Tsitoura E. Wells A.U., Karagiannis K. et al. submitted miR-29a expression inversely correlated with COL1A1 expression Spearman correlation within IPF group r =-0.418 P= 0.005 **: p<0.005 COL1A1 expression is strongly associated with CPI multivariate analysis COL1A1 CPI Tsitoura E. P-value 0.005** a sense of deep satisfaction in completing this task, which Copyright © 2014 by the American Thoracic Society Stochastic Age-related Epigenetic Drift in the Pathogenesis of Idiopathic Pulmonary Fibrosis Idiopathic pulmonary fibrosis (IPF) is a progressive, irreversible, and lethal aging-associated lung disease of unknown etiology (1). Its pathogenic mechanisms are unknown, but it has been proposed that involve the convergence of a (variable) combination of risk genetic factors (common polymorphisms and/or rare variants) affecting the epithelial integrity, with some accelerated aging processes (e.g., exaggerated telomere shortening, mitochondrial dysfunction, cell senescence) and epigenetic modifications that influence the behavior of both alveolar epithelial cells and fibroblasts (2). Epigenetic mechanisms play a key role in biological processes at the level of chromatin structure and organization and include DNA methylation (DNAm), post-translational modifications of 1328 histone tails, and noncoding RNA. Under physiological conditions, the epigenome ultimately determines the silencing or activation of gene expression in a temporally coordinated way, and its dysregulation contributes to a variety of human diseases, including IPF (3, 4). DNAm, a key epigenetic mark, occurs by thecovalent addition of a methyl group to a cytosine, usually in the context of the symmetrical CpG dinucleotides. DNAm is crucial in a vast array of processes, including gene expression, reprogramming, and stability; genomic imprinting; cell differentiation; alternative splicing; and DNA repair. Importantly, emerging evidence indicates that the genomic landscapeof DNAm ismodified asafunction of age. In thiscontext, American Journal of Respiratory and Critical Care Medicine Volume 190 Number 12 | December 15 2014 Selman, Pardo. AJRCCM 2014 Epigenetic modifiers of lung diseases: preclinical studies Comer SB, et al. PPT 2015 Eur Respir J 2015 Collaborators Laboratory of Cellular and Molecular pneumonology •Katerina Antoniou Royal Brompton Hospital and Harefield NHS Trust Prof Athol Wells Elizabeth Renzoni Eliza Tsitoura, Post doc Hiroe Sato Researcher George Margaritopoulos, PhD Ismini Lasithiotaki Kostas Karagiannis Athanasia Proklou Laboratory of clinical Virology Prof Giogros Sourvinos Eleni Bibaki Evi Vlachava Stella Sarantoulaki Melina Tseliou Stelios Michelakis Nectaria Goulidaki Eirini Varsamidi Chryssa Kokkinaki Eirini Charalambous