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Genetics & Epigenetics in IPF

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 ” ?.

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Genetics & Epigenetics in IPF

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  1. 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

  2. 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.

  3. Common Genetic Variants in IPF associated with IPFrisk or progression • Variants in several genes related to inflammation and immuneresponse, 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

  4. Surfactant protein C mutations • heterozygous missense SFTPC mutation (L188Q): mutated protein precursor that accumulatesin the endoplasmic reticulum and causes endoplasmicreticulum stress in pts. • This protein accumulationactivates the unfolded protein response, a cascade ofevents that, although designed to protect the cell, couldlead to alveolar epithelial cell apoptosis in cases of longtermor severe activation. Thomas AQ, Lane K, Phillips J 3rd, et al. AJRCCM 2002

  5. In 2011: MUC5B gene • A common polymorphism in the promoter of theMUC5B gene has been associated with bothsporadicand familial IPF. • Associated with a 20-fold increased risk of IPFin subjects that were homozygous for thepolymorphism and a 7-fold increased risk inheterozygous subjects. • At least one copy of the promoterpolymorphism was present in 34-38% of IPFsubjects compared with 9% of healthycontrols. • Seibold et al. NEJM 2011 • Zhang Y, et al. NEJM 2011

  6. The polymorphism was shownto lead to markedly increased MUC5Bexpression in the lung MUC5B distributionin the cytoplasm of the secretory columnar cellsof the bronchi and larger proximal bronchioles in a specimenof lung tissue. Dense accumulation of MUC5B: In areas of microscopical honeycombing and involved patchystaining of the metaplastic epithelia lining the honeycombcysts (Panel B). Accumulation was also observed in the mucous plugs within the cysts (Panel C). Control IPF

  7. Hypothesis • It is not known whetherthis 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

  8. 7% 52% 41% Participants with ILA were older, increased smoking history % more respiratory symptoms

  9. 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 theminor rs35705950 allele were increased by a factorof 2.8, with a frequency of 10.5% for theminor allele in the population. • About one quarterof the participants with ILA had CT abnormalities that were diagnosticof pulmonary fibrosis. • in theseparticipants the odds of having each copy of theminor rs35705950 allele were increased by afactorof 6.3.

  10. The INSPIRE cohort was used to model theassociation of the MUC5B genotype • with survival, accounting for the effectMMP-7blood concentration • and other demographic and clinicalcovariates. Peljto Anna, et al.

  11. MUC5B Promoter SNP is Associated with Improved Survival [INSPIRE; N=438; 73 deaths] P<0.001 Hazard Ratio (95% CI) Probability of Survival 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

  12. Results • Theobserved association ofMUC5Bwith survivalwasindependent 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.

  13. 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.

  14. 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.

  15. Second Genome Association Study • 5 loci achieved genome-wide significance, including four SNPs on chromosome 11p15 and one on 17q21. • Among the11p15 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 anindependent risk locus. • Similar to MUC5B rs35705950, IPF cases with the TOLLIP risk allele (the majorallele) had decreased mortality compared to minor allele carriers. Noth I, Zhang Y, Ma SF, et al. Lancet Respir Med 2013

  16. 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!

  17. 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

  18. Spagnolo P, et al. Lancet Respir 2014

  19. The hallmarks of aging are present in IPF lung • Altered intercellular communication • Genomic instability • Telomere attrition • Epigenetic alterations • Loss of proteostasis • Deregulation of nutrient sensing • Mitochondrial dysfunction • Cellular senescence • Stem cell exhaustion Lopez-Otin, Cell 2013 Thannickal, Biogerontology 2013; Selman, Pardo, AJRCCM 2014

  20. UNIVERSITY OF CRETE SCHOOL OF MEDICINE Spagnolo P, et al. Lancet Respir 2014

  21. UNIVERSITY OF CRETE SCHOOL OF MEDICINE

  22. UNIVERSITY OF CRETE SCHOOL OF MEDICINE Eur Respir J 2015

  23. 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

  24. 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

  25. Expert Opin Drug Discov 2014 1est level 2nd level 3rd level

  26. UNIVERSITY OF CRETE SCHOOL OF MEDICINE Nucleosomes are the basic building block of Chromatin Ubiquitilation U H2Β H2A H4 H3 Rodenhiser& Mann CMAJ 2006 174:341

  27. Epi-miRNAs: microRNAs and epigenetics • Investigations revealed that certain miRNAs (epi-miRNAs) 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

  28. 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. • 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. Apoptosis Inflammation Proliferation Development Angiogenesis Differentiation Stem cell maintenance Motility Metabolism

  29. 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

  30. Impaired DNA methylation studies in IPF CASZ1 Tzouvelekis A, Kaminski N. Biochem Cell Biol 2015

  31. Aberrant histone modifications studies in IPF sirtuin Tzouvelekis A, Kaminski N. Biochem Cell Biol 2015

  32. Noncoding RNA regulation IPF studies Tzouvelekis A, Kaminski N. Biochem Cell Biol 2015

  33. TGF-b mediated EMT and fibroblast activation-proliferation is reflected in the miRNAs affected in IPF miR-21 Targets tumor suppressors/promotes EMT miR-154 cluster miR-155 Targets Inhibitors of the WNT pathway fibroblast proliferation Proinflammatory Activates WNT/ Targets SMADS TGF-b Let-7d miR-31 Targets HMG2 allowing TWIST and SNAI EMT Targets Integrin-a,Rhoa Inhibis fibroblast proliferation migration miR-29 miR-23b cluster miR-200 Targets SMADS Regulating TGFb Induced genes Targets ECM associated types of Collagen Laminins and Integrins ZEB1, ZEB2 Promotes EMT by Tsitoura E.

  34. The expression of most miRNAs tested in BALF cells from IPF patients was down-regulated relative to controls - excluding mir-210 mirRNA expression results • miR-29a and miR-185 were significantly down-regulated in IPF Tsitoura E. Wells A.U., Karagiannis K. et al. submitted

  35. 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 Tsitoura E.

  36. Selman, Pardo. AJRCCM 2014

  37. Epigenetic modifiers of lung diseases: preclinical studies Comer SB, et al. PPT 2015

  38. Eur Respir J 2015

  39. Collaborators Laboratory of Cellular and Molecular pneumonology • Katerina Antoniou Eliza Tsitoura, Post doc Researcher George Margaritopoulos, PhD Ismini Lasithiotaki Kostas Karagiannis Athanasia Proklou Eleni Bibaki Stella Sarantoulaki Stelios Michelakis Eirini Varsamidi Eirini Charalambous Royal Brompton Hospital and Harefield NHS Trust Prof Athol Wells Elizabeth Renzoni Hiroe Sato Laboratory of clinical Virology Prof Giogros Sourvinos Evi Vlachava Melina Tseliou Nectaria Goulidaki Chryssa Kokkinaki

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