Transcript Διαφάνεια 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
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 :
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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.
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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
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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
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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
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SCHOOL OF MEDICINE
Spagnolo P, et al. Lancet Respir 2014
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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