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CD-EPI.CENTER
WHEN THE ENVIRONMENT MEETS
THE GENETIC!
Medicel Meeting
Palermo – April 26th 2014
WHAT WE KNOW:
•
•
CD prevalence is increasing at unexpected rate in the last 20 years:
a series of environmental factors do influence the phenotype of CD.
Several reports point to prenatal and early life factors as a possible
contribution to the epigenetic expression of genes:
― Pre-conceptional status: Offspring of affected fathers have an
higher risk to develop CD, The Carter effect.
― Pregnancy: Ab anti-Tgase in placenta trophoblasts may be
associated to unfavourable outcome of pregnancy.Women with
untreated CD, but not with treated CD has higher risk of SGA
infants.
― Season of birth: Summer birth was associated with a small
increased risk of later CD
― Mode of delivery: An association between elective cesarean
delivery and later CD was reported
― Perinatal events: Breast Feeding appers to delay the onset of
symptoms of CD, A correlation with exposure to neonatal
infections (and being small for gestational age was also reported.
EPIGENETICS
•
Persistent changes in tissue structure
and function, that are induced during the
development of the embryo and fetus,
play an important role in determining the risk of non communicable diseases.
•
The process that underlies the induction of differential risk of disease by
variation in the prenatal environment reflects environmental cues acting
through developmental plasticity, which generate a range of genotypes from a
single genome.
•
Gene-environment interactions are centrally involved in our health but also
in the susceptibility to diseases and these influences are likely to be mediated, to a
large extent, via epigenetic regulatory phenomena.
•
Recent findings show that altered epigenetic regulation of specific genes is
central to the process by which different phenotypes are generated and hence
differential risk of disease rises.
EPIGENETIC MECHANISMS
•The missing heredity od CD could be explained by the action of epigenetic
mechanisms and by their interaction between genes and environmental risk
factors.
•Epigenetic modifications are heritable changes in gene expression without
changes in DNA sequence
•DNA methylation
•Histone modifications (PTMs)
•MicroRNA regulation
DNA METHYLATION
• DNA methylation has been implicated in the control of several cellular
processes including differentiation, gene regulation, development, genomic
imprinting and X-chromosome inactivation.
• Methylated cytosine residues at CpG dinucleotides are commonly
associated with gene repression; conversely, strategic loss of methylation
during development could lead to activation of lineage-specific genes.
• The overall effect of genome-wide de-methylation is to produce pluripotent
cells in which all genes are potentially transcriptionally active. Loss of
pluripotency with cell differentiation and the establishment of adult tissue
function are dependent by changes in the methylation status of individual gene
promoters at different times in development
HISTONE MODIFICATIONS
(PTMS)
• Include acetylation, methylation, phosphorylation, ADP-ribosylation,
ubiquitination, and biotinylation are in general, dynamic, highly regulated,
and transient, so as to adapt to physiological changes in the cell.
• Although chromatin modifications have been divided into those that
correlate with activation and those that correlate with repression of
transcription, the truth is likely to be that any given modification can
activate or repress genes depending on the context.
• The research on the impact of histone modification placental gene
expression may allow to gain a better understanding of important
phenomena, such as tolerance maternal-fetal, and placental growth and
differentiation, and try to explain the effects that environmental factors
have on these processes.
MICRO-RNA REGULATION
• One of the most studied mechanisms of gene regulation in these years is
represented by micro-RNAs (miRNAs).
• MicroRNAs are important regulators of gene expression therefore, it is
easy to conceive that protein overexpression resulting from defective
miRNA-based mRNA regulation may compromise normal cell function and
cause genetic diseases.
• Qualitative and the quantitative expressions of miRNAs, are expected to
exert a profound regulatory influence on the transcriptome, thus the
accurate profiling of miRNA expression represents an important tool to
investigate physiological and pathological states.
WHAT WE PLAN TO DO:
Aim of this project is to evaluate the combined influences
of genetic, epigenetic and environmental factors in the
development of CD in at risk families.
The experimental design involves the articulation in the following
points:
• estimate and validate epidemiological and individual risk factors
• correlate this factors with the outcome and the genomic profile of
the cases
• correlate the outcome and the risk factors (including genomic)
with eventual epigenetic profiles of cases and non-cases.
How we will perform the study....
RECRUITMENT!
NEOCEL cohort:
―300 newborns from at risk families with one proband
followed for 5 years, with known outcome.
From this cohort we have stored:
―sera and DNA from probands, fathers and mothers,
―cord blood, and PBL from the newborns (several
samples longitudinally over time).
How we will perform the study....
RECRUITMENT!
• Perspective new cohort (CD-EPI.CENTER):
―500 families with one celiac proband at
moment of a new pregnancy
• In 3 years 250-300 newborns are expected. For this new
cohort we will collect:
―PBL from parents and probands before conception
(500 samples)
―Samples of placenta (250-300 samples)
―Umbilical cord (250-300 samples)
How we will perform the study....
EXPERIMENTAL DESIGN!
We propose to study the mechanisms of epigenetic regulation in
the 64 genes associated with CD, and in the additional genes
regulated by the main pathways involved in CD pathogenesis.
We will investigate:
• the DNA methylation status of candidates genes and the
transmission inside the family
• the expression levels and activities of histone-modifying
enzymes
• the genomic distribution of particular histone PTMs
• the miRNA expression pattern in PBL and the transmission
inside the family
• the possible variations in epigenetic pattern in relationship to
the risk factors listed above
The PHASE 1: ESTIMATE OF RISK
FACTORS
Our first cohort is the Regional Registry of Coeliac Disease
(8000 Celiacs), we will analyze, in a case control design,
the possible correlation between onset of CD and the
following risk factors:
•
•
•
•
Sex
First Degree Affected
Season of Birth
Phenotype and associated condition
The PHASE 2: NEOCEL COHORT
300 Infants :Followed since birth, with know outcome at 5 years
to validate the previous risk factors, adding the estimate of the following
environmental factors:
• Pregnancy
• Outcome of delivery
• Birth Weight
• Perinatal events
• Breast feeding
• Early infectious events
We plan to analyze 20 families in which the child has developed celiac disease
and compare the data with 20 families in which no son developed celiac disease.
In this cohort we will investigate:
•
the status of DNA methylation of candidates genes and the transmission
inside the family
•
the possible variations in DNA methylation patter in relationship to the risk
factors listed above
•
the expression levels and activities of histone-modifying enzymes
•
the genomic distribution of particular histone PTMs
The PHASE 3: CD-EPICENTER
COHORT
• Also for this cohort we will estimate the environmental factors listed
above.
We aim to perform a comprehensive analysis to estimate the Pre-Natal
risk factors and genomic and epigenetic profiles in a subsample
with higher risk factors
• We plan to analyze in at least 48 families with the more significant risk
factors, suggested by the analysis performed in the previous phases of
the study.
• The comparative epigenetic analysis of the placenta might
provide a first evidence of the direct action of environmental
factors in the modulation of genetic expression pattern in CD.
CONCLUDING…
• We demonstrated that the gene expression analysis of candidate genes, allow
to discriminate between CD and controls. Now, our idea is to understand if
these differences may be due to epigenetic mechanisms, and if they can be
triggered by environmental factors.
• Inherited epigenetic changes have been proposed as an explanation for the
‘‘missing heritability,’’ meaning inherited causes of risk of complex genetic
diseases as CD that have not yet been identified in GWAS.
• The prospective CD-EPICENTER cohort enrolled in this study will contribute
to understand the weight of risk factors in a population of CD cases which is
not fully homogeneous.