CD-EPI.CENTERCD-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. 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. 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.

DNA methylation Histone modifications (PTMs) MicroRNA regulation DNA methylation Histone modifications (PTMs) MicroRNA regulation 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 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 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 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. 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. 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. 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). 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 newborns are expected. For this new cohort we will collect: ― PBL from parents and probands before conception (500 samples) ― Samples of placenta ( samples) ― Umbilical cord ( samples) Perspective new cohort (CD-EPI.CENTER): ― 500 families with one celiac proband at moment of a new pregnancy In 3 years newborns are expected. For this new cohort we will collect: ― PBL from parents and probands before conception (500 samples) ― Samples of placenta ( samples) ― Umbilical cord ( 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 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 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

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 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 2: NEOCEL COHORT

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. 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. 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.