The project tries to answer the fundamental question about the relationship between the degree of immaturity and the epigenic and gene expression profile as well as their function defined as proteome. Moreover, it represents an approach to should shed light on the pathomechanism of late complications of prematurity.
The project objectives:
Methods and approach
- To define in extremely preterm infants the extent of the relationship between the degree of immaturity and the epigenic and gene expression profile as well as their function defined as proteome.
- To asses if in the cohort of preterm infants of the same gestational age there is a dynamic time dependent change of methylation patterns leading to parallel changes in the activity of metabolic pathways. Also to identify environmental factors which might be associated with these changes.
- To define the potential impact of the epigenome of the preterm baby as well as its proteome on the risk of the development of short and long term complications of prematurity.
- To asses association of expression of whole genome with the occurrence and degree of severity of complications of prematurity in the animal model.
The study consists of two parts: animal experiments (carried out in Norway) and a clinical study taking place in neonatal wards (carried out in Poland).
In the experimental part of the project, the standardized mouse model that mimics the clinical entity BPD shall be employed. Such models are well described in the literature. Time pregnant mice are maintained on an ad libitum diet of standard chow and water. Within 12 hours of birth, pups are pooled and randomized to exposure to either 80% oxygen or room air (21%) for 28 days. Oxygen levels are continuously monitored and carbon dioxide levels will be spot checked at the outset to ensure normal ambient carbon dioxide level within the chamber. Nursing dams are rotated between the two groups every 24 hours to prevent oxygen toxicity in the dams. After 28 days of hyperoxia somatic growth and lung development is impaired. Gene expression of several well-known BPD biomarkers changes such as TGF-β which is increased and VEGF-α which decreases. The expression of a number of genes regulating biological processes is altered. Many of these pathways are identical to those we have found in our own previous studies exposing newborn mice to a brief hyperoxic exposure (30 min) following hypoxia (Wollen et al in preparation).
In clinical condition lung maturation is enhanced by administering antenatal steroids to the fetus. We have recently shown that such therapy up-regulates four genes, all related to cancer or inflammation. Moreover, seven gene pathways were up-regulated at day 5. These pathways are associated with cell growth, cell cycle regulation, metabolism, and apoptosis. (Saugstad et al, submitted). We therefore want to expose mice fetuses to antenatal steroids equivalent to the human exposure.
In this study we specifically want to study whether a long term hyperoxic exposure inducing a BPD like condition in newborn mice could lead to epigenetic changes. The following groups of animals will be tested:
1. Mice exposed to 80% oxygen from birth to day 28 (N=40)
2. Mice exposed to 21% oxygen from birth to day 28 (N= 40)
Group 1 and 2 will be divided into subgroups testing out the effect of exposure to antenatal steroids.
Animals will be sacrificed and tissue from the eyes (retina) and lungs will be collected for laboratory evaluation. Moreover, blood will also be obtained to assess if leukocyte expression profiles reflect what is found in the tissues. Collected materials will be transferred to the Department of Medical Genetics, Chair of Pediatrics in Jagiellonian University, Medical College (JU-MC) in Cracow.
These experiments are designed to mimic clinical conditions as closely as possible. Therefore, they may give us valuable information related to the clinical handling of premature babies.
In the second part of the project, a prospective cohort study shall be performed. The study will include 120 preterm infants born <= 30 week of gestation. The neonatal part of the project will be performed in two units: Department of Neonatology, Medical University of Warsaw, and Neonatal Intensive Care Unit of the Department of Pediatrics JU-MC. Both partners will include 60 newborns.
During delivery 1 ml of umbilical cord blood and 5 cm of umbilical cord will be collected. The samples will be placed in RNA/DNALater solutions and transferred to the Department of Medical Genetics Chair of Pediatrics, JU-MC in Cracow or deeply frozen (-700C) and transferred to the OMICRON laboratory of the Jagiellonian University in Cracow. Next, all the subjects enrolled in the study will undergo continuous observations. During program implementation, the same principles of oxygen therapy will be employed to all the children. Special, continuous monitoring (spO2, oxygen concentration in breathing mixture and mean airway pressure) will be carried out in all the children for the first 4 weeks of life. The results of these measurements will be computer-registered. Moreover, detailed information including type of feeding, route of delivery, total protein, fat and glucose intake, energy intake will be collected in all subjects. Blood samples will be drawn from all the study participants for laboratory evaluation on the 7th day of life and at the 36 weeks postmenstrual age.
The collected animal and human samples will be subjected to the following tests: evaluation of DNA methylation (epigentics), whole genome expression study by microarray technique, Blood protein spectrum evaluation (proteome) by the iTRAQ method for relative quantitation and nano-LC-MS/MS technique
The study of epigenetics and proteomics of complex diseases and traits is challenging, as epigenetic alterations or aberrations are tissue specific and undergo dynamic changes in response to the cellular environment and various other stimuli. These characteristics have created two substantial challenges when studying complex diseases: the need for a specific tissue (related to the particular disease) to investigate; and the need for multiple tissue types to be collected at different time points.
The design of the study allows the combination of tissue specific experimental data from the animal model with clinical data. Moreover, the clinical study design permits sampling at different time points. The study shall employ various modern techniques, which shall allow the reliable assessment of practically the whole pathway from genome methylation, RNA expression to proteome.
Description of the Project Plan
In order to manage the progress of the collaborative research towards the project’s objectives, the scientific, innovation-related and managerial activities have been broken down into seven coherent work packages. Each work package has its own defined deliverables and its own defined milestones to reach within the planned time and agreed budgetary limits. For each work package one of the participants has been assigned as the responsible partner.
The study will be started simultaneously in two countries. The Norwegian partner will start with Work Package 1 (WP1) – animal experiments. Experimental models for oxygen related diseases in premature newborn babies will be used. The groups of animals will be exposed to 21% or 80% oxygen from birth to day 28. Next, animals will be sacrificed and tissue from the eyes (retina), lungs and blood will be collected for laboratory evaluation. The goal of this part is to collect samples from 80 animals (40 in each group).
During the same time Polish partners will conduct a study in neonatal units (WP2). We have designed a prospective cohort study. 120 newborns will be enrolled during a 2 year time period. Detailed data concerning the risk factors and prevalence of complications of prematurity will be noted, and what is also important, data from medical monitors will be recorded in real time manner. 240 neonatal blood samples will be collected during this WP. Samples from the animal studies (delivered by WP1) and samples from human studies (delivered by WP2) will be used for epigenome, gene expression (WP3) and proteome (WP4) evaluation.
WP3 will be carried out in the Department of Medical Genetics Chair of Pediatrics, JU-MC in Cracow. Samples will be evaluated progressively when they arrive. 240 animal experiments, 240 gene expression experiments and 240 gene methylation analyses are planned. Moreover, to confirm results of gene expression studies, at the end of this WP, RT-PCR experiments are planned.
WP4 will be carried out in the OMICRON laboratories. Samples will be evaluated progressively when they arrive.120 samples obtained from 60 children treated in MUW will be the subject of a proteome evaluation by iTRAQ method. 120 samples obtained from 60 children treated in the NICU of Department of Pediatrics will be used for the hyperoxia experiment. Blood mononuclear cells (BMC) will be extracted from each sample and next divided into two subsamples. Subsamples from the each patient will be cultured in normoxic conditions (subsample 1) or hyperoxic conditions (subsample 2). Again, the iTRAQ method will by utilized to explore the differences in the protein composition in both subsamples. Data gathered during animal and experimental studies will be the subject of statistical evaluation (WP5). The microarray experiments as well the proteome experiments will deliver an extreme number of raw data. The evaluation will be performed by the Bioinformatics core facility at the University of Oslo. The final step of the project will be data synthesis and dissemination of the project’s results by presentations during international and national meetings and scientific publications (WP6).
To manage the project and to ensure that the consortium will reach its objectives on time and within the budget the WP7 is planned. It will consist of partner meetings, periodical assessments of the project results (reports), external audits, and operational management of the project on time and within the budget the WP7 is planned. It will consist of partner meetings, periodical assessments of the project results (reports), external audits, and operational management of the project on a day-to-day basis.