Neuroepigenetics of preterm white matter injury

Introduction: Preterm birth is increasing worldwide and is a major cause of neonatal death.\ud
Survivors are at increased risk of neurodisability, cognitive, social and psychiatric disorders in\ud
later life. Alterations to the white matter can be assessed using diffusion tensor imaging (DTI)\ud
MRI and are associated with poor neurodevelopmental outcome. The pathogenesis of white\ud
matter injury is multifactorial and several clinical risk and resilience factors have been\ud
identified. DNA methylation (DNAm) is an epigenetic process which links stressful early life\ud
experience to later life disease and is associated with normal brain development, neuronal\ud
processes and neurological disease. Several studies have shown DNAm is altered by the\ud
perinatal environment, however its role in preterm white mater injury is yet to be\ud
investigated.\ud
Aims: 1. To examine the relationship between preterm birth and white matter integrity 2. To\ud
investigate the effect of neuroprotective treatments and deleterious clinical states on white\ud
matter integrity in preterm infants 3. To assess the best DTI method of quantifying white\ud
matter integrity in a neonatal population 4. To investigate the effect of preterm birth on DNA\ud
methylation and 5. To determine the clinical and imaging factors that contribute to the\ud
variance in DNA Methylation caused by preterm birth\ud
Methods: DTI data was acquired from preterm infants (< 32 weeks’ gestation or < 1500 grams\ud
at birth) at term equivalent age (TEA) and term controls (> 37 weeks’ gestation at birth).\ud
Region-of-interests (ROI) and tract-averaged methods of DTI analysis were performed to\ud
obtain measurements of fractional anisotropy (FA) and mean diffusivity (MD) in the genu of\ud
corpus callosum, posterior limb of internal capsule and centrum semiovale. Clinical data was\ud
collected for all infants and the effect of prematurity, neuroprotective agents and clinical risk\ud
factors on white matter integrity were analysed. 8 major white matter tracts were segmented\ud
using probabilistic neighbourhood tractography (PNT), a tract-averaged technique which also\ud
allowed the calculation of tract shape. The two DTI techniques were compared to evaluate\ud
agreement between results. DNA was collected from preterm infants and term controls at\ud
TEA, and a genome-wide analysis of DNAm was performed. DTI parameters from probabilistic\ud
neighborhood tractography (PNT) methodology and clinical risk and resilience factors were\ud
used to inform a principal components analysis to investigate the contribution of white\ud
matter integrity and clinical variables to variance in DNAm.\ud
Results: FA and MD were significantly affected by preterm birth on ROI analysis. In addition,\ud
DTI parameters were affected by clinical factors that included antenatal magnesium sulphate,\ud
histological chorioamnionitis and bronchopulmonary dysplasia. Evaluation of DTI\ud
methodology revealed good accuracy in repeated ROI measurements but limited agreement\ud
with tract-averaged values. Differential methylation was found within 25 gene bodies and 58\ud
promoters of protein-coding genes in preterm infants, compared with controls. 10 of these\ud
genes have a documented association with neural function or neurological disease.\ud
Differences detected in the array were validated with pyrosequencing which captured\ud
additional differentially methylated CpGs. Ninety-five percent of the variance in DNAm in\ud
preterm infants was explained by 23 principal components (PC); corticospinal tract shape\ud
associated with 6th PC, and gender and early nutritional exposure associated with the 7th PC.\ud
Conclusions: Preterm birth is associated with alterations in white matter integrity which is\ud
modifiable by clinical risk factors and neuroprotective agents. ROI analysis may not provide\ud
sufficient representation of white matter tracts in their entirety. Prematurity is related to\ud
alterations in the methylome at sites that influence neural development and function.\ud
Differential methylation analysis has identified several promising candidate genes for future\ud
work and contributed to the understanding of the pathogenesis of preterm brain injury