Variability in diffusion kurtosis imaging: Impact on study design, statistical power and interpretation.

Diffusion kurtosis imaging (DKI) is an emerging technique with the potential to quantify properties of tissue microstructure that may not be observable using diffusion tensor imaging (DTI). In order to help design DKI studies and improve interpretation of DKI results, we employed statistical power analysis to characterize three aspects of variability in four DKI parameters; the mean diffusivity, fractional anisotropy, mean kurtosis, and radial kurtosis. First, we quantified the variability in terms of the group size required to obtain a statistical power of 0.9. Second, we investigated the relative contribution of imaging and post-processing noise to the total variance, in order to estimate the benefits of longer scan times versus the inclusion of more subjects. Third, we evaluated the potential benefit of including additional covariates such as the size of the structure when testing for differences in group means. The analysis was performed in three major white matter structures of the brain: the superior cingulum, the corticospinal tract, and the mid-sagittal corpus callosum, extracted using diffusion tensor tractography and DKI data acquired in a healthy cohort. The results showed heterogeneous variability across and within the white matter structures. Thus, the statistical power varies depending on parameter and location, which is important to consider if a pathogenesis pattern is inferred from DKI data. In the data presented, inter-subject differences contributed more than imaging noise to the total variability, making it more efficient to include more subjects rather than extending the scan-time per subject. Finally, strong correlations between DKI parameters and the structure size were found for the cingulum and corpus callosum. Structure size should thus be considered when quantifying DKI parameters, either to control for its potentially confounding effect, or as a means of reducing unexplained variance

Neuroepigenetics of preterm white matter injury

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

Genetic determinants of white matter integrity in bipolar disorder

Bipolar disorder is a heritable psychiatric disorder, and several of the genes associated with bipolar disorder and related psychotic disorders are involved in the development and maintenance of white matter in the brain. Patients with bipolar disorder have an increased incidence of white matter hyper-intensities, and quantitative brain imaging studies collectively indicate subtle decreases in white matter density and integrity in bipolar patients. This suggests that genetic vulnerability to psychosis may manifest itself as reduced white matter integrity, and that white matter integrity is an endophenotype of bipolar disorder. This thesis comprises a series of studies designed to test the role of white matter in genetic risk to bipolar disorder by analysis of diffusion tensor imaging (DTI) data in the Bipolar Family Study. Various established analysis methods for DTI, including whole-brain voxel-based statistics, tract-based spatial statistics (TBSS) and probabilistic neighbourhood tractography, were applied with fractional anisotropy (FA) as the outcome measure. Widespread but subtle white matter integrity reductions were found in unaffected relatives of patients with bipolar disorder, whilst more localised reductions were associated with cyclothymic temperament. Next, the relation of white matter to four of the most prominent psychosis candidate genes, NRG1, ErbB4, DISC1 and ZNF804A, was investigated. A core haplotype in NRG1, and three of the four key single nucleotide polymorphisms (SNPs) within it, showed an association with FA in the anterior thalamic radiations and the uncinate fasciculi. For the three SNPs considered in ErbB4, results were inconclusive, but this was consistent with the background literature. Most notable however, was a clear association of a non-synonymous DISC1 SNP, Ser704Cys, with FA extending over most of the white matter in the TBSS and voxel-based analyses. Finally, FA was not associated with a genome-wide supported risk SNP in ZNF804A, a finding which could not be attributed to a lack of statistical power, and which contradicts a strong, but previously untested hypothesis. Whilst the above results need corroboration from independent studies, other studies are needed to address the cellular and molecular basis of these findings. Overall, this work provides strong support for the role of white matter integrity in genetic vulnerability to bipolar disorder and the wider psychosis spectrum and encourages its future use as an endophenotype

Investigation into DNAm and brain structural and connectomic covariance: a life course approach

Early life environmental stress, indexed by perinatal factors such as birth weight and gestational age, is associated with differences in brain structure and connectivity in early life, as well as being associated with a range of neurodevelopmental, psychiatric and cognitive outcomes, from childhood and into adulthood. While studies have investigated how these early life factor influence brain structure in early life, few have investigated this in older age. The molecular underpinnings of these relationships are not well understood. DNA methylation is an epigenetic mechanism that regulates gene expression; it is developmentally dynamic and responsive to environmental factors, making it a promising candidate for providing mechanistic insight into how early life stressors exert their effects. The aims of this thesis are as follows: to better characterise the associations between birth weight and brain structure and connectivity in later-life; to evaluate the evidence that DNAm is implicated in brain structure and function; to investigate the impact of gestational age at birth on the neonatal methylome and its association with brain connectivity. In the first study, I investigated the associations between variation in normal birth weight and measures of brain structure and connectivity in participants aged 73 years from the Lothian Birth Cohort 1936. Larger birth weight was associated with larger brain volume, and with regional cortical surface area, but not with white matter microstructure. This relationship between birth weight and brain size did not appear to be related to the degree of atrophy that had taken place. Early life growth is likely to be associated with brain tissue reserve, in evidence in later life. In the second study, I conducted a systematic review to evaluate the evidence linking DNA methylation to brain structure and function across the life-course. Sixty studies, encompassing both health and disease contexts, were identified. Together, these studies indicated that differential DNAm is associated with brain structure and function for 8 categories of disease across the life course, although uncertainties remain. Modest consistency between DNAm and neuroimaging features precluded the possibility of quantitative synthesis. I identified potential sources of bias in existing literature, enabling the development of guidelines that could reduce methodological heterogeneity in imaging-DNAm studies. Finally, I identified a DNAm signature of gestational age in neonatal saliva samples and tested its association with brain white matter microstructure. Participants were neonates, born preterm or term, recruited to the Theirworld Edinburgh Birth Cohort. There was widespread differential methylation associated with gestational age at birth, at term equivalent age. Several genes were identified that have previously been implicated in association with gestational age in cord blood, and with disorders known to contribute to the aetiology of preterm birth, such as pre-eclampsia. An epigenome-wide variable of the DNAm signature was associated with white matter microstructure, suggesting that DNAm contributes to white matter dysconnectivity in the neonatal period. This thesis provides evidence that early life exerts an effect on brain structure into later life, that DNAm and MRI neuroimaging are associated across the life-course and in a range of health and disease contexts, and that DNAm is profoundly altered in association with variation in gestational age and that this may contribute to white matter connectivity in the neonatal period

Diffusion imaging and tractography in the paediatric neurosurgical population

Diffusion MRI uses magnetic field gradients to sensitise a MR sequence to in vivo water diffusion. Application of these gradients in specific directions (20 in this work) enables a 3D representation of diffusion on a voxel basis. Quantitative diffusion measures are derived; using the voxel maximal diffusion direction and linking neighbouring voxels iteratively based on this creates a visual construct of the white matter: tractography. It is not possible, currently, to non-invasively determine the histological nature of an intracranial tumour. We recruited paediatric patients with radiological evidence of such lesions from April 2006 to January 2008 and retrospectively to August 2003. We used diffusion MR metrics to discriminate paediatric central nervous system tumours based on existing histological diagnoses. Using apparent diffusion coefficient histograms, common posterior fossa childhood tumours were differentiated with 93% success; Primitive neuroectodermal tumours (PNET) and supratentorial atypical teratoid rhabdoid tumours (ATRT) were separated in 100% of cases. Development of these methods with a larger population may facilitate the obviation of surgical biopsy and its attendant risks. Diffusion data was used to reconstruct the cerebellar white matter anatomy using tractography. Initially a population of normal subjects were investigated using single region of interest (ROI) analysis. DTI metrics were implemented, demonstrating the existence of white matter asymmetry where lateralisation corresponded to handedness in 17 right-handed subjects. To asses functional significance of changes in DTI metrics; clinical cerebellar dysfunction was correlated with changes in cerebellar white matter DTI metrics in a patient population with posterior fossa tumours and with the normal population. Fractional anisotropy of the tracts was reduced in patients with tumours d clinical cerebellar signs as compared to healthy individuals. This work demonstrates that diffusion MRI and tractography metrics may enable discrimination of paediatric CNS tumour type and are related to the functional integrity of cerebellar white matter tracts

Effect of preterm birth on white matter tracts and infant cognition

Preterm birth (defined as birth before 37 weeks) is a leading cause of neurocognitive impairment in childhood, including difficulties in social cognition and executive function. Microstructural divergence from typical brain development in the preterm brain can be quantified using diffusion magnetic resonance imaging (dMRI) tractography during the neonatal period. The relationship between dMRI tractography metrics and later cognitive difficulties remains inconclusive. A general measure of white matter microstructure (gWM) offers a neural basis for cognitive processes in adults, however it remains unclear when gWM is first detectable in the developmental trajectory. Eye-tracking is a technique which assesses eye-gaze behaviour in response to visual stimuli, which permits inference about underlying cognitive processes, such as social cognition and executive function in infancy. The primary aims of this thesis were to test the hypotheses: dMRI tractography reveals significant differences in tract-average fractional anisotropy (FA) and mean diffusivity (MD) between preterm and term infants, and variance in tract-average FA and MD is shared across major tracts. Secondly, infants born preterm have altered social cognition and executive function compared to term born peers, assessed by eye-tracking and finally, neonatal MRI gWM is associated with cognitive function in infancy. Preterm (birth weight ≤ 1500g) and term infants (born ≥ 37 weeks’ post-menstrual age [PMA]) were recruited and underwent a MRI scan at term equivalent age (between 38 - 42 weeks’ PMA) and an eye-tracking assessment six to nine months later. Preterm infants were assessed at two years using the Bayley Scales of Infant and Toddler Development, Third Edition (BSID-III). dMRI tractography metrics were generated using probabilistic neighbourhood tractography (PNT) in eight pre-defined tracts-of-interest. Principal component analyses (PCA) were used to determine the correlations between the eight tracts-of-interest for four tract-averaged water diffusion parameters. dMRI metrics were compared to the eye-tracking performance and two year outcome data. Quantitative microstructural changes were identifiable within the preterm brain when compared to infants born at term. PCA revealed a single variable that accounts for nearly 50% of shared variance between tracts-of-interest, and all tracts showed positive loadings. Eye-tracking revealed group-wise differences in infant social cognition, attributable to preterm birth, but executive functions inferred from eye-tracking did not differ between groups. dMRI tractography metrics within the neonatal period did not relate to later outcome measures. This thesis shows that variance in dMRI parameters is substantially shared across white matter tracts of the developing brain and suggests that anatomical foundations of later intelligence are present by term equivalent age. Social cognition is altered by preterm birth, however social cognitive ability in infancy is independent of gWM