The aberrant glycosylation of α-dystroglycan is associated with a subset of clinically heterogeneous muscular dystrophies collectively referred to as dystroglycanopathies. Mutations in seven genes are currently known to result in these autosomal recessive disorders. To define the mutation frequency and clinical phenotypes associated with mutations in five of these genes, I analysed a large cohort of patients. This study redefined the clinical spectrum associated with mutations in POMT1, POMT2, POMGNT1, FKTN and LARGE. Mutations in these genes were detected in approximately a third of all cases indicating that, after the exclusion of FKRP involvement, the majority of patients with a dystroglycanopathy harbour mutations in novel genes. In order to identify novel genes in these disorders I applied a candidate gene approach to the whole genome. Genes known to be causative in dystroglycanopathy animal models and those predicted to function within the pathway of dystroglycan glycosylation were investigated. Mutation screening indicated that defects in GYLTL1B, β3GNT1, WWP1, GYG1, GYG2, MGAT5B and DAG1 are not frequent causes of human dystroglycanopathy. In order to evaluate the function of a subset of candidate genes, I optimised a cell culture model of dystroglycanopathy. Using a ribonucleotide interference based strategy, the expression of candidate genes were modulated in a mouse myogenic cell line. The down regulation of β3gnt1 induced a marked reduction in the glyco-epitope present on α-dystroglycan. This finding confirms β3gnt1 as a key enzyme functioning in the pathway of α-dystroglycan glycosylation. In addition, I combined the use of high density single nucleotide polymorphism genotyping platforms, expression microarrays and sequencing data to investigate the cause of disease in a small consanguineous pedigree. In summary, this data set has refined genotype-phenotype correlations of known causative genes as well as investigated the involvement of novel loci in muscular dystrophies with defective glycosylation of dystroglycan.

22q11DS is the most common microdeletion syndrome in humans, causing a range of phenotypes associated with abnormal pharyngeal development. TBX1 is considered the major genetic determinant of the syndrome. Deletion of Tbx1 in animal models phenocopies the patient manifestations. In an effort to understand the pathways involved in these processes, potential Tbx1 effectors previously determined by microarray screens were studied. Smad7 is an inhibitor of the TGF-/BMP pathways and to date several TGF-/BMP signalling genes have been described in heart development. In this thesis Smad7 was found expressed in pharyngeal and cardiovascular structures during mouse embryogenesis while a gene-trap mouse model displayed a partially penetrant 22q11DS-like phenotype. Furthermore, Tbx1 and Smad7 were found to interact synergistically towards arch artery morphogenesis. Hes1 is a transcriptional repressor, part of the Notch signalling pathway, which produces craniofacial and cardiovascular anomalies when deactivated. Expression analysis undertaken demonstrated that Hes1 overlaps with Tbx1 in the embryonic pharynx and it is diminished in Tbx1-/- embryos. A conditional mutagenesis approach revealed non cell autonomous actions for Hes1 in the ectoderm affecting great vessel and eye development, while Hes1 was required in both the ectodermal and neural crest lineages for thymus morphogenesis. Slit2 is a secreted protein that regulates axonal guidance, migration, outgrowth and branching. Recent evidence suggests a possible mechanism by which Tbx1 may regulate neural crest patterning through the Slit/Robo pathway. Slit1 and Slit2 were here shown to share expression domains with Tbx1 in the embryonic pharynx. Nevertheless, neither the Slit2 nor the Slit1;Slit2 mutation produced a 22q11DS-like phenotype while double mutation for Tbx1;Slit2 did not enhance the Tbx1 haploinsufficient phenotype. Altogether, this thesis provides data clarifying a subset of Tbx1 mechanisms important for cardiovascular development and identifies Smad7 as a Tbx1 regulated gene involved in great vessel morphogenesis.

This thesis aims to examine whether government policies in the early years are likely to influence health inequalities, using information derived from a range of sources to contribute to the 'jigsaw of evidence', and focussing on two case studies featuring high on the Labour government agenda: the first a measure of health (unintentional injury) and the second a policy (childcare). Scoping reviews were used to map areas requiring further research for each case study. A review of policy documents helped set the policy context, and secondary datasets were utilised to describe prevalence, trends and inequalities. Following this some of the less researched associations identified in the scoping reviews were explored, using data from the Millennium Cohort Study (MCS). An analysis investigating whether the home environment lay on the causal pathway between socio-economic circumstances (SECs) and unintentional injuries found that controlling for a number of proxy measures for housing quality and safety equipment use did not alter the social gradient in injuries occurring in the home. A second analysis exploring a measure of main childcare use in relation to injuries found that infants (aged 9 months) from more advantaged SECs were less likely to have been unintentionally injured (anywhere) since birth if they were looked after in formal childcare (compared to those looked after only by a parent). In contrast, those from less advantaged SECs appeared to be at greater risk of injury. Informal childcare was associated with an increased risk of injury between 9 months and 3 years, overall and in less advantaged SECs. Two further analyses explored breastfeeding and overweight in relation to childcare use. Informal and formal childcare were both associated with a reduced likelihood of breastfeeding, and whilst this remained the case for informal childcare across all social groups, for formal childcare the reduced likelihood of breastfeeding was only observed in those from more advantaged groups. Children who were looked after in informal childcare appeared to be more likely to be overweight at 3 years than those only looked after by a parent, particularly if they were from more advantaged SECs. These results were then synthesised in light of current and potential policy. The analyses were conducted with observational data and using proxy measures for the home environment and main childcare use, therefore further research is required. Findings imply that improvements to the home environment may not necessarily influence inequalities in injuries (although are likely to benefit other areas of health and wellbeing). Main childcare type appeared to be associated with better health outcomes for some social groups but not for others; further research is required to ascertain how these benefits can be recognised for all groups, for example through improved training and guidance for childcare professionals. This thesis demonstrates the use of epidemiological information for contributing to the 'jigsaw of evidence', but also the complexities of considering policy impacts on health inequalities.

My MD (Res) thesis is based on a research study describing the characteristics and developmental trajectories of a high risk cohort of children and young people presenting to my specialist clinical service with sexually abusive behaviour and emerging severe personality disorder traits. At the time I commenced the research study there was considerable public interest in the early origins of serious adult offending, including sexual offending and adult personality disorder. The research study was funded by the Home Office DSPD (Dangerous People with Severe Personality Disorder) Programme and supported by the Department of Health and the National Offender Management Service. It was hoped that my study could throw some light on these issues and that the results might provide supportive evidence for current government policies on early intervention to prevent antisocial behaviour. The MD(Res) thesis describes this background to the study and the rationale for beginning the research as well as its' scientific value. A full literature review sets out the evidence base for undertaking the study and informs the research aims, objectives and hypotheses. The findings of the MD(Res) research study inform my conclusions and the limitations of the study are fully acknowledged. Clinical, Policy and Research Recommendations are made stemming from the study conclusions.

Facial emotion recognition and theory of mind abilities are important aspects of social cognition. Genes within the X chromosome may influence these abilities as males show increased vulnerability to impaired social cognition compared to females. An influence of a single nucleotide polymorphism (SNP), rs7055196 (found within the X-linked EFHC2 gene), on facial fear recognition abilities has recently been reported in Turner Syndrome. This thesis explores the influence of SNP rs7055196 on aspects of social cognition in healthy males. Males possessing the G allele showed poorer facial fear recognition accuracy compared to males possessing the A allele. This group difference in fear recognition accuracy was not due to a difference in gaze fixations made to the eye or mouth regions. Males possessing the G allele also showed smaller N170 amplitudes in response to faces compared to males possessing the A allele. These results suggest males possessing the A allele may use a more holistic / configural face processing mechanism compared to males possessing the G allele, and this difference may account for the difference in fear recognition accuracy between the groups. Males possessing the G allele were also less accurate at inferring others’ mental states during the Reading the Mind in the Eyes task, and showed reduced activity in the right superior temporal gyrus, left inferior parietal lobule and left cingulate gyrus during this task compared to males possessing the A allele. SNP rs7055196 may therefore also influence theory of mind abilities, with males possessing the A allele showing better theory of mind than those possessing the G allele. This result may reflect higher empathising abilities in the males possessing the A allele. These results suggest an influence of SNP rs7055196 on social cognitive abilities in males. This may help to explain the sex difference in vulnerability to impaired social cognition.

The 22q11 deletion syndrome (22q11DS/DiGeorge Syndrome [DGS]) is a congenital disorder with complex aetiology including cardiovascular, thymic/parathyroid, craniofacial and neuro-behavioural phenotypes. These arise via abnormal development of embryonic structures including the pharyngeal arch/artery apparatus and the secondary heart field. Large (3Mb) hemizygous deletions of 22q11containing the TBX1 transcription factor are found in most human cases. Animal models and non-deleted patient data suggest haploinsufficiency of TBX1 is the major underlying cause of 22q11DS. To investigate the role of Tbx1 in cardiovascular development, putative transcriptional targets were previously identified using microarray. This thesis examines the role of two such targets, the Cyp26 gene family and Hes1. These genes are known to be involved, respectively, in the retinoic acid (RA) and Notch-signalling pathways. Both pathways are important in pharyngeal/cardiovascular development. Control of RA homeostasis/dosage is required for normal development and the Cyp26 enzymes metabolise RA to less active forms. Embryonic Cyp26 genes have altered expression in Tbx1-/-mice. This project investigated the effect of chemically blocking Cyp26 function upon pharyngeal/cardiovascular development in the chick. Furthermore, a mutant mouse model was used to establish whether loss of Cyp26b1function could result in the 22q11DS phenocopy observed. Finally, epistasis experiments ascertained whether a genetic interaction exists between Tbx1 andCyp26b1. The transcriptional repressor Hes1 is required for normal pharyngeal/cardiovascular development in the mouse. This thesis presents data showing a conserved role for her6 (zebrafish homologue) in zebrafish pharyngeal development and verification of a tbx1/her6 genetic interaction during pharyngeal development. Overall, this work provides further evidence that Tbx1 co-ordinates a number of signalling pathways in pharyngeal/cardiovascular development. This data refines the role of Tbx1 and RA-regulatory genes in 22q11DS cardiovascular phenotypes and corroborates the importance of an interaction between tbx1 and her6 (Hes1) in pharyngeal development.

Context: Intrauterine and postnatal growth influences future risks for metabolic syndrome. Body size and blood pressure (BP) are polygenic traits. The role for genetic variations in growth hormone (GH)-insulin-like growth factor (IGF) axis genes on intrauterine and early childhood growth and blood pressure, as well as gene loci identified through Genome Wide Association Studies (GWAS), are unclear. Objective: To determine whether common variations in the genes of the GH-IGF axis associate with antenatal growth and birth size and play a role in the determination of body size and blood pressure at 3 years of age. Study design: Pregnant women from white European families were recruited by the University College London Fetal Growth Study (n = 774). Fetal growth was measured by ultrasonography at each trimester. Postnatal growth data were collected prospectively at 6 months, 1 year, 2 years and 3 years of age. BP was measured at 3 years of age. Genotyping was performed by a combination of restriction fragment length polymorphism analysis, KBioscience Competitive Allele specific PCR genotyping System (KASP) analysis and multiplex polymerase chain reaction (PCR). Results: The GHR exon 3 deletion genotype was significantly associated with birth weight and placental weight. IGF1 SNPs did not demonstrate significant consistent longitudinal association with parameters investigated. IGF2 SNPs were significantly associated with intrauterine growth (rs680), birth weight (rs680), placental weight (rs680) and BP (rs3842759) at 3 years of age. Several SNPs in genes found to be associated with adult BMI and BP from GWAS were significantly associated with early childhood size (MTCH2, SH2B3), body composition (SH2B1, TMEM18) and BP (FTO). Conclusion: These data suggest that several polymorphisms in the GH-IGF axis and in GWAS-identified genes for adult BMI and BP are significantly associated with intrauterine and early childhood size and BP at 3 years of age.

Aims: This thesis sets out to assess the long-term speech, language and intellectual abilities of children born very prematurely (less than 33 weeks' gestational age) – and to investigate the neuroanatomical correlates of any deficits identified. Method: The study participants (n=50, mean age 16 years) consisted of a representative sample that was recruited from a prospective follow-up study at University College Hospital (London). This included children with a wide spectrum of brain injuries (identified at birth) matched with a term-born control group (n=30). An extensive battery of standardised assessments was administered, including measures of expressive and receptive language, vocabulary, speech-motor control and intelligence. All participants underwent neuroimaging, including diffusion-weighted MRI (DWI). Functional MRI was also used to determine hemispheric language lateralisation. Potential neural correlates of intelligence, speech and language were explored using manual tracing, voxel-based morphometry and DWI-tractography of relevant white matter pathways, such as the language-associated arcuate fasciculus and the speech-motor fibres of the corticobulbar tract (CBT). Results: In comparison to controls, the preterm group had a lower mean IQ score and increased incidence of speech and language problems. Conventional MRI showed abnormalities in more than half of the preterm children, most commonly within the periventricular and callosal white matter. Global brain white matter volume was also reduced in the preterm sample and explained a substantial proportion (70%) of the variance in their IQ scores. Problems with oromotor control were found to be associated with specific abnormalities in the posterior limb of the internal capsule (which contains the CBT); and the degree of language impairment was linked to a reduction in the volume of interhemispheric connections between the temporal lobes. Conclusions: This study has shown that preterm birth is associated with persistent changes in global, commissural and periventricular white matter (identifiable in adolescence) and has established robust neuroanatomical correlates of long-term outcomes in general intelligence, speech and language abilities.

Background The prevalence, aetiology and optimal management of the behavioural and cognitive difficulties, and circadian rhythm disturbances in children with midline brain abnormalities including isolated growth hormone deficiency (IGHD), isolated optic nerve hypoplasia (ONH) and septo-optic dysplasia (SOD) have to date not been adequately addressed. Aims and Methods This thesis aims to assess the prevalence of cognitive/behavioural problems and circadian rhythm abnormalities in children with midline brain abnormalities, and to further investigate any problems identified using high resolution MRI brain, actigraphy and melatonin profiling. Results Children with IGHD have significant impairments in motor skills and lower cognitive function scores, and children with ONH have significantly higher scores on the child behavioural checklist than controls. Children with SOD have significant sleep abnormalities. In IGHD corticospinal tract and corpus callosum fractional anisotropy (FA) and specific neural volumes are significantly lower than in controls, with neural abnormalities correlating significantly with IQ and motor skills scores. In ONH ventral cingulum, corpus callosum and optic radiation FA are significantly reduced, with right ventral 13 cingulum FA correlating significantly with behavioural assessment scores. In SOD melatonin production was absent in one child in association with a fragmented sleep pattern. Three children had normal melatonin profiles, one with an arrhythmic and two with fragmented sleep patterns. The remaining child had fragmented sleep and a modest increase in daytime melatonin concentrations. Conclusions These studies suggest that the GH-IGF-1 axis plays a role in brain and cognitive development. They also show that children with ONH require behavioural assessment, not previously part of routine clinical care, and that the behavioural abnormalities identified in children with ONH may be related to underlying whiter matter abnormalities. We have also demonstrated that the aetiology of the sleep disturbances found in SOD is complex, and not solely due to abnormal nocturnal melatonin production.

Convulsive Status Epilepticus (CSE) is the most common medical neurological emergency in children. Prolonged seizures are associated with brain injury in both animal models and humans and there is concern about longer term outcomes. A qualitative difference between prolonged and shorter seizures is apparent, but information about the longer term prognosis of CSE remains scarce. Retrospective studies have linked the commonest form of childhood CSE, prolonged febrile seizures (PFS), and temporal lobe epilepsy due to mesial temporal sclerosis (TLE-MTS). This study aimed to prospectively follow a population-representative cohort of children following CSE and look for signs of evolving MTS or other brain injury. Magnetic resonance imaging (MRI) was used to visualise underlying brain abnormalities and identify early signs of injury. To investigate the longitudinal evolution of any injury, MRI investigations were repeated at 6 and 12 months post-CSE and the child’s clinical status monitored throughout. 31.2% of children had an abnormal MRI scan post-CSE. Most of these abnormalities pre-dated the episode of CSE and no clinically significant abnormalities were found in children with PFS. Mean hippocampal volumes were only reduced in the group of children with symptomatic CSE; however 20-30% of all children showed loss of hippocampal volume during the year following CSE. This could represent a precursor to TLE-MTS. Further abnormalities were shown on diffusion tensor imaging in children with PFS. Hippocampal mean diffusivity did not show the usual age dependency and widespread reductions in fractional anisotropy were seen across major white matter tracts. These reductions were apparent at 1 and 6 months post-PFS, but resolved by 1 year. These findings form important evidence that children with non-PFS CSE are also at risk of long-term hippocampal damage and that children with PFS appear to have extensive extra-hippocampal abnormalities. These will be further explored in the body of this thesis.