Paediatrics (RCH) - Theses
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ItemInvestigating the molecular basis of brain malformations causing severe infantile epilepsies( 2023-08)Malformations of cortical development (MCD) encompass a wide spectrum of conditions that result from abnormal brain development, including polymicrogyria, periventricular nodular heterotopia and focal cortical dysplasia. MCD affect ~1 in 2,500 children and can result in epilepsy, developmental delay and cerebral palsy. Infantile epileptic spasms syndrome (IESS) is one of the most common forms of severe epilepsy of infancy and MCD are observed in ~50% of children with IESS. It is thought that the majority of cases of IESS and MCD have an underlying genetic cause. Although multiple molecular pathways have been identified, ~60% of cases still lack a genetic diagnosis after standard clinical diagnostic testing. Genetic diagnoses are important for prognosis, diagnostic testing, genetic counselling and precision medicine. Recent advances in genetic technologies allow for rapid, in-depth interrogation of the molecular mechanisms underlying MCD formation. The hypotheses of this thesis are that the application of high-resolution genomic technologies will facilitate a better understanding of the genetic causes of IESS with MCD and that functional characterisation of genes of interest will assist to identify crucial molecular pathways in the normal development and function of the developing brain. The primary research aims of this project are: to investigate the genetic landscape of IESS due to MCD by utilising brain tissue studies; to develop a deeper understanding of the molecular basis and phenotypic classifiers involved in the pathogenesis of brain somatic SLC35A2 variation and its relationship to mild malformation of oligodendroglial hyperplasia in epilepsy (MOGHE); and to identify molecular mechanisms and biomarkers of MOGHE. The genetic basis of IESS due to MCD was identified in 47/59 (80%) individuals. Germline pathogenic variants were identified in 27/59 (46%) individuals in TSC2 (n=19), DEPDC5 (2), CDKL5 (2), COL4A1 and 6p25.2 deletion (1), NPRL3 (1), FGFR1 (1) and TSC1 (1). Pathogenic brain somatic variants were identified in 21/59 (36%) cases, in SLC35A2 (n=9), PIK3CA (3), MTOR (2), TSC2 (2), AKT3 (2), OFD1 (1), TSC1 (1) and DEPDC5 (1). This included one individual with a two-hit DEPDC5 diagnosis, with a germline DEPDC5 variant and a somatic DEPDC5 variant. Multidisciplinary tools and integrated diagnosis review identified all nine individuals with SLC35A2 variants as having mild malformation of cortical development with oligodendroglial hypoplasia in epilepsy (MOGHE). Multi-omic analysis of MOGHE tissue using single nuclei RNA-sequencing (snRNA-seq) and Tandem Mass Tag 16pro liquid chromatography–mass spectrometry (LC-MS/MS) showed that neurogenesis, neuron projection development, axo-dendritic development and neuron differentiation are dysregulated in MOGHE. snRNA-seq analysis identified oligodendrocyte progenitor cells (OPCs) and dividing oligodendrocyte progenitor cells (cOPCs) as the primary cells exhibiting aberrant expression profiles in lesional MOGHE tissues. ACTB, GNG7, MAP4, NEFL, SEMA3E and SOD1 were identified as potential biomarkers in MOGHE and may serve as disease biomarkers or druggable targets in the future. The findings of this thesis contribute to the knowledge of the genetic landscape of paediatric brain malformations and severe epilepsies of infancy and broaden the understanding of cortical malformation development and epileptogenesis.
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ItemThe Epilepsies of Cerebral Palsy( 2023-05)Epilepsy in children with cerebral palsy (CP) places a significant burden on individuals, society and the health care system. There is limited data on the association between aetiology, neuroimaging, EEG findings and epilepsy syndrome classification for children with CP. I have conducted a population-based study to characterise the epilepsy according to single pathological substrates: white matter injury (WMI) and grey matter injury (GMI), which can be further subclassified to deep GMI, watershed injury or an arterial ischaemic stroke (AIS). I also analysed the prevalence, type and outcomes of epilepsy in children with CP. This will help guide counselling and treatment.
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ItemTuberous sclerosis complex and autism spectrum disorder: phenotypic presentation and associated clinical factors( 2022)Background: Tuberous sclerosis complex (TSC) is an autosomal dominant, multisystem, genetic condition with variable disease severity, due to pathogenic variants in TSC1 or TSC2, which disrupt the mTOR pathway. Neurological morbidity, including seizures and structural brain changes, occur in most people. Seizures commonly present during the first three years of life and infantile spasms occur in around one third of children. Autism spectrum disorder (ASD) and intellectual disability (ID) are reported in half of TSC affected people. When this PhD commenced, it was unclear if ASD in TSC was similar to ASD without a co-existing medical condition, that is ‘idiopathic’ ASD, and if TSC clinical factors contributed to ASD like they impact on ID. Research had shown early seizure-onset, infantile spasms, TSC2 variant, and tubers to be potentially associated with ASD in TSC. However, findings were mixed, and many studies employed weak methods for ASD categorisation. A systematic and comprehensive investigation had not been undertaken. Aims: This thesis aimed to (A) define the autism phenotype in children with TSC and (B) investigate the major clinical factors: (1) seizures and associated factors, (2) genetic factors, (3) structural brain changes, and (4) individual child characteristics, associated with ASD outcome in TSC. Methods: Two systematic reviews with meta-analyses, methodically and comprehensively summarised the literature about (1) the TSC-ASD phenotype and (2) the TSC clinical, EEG, imaging and genetic factors reportedly associated with ASD. The systematic reviews informed development of a cohort study with 50 children which undertook robust ASD assessment and developmental phenotyping in children with TSC and investigated associations between ASD with TSC factors. Results: Systematic review and meta-analysis found 90 percent of children with TSC-ASD had ID, but little was reported about other phenotypic aspects. The cohort study found children with TSC-ASD had moderate ASD severity and typical characteristics, although most had severe ID. Co-occurring language disorder, ADHD, executive dysfunction and externalising behaviours occurred in most, and internalising behaviour in one-third. Systematic review and meta-analysis found significant associations between TSC-ASD and history of seizures (OR 3.79), seizure-onset in infancy (OR 2.65), infantile spasms (OR 3.04), and male sex, (OR 1.62). Seizure-related associations were not significant in the cohort study, but the trends were similar. Incorporation of cohort study data with the meta-analysis led to a significant association between TSC-ASD and TSC2 variant, (OR 1.95). Tuber burden was not associated with TSC-ASD in the systematic review meta-analysis or cohort study. Conclusion: Clarification of the TSC-ASD phenotype enables better counselling for families of affected children and improved early intervention planning. Early life seizures and TSC2 variant are likely important pathways to TSC-ASD. The role of cortical tubers is likely to be indirect, via seizures. Research following large TSC-ASD cohorts from infancy throughout childhood, measuring all relevant variables, will assist with disentangling the relationships. Studies investigating interventions targeting possible aetiological factors will be of significant value, aiding the understanding of causation and potentially improving developmental outcomes.
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ItemUnderstanding the genetic basis and pathogenic mechanism of focal cortical dysplasia( 2021)Focal Cortical Dysplasia (FCD) is one of the most common brain malformations and a frequent cause of intractable epilepsy. Affected individuals often require surgical removal of the affected cortex for seizure control. The European Epilepsy Brain Bank estimates that different subtypes of FCD are the reason for up to 70.6% of epilepsy surgery related to brain malformations. Given the clinical importance of this disorder, there is an urgent need to further understand its biology and disease mechanism. Although FCD was first described 50 years ago, the genetic and pathogenic mechanisms of the disorder remain incompletely understood. We hypothesise that the delineation of disease mechanism underlying FCD will not only improve diagnosis and clinical management, but will also provide novel insights into the mechanisms underpinning human brain development and function. This project applied modern genomic technologies to comprehensively study a unique archive of surgical brain tissues derived from individuals affected by FCD. The first part of this study focussed on understanding the genetic basis of a subtype of FCD, known as bottom-of-sulcus dysplasia (BOSD). BOSD is a highly localised subtype of FCD with the dysplastic features being maximal at the bottom of sulcus. Using deep sequencing, we analysed blood and brain derived gDNA from 20 BOSD brain specimens. We found that brain-specific, pathogenic somatic MTOR variants accounted for 50% of the cases, with an additional two cases caused by heterozygous germline variant in DEPDC5 or NPRL3. These genes are all involved in the mTOR signalling pathway. Further characterisation of four somatic MTOR cases found that the somatic variants were more abundant in the bottom of sulcus compared to the nearby cortex, with a positive correlation with the severity of histopathology. Our study shows that BOSD belongs to an expanding group of “mTORopathies” and highlights that BOSD lesion is highly localised, with well-defined genetic and pathologic substrates. In the second part of this study, we tested the germline + somatic “two-hit” hypothesis in FCD. The “two-hit” hypothesis suggests that individuals with heterozygous germline variant acquired FCD lesion as a result of a somatic second “hit”. In a case study of an individual with FCD and a heterozygous germline DEPDC5 variant, we identified a somatic second “hit” in the same gene. By combining experimental and clinical findings, we showed in this individual that the brain region with the highest somatic variant allele frequency was also the region of seizure onset, suggesting a relationship between the somatic variant and epileptogenicity. We further characterised the brain specimens and found that somatic variant allele frequency positively correlated to the density of dysmorphic neurons. Furthermore, laser capture microdissection showed that the somatic variant was only detected in the dysmorphic neurons and not in the normal neurons. Collectively, we provided supporting evidence for the “two-hit” mechanism and demonstrated the molecular and cellular characteristics of FCD lesion. The third part of this study involved the application of single nucleus RNA-sequencing (snRNA-seq) to study 26 surgical brain specimens resected due to a range of brain malformations including FCD. These brain malformations are closely related and commonly characterised by the presence of abnormal cell types i.e. dysmorphic neurons and balloon cells. We generated 284,891 single nuclei transcriptomes, the largest snRNA-seq dataset for epilepsy brain specimens to date. Using a combination of bioinformatic analysis and molecular characterisation, we identified the probable transcriptional profiles of dysmorphic neurons and balloon cells. Furthermore, we identified commonly perturbed pathways in both cell types, namely calcium signalling pathway and glutamatergic synapse regulation, which may help explain the pathogenic mechanism underlying these different types of closely related brain malformations. In conclusion, this study has provided fundamental insights into the genetic and pathogenic mechanism underlying FCD and related brain malformations. In addition to improving genetic diagnosis and clinical management, these findings have the potential to pave the way for the identification of novel druggable targets by defining the genetic and transcriptomic profiles of FCD.