Paediatrics (RCH) - Theses
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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.