Abstract
Brain development is an intricate process governed by a series of cell proliferation, differentiation, and migration events to form the complex architecture of the human brain. To ensure that correct cellular development is achieved, dosage, location, and timing of gene expression are essential. Perturbations during these developmental stages can result in a plethora of neurodevelopmental disorders (NDDs). Collectively, NDDs are defined as a heterogeneous group of disorders, characterised by deficits in cognition and motor abilities. Investigations into the underlying molecular mechanisms of NDDs has revealed impairments in cell proliferation and differentiation, chromatin remodelling, synaptogenesis, and post-transcriptional regulation. While extensive efforts have been employed to identify these mechanisms, many of the genes involved remain unexplored.
Given the large amount of phenotypic and genetic variability, NDDs are often difficult to diagnose. As a result, many individuals do not have a genetic diagnosis for their disorder. Therefore, this thesis aimed to investigate the molecular and genetic mechanisms underpinning NDDs. To address this aim, three specific objectives were carried out. Firstly, exome and genome data analysis were undertaken to identify causative variants in New Zealand families living with genetically undiagnosed NDDs. From these analyses, three families were provided with a genetic diagnosis for their child’s disorder. Specifically, two of the variants were identified in well-established disease genes (XRCC4, EP300), providing an immediate answer for these families. A third variant was identified in a novel disease gene, MSL2, which encodes a component of the MSL complex involved in chromatin modification. This discovery has now been published as part of an international collaboration.
The remaining objectives of this PhD involved investigating ELAV-Like 2 (ELAVL2), a gene previously identified for its crucial role in brain development. Currently, ELAVL2 has not been associated with NDDs. We have collated an international cohort of 15 patients with de novo variants in ELAVL2, all of whom present with overlapping neurodevelopmental phenotypes. Five truncating variants and two larger-scale gene disruptions have been identified, confirming that haploinsufficiency for ELAVL2 is the likely disease mechanism. However, seven missense variants and a terminal exon frameshift required further molecular investigation. It was hypothesised that these variants also act in a haploinsufficient manner, by disrupting the levels or function of ELAVL2. A cycloheximide chase assay confirmed that several variants reduced the stability of ELAVL2, supporting a haploinsufficiency hypothesis and that these variants are pathogenic. Variants that did not decrease protein stability were studied for their impact on ELAVL2 homodimerisation, an interaction that has not been well documented. Through co-immunoprecipitation, it was confirmed that mammalian ELAVL2 can form a homodimer and that this interaction is upheld in the presence of select patient variants. These results support that variants in ELAVL2 cause a novel NDD, connecting this well-established neuronal protein with the neurodevelopmental phenotypes seen in patients.
Together, this study has contributed to our understanding of the genes and pathways disrupted in NDDs. During these analyses, three New Zealand families were provided with a genetic diagnosis for their child’s disorder. These findings are crucial for the families involved, as they may assist with reproductive planning and clinical management strategies. Further, the identification of MSL2 and ELAVL2 as novel NDD genes has expanded our knowledge of the genes that cause such disorders. The precise molecular mechanisms driving the MSL2 and ELAVL2-related disorders remain to be elucidated.