A Study of Nonsense Mediated mRNA Decay Using Naturally Occurring Genetic Variants and Through the Development of a Synthetic Reporter Transgene

Abstract

The nonsense mediated mRNA decay pathway (NMD) plays an important role in normal brain development. Genetic variation which disrupts genes encoding key NMD pathway members are implicated in neurodevelopmental disorders such as intellectual disability and autism. The mechanism by which deficient NMD results in neurodevelopmental dysfunction, however, remains unknown. Recently, NMD activity has been recognised to vary across cell types, tissue types and even display inter-individual variability. Yet current methods to quantify NMD activity rely on average cell population measurements and thus lack the resolution needed to capture dynamic changes resulting from the cell and tissue heterogeneity of NMD, as well as its developmental complexity. This thesis aims to further understand NMD by, (1) investigating naturally occurring genetic variants which cause neurodevelopmental disorders and (2) by the development of a synthetic NMD reporter transgene with single cell resolution. As part of this thesis three novel variants in genes encoding NMD factors which were identified in patients with neurodevelopmental disorders were characterised. The first of these variants was a synonymous single nucleotide variant (SNV) found in a canonical splice region of UPF3B. This variant was originally classified as a variant of unknown significance (VUS) and as such overlooked regarding pathogenicity. Molecular investigations in this thesis were able to conclusively resolve this variant as being pathogenic and facilitate patient diagnosis. The remaining two variants were identified within UPF2, the first was a novel frameshift variant, which is one of only two SNVs identified to exclusively disrupt UPF2. The second was a large copy number variant (CNV) which resulted in the heterozygous deletion of UPF2 alongside 21 other genes. Investigations into the pathogenicity of these variants supported the involvement of UPF2 in a spectrum of neurodevelopmental disorders which has been concluded from previous studies where UPF2 has been disrupted by large CNV deletions. Within this thesis two versions of a fluorescent NMD reporter transgene which can measure NMD activity at a single cell level were also designed. Both transgenes are composed of a number of expression cassettes in ‘cis’. The most important of these are the Selection, Control and NMD cassettes. The Control and NMD cassettes co-express distinguishable fluorescent proteins allowing for visual and quantitative real-time output of NMD activity. The Selection cassette enables recombination mediated cassette exchange to take place, allowing the entire transgene to be introduced into the Col1a1 locus of germ-line competent transgenic mouse embryonic stem cells (mESCs) or transgenic mouse zygotes. In this thesis I have developed and used experimental pipelines to test the responsiveness of the designed NMD reporter transgenes to NMD inhibition in vitro. Unfortunately, following integration into mESCs neither version of the NMD reporter transgene was completely responsive to changes in cellular NMD activity. One version, however, was used to establish a stable and functional NMD reporter HEK293T cell line. These cells can facilitate highthroughput screening tests for drugs or small compounds which alter NMD activity to drive the development of therapeutics or benefit research. Once the design of an NMD reporter transgene is perfected for use in mESCs or a transgenic mouse line, this technology will provide visual and quantitative tracking of endogenous NMD activity at a single cell level. A possible immediate application would be to track NMD activity across embryonic brain development and into postnatal life. By providing a means to define regions or cell types in the brain most affected by malfunctioning NMD, e.g. due to heritable DNA mutations, the underlying mechanism by which deficient NMD leads to neurodevelopmental dysfunction can be further elucidated. This will support the development and assessment of more targeted therapies for individuals affected with neurodevelopmental disorders due to NMD disrupting genetic variants.