Hidden stitches: RNA cryptic splicing and its role in human disease

A great majority of human genes contain introns: tracts of mostly non-functional sequence that intervene the functional exons. When intron-bearing genes are transcribed into RNA, the introns are removed from the transcript via splicing, a process controlled by a multimolecular assembly called the spliceosome. Although splicing is generally well-regulated, the spliceosome sometimes splices RNA transcripts at sites other than their canonical exon boundaries. This “cryptic” splicing can be a random event, part of an unidentified regulatory process, the effect of a mutation, or the result of other perturbances to the spliceosome’s normal behaviour. In this thesis, I present four reports on the mechanisms underlying certain forms of cryptic splicing. In the first report, an analysis of pathogenic pseudoexons in the DMD gene reveals that each causative mutation falls into a distinct category defined by its proximity to the pseudoexon, and that many DMD pseudoexon splice sites are actively spliced in non-mutant cells. The second report builds on this by constructing a catalogue of over 400 pseudoexon variants from across the human transcriptome and uses this dataset to propose new and revised pseudoexon mutation categories. Like the first report, this second report also finds substantial congruence between pseudoexons and active deep intronic splice sites – including several recursive splice sites – suggesting a causal link between these phenomena. A third report explores how some cryptic exons may provide an explanatory mechanism to connect common genetic variants with their associated population phenotypes and outlines a simple method for discovering new examples. The fourth and final report uses RNA secondary structure modelling to explain why some antisense oligonucleotides can induce partial exon skipping through cryptic splice-site activation. Collectively, these reports present several novel insights into the causes of cryptic splicing and offer suggestions for how future research may build upon these insights

Allopurinol and blood pressure variability following ischemic stroke and transient ischemic attack:a secondary analysis of XILO-FIST

Blood Pressure Variability (BPV) is associated with cardiovascular risk and serum uric acid level. We investigated whether BPV was lowered by allopurinol and whether it was related to neuroimaging markers of cerebral small vessel disease (CSVD) and cognition. We used data from a randomised, double-blind, placebo-controlled trial of two years allopurinol treatment after recent ischemic stroke or transient ischemic attack. Visit-to-visit BPV was assessed using brachial blood pressure (BP) recordings. Short-term BPV was assessed using ambulatory BP monitoring (ABPM) performed at 4 weeks and 2 years. Brain MRI was performed at baseline and 2 years. BPV measures were compared between the allopurinol and placebo groups, and with CSVD and cognition. 409 participants (205 allopurinol; 204 placebo) were included in the visit-to-visit BPV analyses. There were no significant differences found between placebo and allopurinol groups for any measure of visit-to-visit BPV. 196 participants were included in analyses of short-term BPV at week 4. Two measures were reduced by allopurinol: the standard deviation (SD) of systolic BP (by 1.30 mmHg (95% confidence interval (CI) 0.18-2.42, p = 0.023)); and the average real variability (ARV) of systolic BP (by 1.31 mmHg (95% CI 0.31-2.32, p = 0.011)). There were no differences in other measures at week 4 or in any measure at 2 years, and BPV was not associated with CSVD or cognition. Allopurinol treatment did not affect visit-to-visit BPV in people with recent ischemic stroke or TIA. Two BPV measures were reduced at week 4 by allopurinol but not at 2 years.</p

Xanthine oxidase inhibition and white matter hyperintensity progression following ischaemic stroke and transient ischaemic attack (XILO-FIST): a multicentre, double-blinded, randomised, placebo-controlled trial

Acknowledgments This work was supported by the Stroke Association and British Heart Foundation [grant number TSA BHF 2013/01]. The work of Dr David Dickie and Dr Terry Quinn is funded by the Stroke Association. We would like to thank Christine McAlpine, Ruth Graham, Glasgow Royal Infirmary, UK; Lauren Pearce, Royal United Hospital, UK; Caroline Fornolles, Louise Tate, Frances Justin, Luton and Dunstable University Hospital, UK; Dean Waugh, Leeds Teaching Hospitals NHS Trust, UK; Donal Concannon, Altnagelvin Hospital, UK; Sharon Tysoe, Nina Francia, Nisha Menon, Raji Prabakaran, Southend University Hospital, UK; Amy Ashton, Caroline Watchurst, Marilena Marinescu, Sabaa Obarey, Scheherazade Feerick, University College London NHS Foundation Trust, UK; and Janice Irvine, Sandra Williams, and German Guzman Gutierrez, Aberdeen Royal Infirmary, UK; Caroline Fox and Joanne Topliffe, Broomfield Hospital, Essex, UK.Peer reviewedPublisher PD

Xanthine oxidase inhibition and white matter hyperintensity progression following ischaemic stroke and transient ischaemic attack (XILO-FIST): a multicentre, double-blinded, randomised, placebo-controlled trial

Background: People who experience an ischaemic stroke are at risk of recurrent vascular events, progression of cerebrovascular disease, and cognitive decline. We assessed whether allopurinol, a xanthine oxidase inhibitor, reduced white matter hyperintensity (WMH) progression and blood pressure (BP) following ischaemic stroke or transient ischaemic attack (TIA). Methods: In this multicentre, prospective, randomised, double-blinded, placebo-controlled trial conducted in 22 stroke units in the United Kingdom, we randomly assigned participants within 30-days of ischaemic stroke or TIA to receive oral allopurinol 300 mg twice daily or placebo for 104 weeks. All participants had brain MRI performed at baseline and week 104 and ambulatory blood pressure monitoring at baseline, week 4 and week 104. The primary outcome was the WMH Rotterdam Progression Score (RPS) at week 104. Analyses were by intention to treat. Participants who received at least one dose of allopurinol or placebo were included in the safety analysis. This trial is registered with ClinicalTrials.gov, NCT02122718. Findings: Between 25th May 2015 and the 29th November 2018, 464 participants were enrolled (232 per group). A total of 372 (189 with placebo and 183 with allopurinol) attended for week 104 MRI and were included in analysis of the primary outcome. The RPS at week 104 was 1.3 (SD 1.8) with allopurinol and 1.5 (SD 1.9) with placebo (between group difference −0.17, 95% CI −0.52 to 0.17, p = 0.33). Serious adverse events were reported in 73 (32%) participants with allopurinol and in 64 (28%) with placebo. There was one potentially treatment related death in the allopurinol group. Interpretation: Allopurinol use did not reduce WMH progression in people with recent ischaemic stroke or TIA and is unlikely to reduce the risk of stroke in unselected people. Funding: The British Heart Foundation and the UK Stroke Association

Splicing behaviour and exotic mutations in the DMD gene

DMD is the largest gene in the human genome, spanning over 2.2Mb of the X chromosome, and more than 99% of the gene content is intronic sequence. DMD encodes dystrophin, a crucial protein for protecting muscle fibres from mechanical damage. Mutations to DMD can cause any one of a family of diseases, known as the dystrophinopathies. The most severe of these is Duchenne muscular dystrophy, a progressive and global muscle wasting disease that is fatal to affected males in early life. Therapies for dystrophinopathies have progressed substantially in recent years, but at present there is no cure. The projects comprising my MPhil research aim to improve our understanding of splicing and mutation in DMD transcripts. DMD splicing is necessarily complex due to the size of the gene and its multiple spliceoforms. As such, the full effects of a given mutation within the gene can be unpredictable. Because the first step of describing a mutation should be elucidation of the sequence, a new method (Fractal PCR) has been devised for efficiently determining the intronic breakpoints of whole-exon deletions in DMD. Existing research into DMD and NF1 pseudoexons was used to inform PCR designs, and this strategy successfully discovered rare alternative transcripts of these two genes in normal human RNA. For the bioinformatics component of this project, results were compiled for hundreds of putative exon-skipping antisense oligomers (AOs) and a search was conducted for patterns in the splice factor (SF) motifs targeted or avoided by the most effective of these molecules. The intent of this work was to generate a predictive model for optimal AO design. While the power of this model was equivocal in some regards, the characteristics of the SF motifs identified as targets unexpectedly point to a clear link between DMD transcript splicing and myotonic dystrophy

The art of juncture – transformations of Irish traditional music

This thesis examines 'transformations' of Irish traditional music represented in speech, notation and writing in historical and contemporary contexts. Instances of these three modes of transformation are examined, and their structure, role and motivations in the context of Irish traditional dance music are interrogated, drawing on cognitive linguistics, structuralist and post-structuralist discourse. The thesis illustrates the way that the community supporting this set of performance practices engages in the creative use of language and signs to frame their own music making. Fundamental to these processes is the use of metaphor, as understood in the tradition of scholars such as George Lakoff, Mark Turner and Mark Johnston. The development and use of these transformations by traditional musicians occur in a context dominated by the power of language and symbols. This development is motivated by a number of societal and aesthetic factors as well as the contemporary need to frame this music culture in the contemporary world. The way in which the nature of these transformations frames and shapes the discourses of the music community, as well as the music itself, is highlighted and examined

Allopurinol and Blood Pressure Variability Following Ischemic Stroke and Transient Ischemic Attack: A Secondary Analysis of XILO-FIST

Background Blood Pressure Variability (BPV) is associated with cardiovascular risk and serum uric acid level. We investigated whether BPV is lowered by allopurinol and whether it is related to markers of cerebral small vessel disease.Methods We used data from a randomised, double-blind, placebo-controlled trial of two years allopurinol treatment after recent ischemic stroke or transient ischaemic attack. Visit-to-visit BPV was assessed using brachial blood pressure (BP) recordings. Short-term BPV was assessed using ambulatory BP monitoring (ABPM) performed at 4 weeks and 2 years. Brain MRI was performed at baseline and 2 years. BPV measures were compared between the allopurinol and placebo groups and with white matter hyperintensity progression.Results 409 participants were included (205 allopurinol; 204 placebo) were included in analyses of visit-to-visit BPV and there were no significant differences between groups. 196 participants were included in analyses of short-term BPV at week 4. Two measures were reduced by allopurinol: the standard deviation (SD) of systolic BP (by 1.30mmHg (95% confidence interval (CI) 0.18–2.42, p=0.023)); and the average real variability (ARV) of systolic BP (by 1.31mmHg (95% CI 0.31–2.32, p=0.011)). There were no differences in other measures at week 4 or in any measure at 2 years.Conclusions Allopurinol treatment did not affect visit-to-visit BPV in people with recent ischemic stroke or TIA. Two BPV measures were reduced at week 4 by allopurinol but not at 2 years. Allopurinol is unlikely to lead to an important reduction in BPV in people with ischemic stroke or TIA