Investigating the Pathogenic Mechanism of Expanded Polyalanine Tract Mutations in the ARX Homeobox Transcription Factor causing Intellectual Disability

Intellectual disability (ID) is a highly prevalent disorder that affects 1-3% of the population, with ~ 100 causative genes mapped to the X-chromosome. The Aristaless-related homeobox gene (ARX) is an important transcription factor with critical roles in development, particularly in the developing brain. Variants are not well tolerated within ARX, with missense mutations resulting in phenotypes that always involve ID with frequent comorbidities of epilepsy, infantile spasms, hand dystonia, autism or dysarthria. Historically, it was thought that only males were affected by mutations in ARX due to their single X-chromosome. In this thesis, we describe a family with multiple affected individuals, including two females with a novel insertion mutation within ARX. We furthermore review the reported phenotype of females with mutations in ARX and highlight the importance of screening ARX in both male and female patients with ID and seizures. The majority of patients with ARX mutations are affected by expansion mutations in polyalanine tract 1 and 2 within the protein, giving rise to ID with or without epilepsy and movement disorders of varying severity. Mice modelling the two most frequent polyalanine expansion mutations (Arx(GCG)7 referred to as PA1 and Arx432-455dup24 referred to as PA2) recapitulate many of the clinical features seen in humans (Kitamura et al. 2009, Jackson et al. Submitted 2017). To dissect the molecular basis of different polyalanine expansions in vivo, we used 12.5 dpc brain samples from PA1 and PA2 mice to analyse disruptions in gene expression in the developing forebrain to capture the primary disruption leading to the developmental phenotypes caused by these mutations. A greater number of genes deregulated in the more severe PA1 mice was shown, with the majority of genes also perturbed in the milder PA2 mice, but failed to reach significance compared to WT at this early stage of development. We saw a significant overlap with a number of known direct targets of ARX (5%) and genes implicated in ID, epilepsy and autism (12%). From my analysis, I predict a core pathway of transcription regulators as potential drivers of the ID and infantile spasms in patients with ARX polyalanine expansion mutations. Next, I investigated the mechanisms driving the partial loss of function. My data indicates this reduced function does not occur through disruptions of binding to DNA or protein interactors in relation to the region of ARX spanning both polyalanine tract 1 and 2. However, in this thesis, I demonstrate a marked reduction in polyalanine mutant protein may be the contributing factor to disease manifestation. Transcription activity assays indicated ARX responds in a dose-depend manner, and greater reduction in protein leads to an increased severity of the disease. Investigations into the molecular mechanism contributing to this reduction in protein level show no significant change in protein stability (in vitro). Instead, initial studies indicate inefficiency of translation resulting in reduced protein abundance. From my data and other previous studies, I discuss the likelihood of a multiple hit model contributing to the partial loss of function and leading to the variability of clinical presentations.Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 201

Myrtaceous shrub species respond to long-term decreasing groundwater levels on the Gnangara Groundwater Mound, northern Swan Coastal Plain

Analysis of four vegetation transects that were established on the Bassendean Dune System, northern Swan Coastal Plain classified 42 native plant species into four ‘habitat’ groups based on their preferred soil moisture regimes (Havel 1968). Using adult abundance and distribution data from three of these transects and an additional transect established in 1976, we investigated the various ecological responses of myrtaceous shrub species representing the four habitat groups to long-term (20-30 yr) decreasing groundwater and soil moisture levels. Myrtaceous shrubs were chosen for analysis because of their widespread occurrence and diversity on the Swan Coastal Plain, and because Myrtaceae is the only plant family to be represented in all four of Havel’s habitat categories. Myrtaceous species ‘tolerant of excessive wetness’ (Astartea fascicularis, Hypocalymma angustifolium, Pericalymma ellipticum, Regelia ciliata) are all shallow rooted (rooting depth < 1m), occur in winter-wet depressions, and displayed the greatest reduction in population size in response to decreasing groundwater levels. Species cateogorised as displaying ‘maximum development on dry sites’ (Eremaea pauciflora, Melaleuca scabra, Scholtzia involucrata) commonly occurred on the upper and mid-slopes of the transects, are deeper-rooted and varied in their population response to long-term declines in water availability. All three species probably rely to some extent on accessing soil moisture at depth (2-6 m) during summer drought. The scenario was similar for species ‘optimum on moist sites’ (M. seriata) and for species ‘without clear-cut site preferences’ (Calytrix flavescens). In the context of Havel’s four habitat categories, whether a particular habitat preference is ‘wet’ or ‘dry’ refers to availability of soil moisture, although to what soil depth and moisture levels are uncertain. Habitat preferences, based on soil moisture availability, may have no relevance to a species preferred groundwater regime and hence response to decreasing groundwater levels. A species’ groundwater requirements during periods of drought are dependent on their position in the landscape, summer groundwater depth and the species rooting depth

Long-term changes in vigour and distribution of Banksia and Melaleuca overstorey species on the Swan Coastal Plain

Long-term changes in vigour and distribution of the dominant Banksia (5 species) and Melaleuca (1 species) overstorey species were examined within four vegetation transects overlying the Gnangara Groundwater Mound, a superficial unconfined shallow aquifer on the northern Swan Coastal Plain, Western Australia. All transects were positioned along topographical gradients and monitored over a 20-30 year period. The two co-dominant overstorey species (Banksia attenuata and B. menziesii) inhabited a range of topographical positions within the landscape, from dune crest to low lying areas, with only B. attenuata increasing its distribution (moving further downslope) within the transects over time. Both species displayed a reduction in vigour, as indicated by foliage condition, during the monitored period. Species commonly inhabiting low-lying winter-wet areas (e.g. Banksia littoralis, Melaleuca preissiana) showed the greatest loss of tree vigour in response to declining groundwater levels, with B. littoralis replaced by the more drought tolerant B. prionotes. M. preissiana populations were overall more resilient to altered groundwater regimes, responding over a much greater time period (many decades) than B. littoralis (<10 years). Overall, changes in species distribution and vigour were primarily caused by long-term declines in groundwater levels resulting from the cumulative effects of abstraction and below average annual rainfall (low groundwater recharge). Long-term distribution trends and overall observed reductions in population vigour within the transects may be a function of the species’ dependency on groundwater to fulfil its water requirements. This may explain declining vigour and tree numbers of B. ilicifolia on the Gnangara Groundwater Mound, as this species is considered an important indicator of significant long- and short-term reductions in groundwater levels

Towards a reliable repeated-measures beads task for assessing the jumping to conclusions bias

© 2017 Elsevier BV. This manuscript version is made available under the CC-BY-NC-ND 4.0 license: http://creativecommons.org/licenses/by-nc-nd/4.0/ This author accepted manuscript is made available following 12 month embargo from date of publication (April 2018) in accordance with the publisher’s archiving policyThe jumping to conclusions bias (JTC), in which some people gather less information than others before making a decision, has been linked to delusions in psychosis. JTC is usually identified via the beads task, in which a sequence of beads (the “target” sequence) is used to measure the amount of evidence participants require before making a decision. Yet, despite its common use, the reliability of the task has never been properly investigated. We investigated its reliability, and tested an alternate version which used distractor sequences to obfuscate the target sequence. Healthy participants (N = 212) were randomised into two groups. One group completed ten trials using the target sequence, while the other completed ten trials of the target sequence and three distractor sequences. Our data indicated the standard task may not be reliable over repeated measures, but that by including distractor sequences, the task becomes more believable, repeatable, and reliable. Additionally, excluding first-trial data (a “silent” practice trial) also improves repeatability. These improvements to the task are relevant to single trial studies, and will be especially useful to repeated-measures longitudinal, experimental, and treatment studies. Such repeated-measures studies are important for investigating the causal link between JTC and delusions

Sex recognition by odour and variation in the uropygial gland secretion in starlings

1. Although a growing body of evidence supports that olfaction based on chemical compounds emitted by birds may play a role in individual recognition, the possible role of chemical cues in sexual selection of birds has been only preliminarily studied.2. We investigated for the first time whether a passerine bird, the spotless starling Sturnus unicolor, was able to discriminate the sex of conspecifics by using olfactory cues and whether the size and secretion composition of the uropygial gland convey information on sex, age and reproductive status in this species.3. We performed a blind choice experiment during mating, and we found that starlings were able to discriminate the sex of conspecifics by using chemical cues alone. Both male and female starlings preferred male scents. Furthermore, the analysis of the chemical composition of the uropygial gland secretion by using gas chromatography–mass spectrometry (GC–MS) revealed differences between sexes, ages and reproductive status.4. In conclusion, our study reveals for first time that a passerine species can discriminate the sex of conspecifics by relying on chemical cues and suggests that the uropygial gland secretion may potentially function as a chemical signal used in mate choice and/or intrasexual competition in this species.This research was funded by the Spanish Ministry of Education and Science ⁄ FEDER (CGL2008-00718) and PIE 200930I029 to J. M. Avilés and D. Parejo.The study was conducted under licence of the Junta de Andalucía GC–MS analyses were performed by Dr. Rafael Núñez at the Scientific Instrumentation Service (EEZ, CSIC) (Granada, Spain).Peer reviewe

In depth analysis of the Sox4 gene locus that consists of sense and natural antisense transcripts

Available online 17 February 2016SRY (Sex Determining Region Y)-Box 4 or Sox4 is an important regulator of the pan-neuronal gene expression during post-mitotic cell differentiation within the mammalian brain. Sox4 gene locus has been previously characterized with multiple sense and overlapping natural antisense transcripts [1], [2]. Here we provide accompanying data on various analyses performed and described in Ling et al. [2]. The data include a detail description of various features found at Sox4 gene locus, additional experimental data derived from RNA-Fluorescence in situ Hybridization (RNA-FISH), Western blotting, strand-specific reverse-transcription quantitative polymerase chain reaction (RT-qPCR), gain-of-function and in situ hybridization (ISH) experiments. All the additional data provided here support the existence of an endogenous small interfering- or PIWI interacting-like small RNA known as Sox4_sir3, which origin was found within the overlapping region consisting of a sense and a natural antisense transcript known as Sox4ot1.King-Hwa Ling, Peter J. Brautigan, Sarah Moore, Rachel Fraser, Melody Pui-Yee Leong, Jia-Wen Leong, Shahidee Zainal Abidin, Han-Chung Lee, Pike-See Cheah, Joy M. Raison, Milena Babic, Young Kyung Lee, Tasman Daish, Deidre M. Mattiske, Jeffrey R. Mann, David L. Adelson, Paul Q. Thomas, Christopher N. Hahn, Hamish S.Scot

Derivation of an endogenous small RNA from double-stranded Sox4 sense and natural antisense transcripts in the mouse brain

Natural antisense transcripts (NATs) are involved in cellular development and regulatory processes. Multiple NATs at the Sox4 gene locus are spatiotemporally regulated throughout murine cerebral corticogenesis. In the study, we evaluated the potential functional role of Sox4 NATs at Sox4 gene locus. We demonstrated Sox4 sense and NATs formed dsRNA aggregates in the cytoplasm of brain cells. Over expression of Sox4 NATs in NIH/3T3 cells generally did not alter the level of Sox4 mRNA expression or protein translation. Upregulation of a Sox4 NAT known as Sox4ot1 led to the production of a novel small RNA, Sox4_sir3. Its biogenesis is Dicer1-dependent and has characteristics resemble piRNA. Expression of Sox4_sir3 was observed in the marginal and germinative zones of the developing and postnatal brains suggesting a potential role in regulating neurogenesis. We proposed that Sox4 sense-NATs serve as Dicer1-dependent templates to produce a novel endo-siRNA- or piRNA-like Sox4_sir3

Unraveling the pathogenesis of ARX polyalanine tract variants using a clinical and molecular interfacing approach

The Aristaless-related homeobox (ARX) gene is implicated in intellectual disability with the most frequent pathogenic mutations leading to expansions of the first two polyalanine tracts. Here, we describe analysis of the ARX gene outlining the approaches in the Australian and Portuguese setting, using an integrated clinical and molecular strategy. We report variants in the ARX gene detected in 19 patients belonging to 17 families. Seven pathogenic variants, being expansion mutations in both polyalanine tract 1 and tract 2, were identifyed, including a novel mutation in polyalanine tract 1 that expands the first tract to 20 alanines. This precise number of alanines is sufficient to cause pathogenicity when expanded in polyalanine tract 2. Five cases presented a probably non-pathogenic variant, including the novel HGVS: c.441_455del, classified as unlikely disease causing, consistent with reports that suggest that in frame deletions in polyalanine stretches of ARX rarely cause intellectual disability. In addition, we identified five cases with a variant of unclear pathogenic significance. Owing to the inconsistent ARX variants description, publications were reviewed and ARX variant classifications were standardized and detailed unambiguously according to recommendations of the Human Genome Variation Society. In the absence of a pathognomonic clinical feature, we propose that molecular analysis of the ARX gene should be included in routine diagnostic practice in individuals with either nonsyndromic or syndromic intellectual disability. A definitive diagnosis of ARX-related disorders is crucial for an adequate clinical follow-up and accurate genetic counseling of at-risk family members.Unit for Multidisciplinary Research in Biomedicine, UMIB, ICBAS-UP, Porto, Portugal was funded by FEDER funds of the Operational Program for Competitiveness Factors – COMPETE through FCT – Foundation for Science and Technology under the project: Fcomp-01-0124-FEDER-015896. The Neurogenetics research program in the Department of Paediatrics, University of Adelaide, Australia was funded by the Australian National Health and Medical Research Council (Grant No. 1063025). C. S. is supported Australian Research Council (Future Fellowship FT120100086

In depth analysis of the Sox4 gene locus that consists of sense and natural antisense transcripts

SRY (Sex Determining Region Y)-Box 4 or Sox4 is an important regulator of the pan-neuronal gene expression during post-mitotic cell differentiation within the mammalian brain. Sox4 gene locus has been previously characterized with multiple sense and overlapping natural antisense transcripts [1] and [2]. Here we provide accompanying data on various analyses performed and described in Ling et al. [2]. The data include a detail description of various features found at Sox4 gene locus, additional experimental data derived from RNA-Fluorescence in situ Hybridization (RNA-FISH), Western blotting, strand-specific reverse-transcription quantitative polymerase chain reaction (RT-qPCR), gain-of-function and in situ hybridization (ISH) experiments. All the additional data provided here support the existence of an endogenous small interfering- or PIWI interacting-like small RNA known as Sox4_sir3, which origin was found within the overlapping region consisting of a sense and a natural antisense transcript known as Sox4ot1