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

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.Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 201

De Novo Loss-of-Function Mutations in USP9X Cause a Female-Specific Recognizable Syndrome with Developmental Delay and Congenital Malformations

Mutations in more than a hundred genes have been reported to cause X-linked recessive intellectual disability (ID) mainly in males. In contrast, the number of identified X-linked genes in which de novo mutations specifically cause ID in females is limited. Here, we report 17 females with de novo loss-of-function mutations in USP9X, encoding a highly conserved deubiquitinating enzyme. The females in our study have a specific phenotype that includes ID/developmental delay (DD), characteristic facial features, short stature, and distinct congenital malformations comprising choanal atresia, anal abnormalities, post-axial polydactyly, heart defects, hypomastia, cleft palate/bifid uvula, progressive scoliosis, and structural brain abnormalities. Four females from our cohort were identified by targeted genetic testing because their phenotype was suggestive for USP9X mutations. In several females, pigment changes along Blaschko lines and body asymmetry were observed, which is probably related to differential (escape from) X-inactivation between tissues. Expression studies on both mRNA and protein level in affected-female-derived fibroblasts showed significant reduction of USP9X level, confirming the loss-of-function effect of the identified mutations. Given that some features of affected females are also reported in known ciliopathy syndromes, we examined the role of USP9X in the primary cilium and found that endogenous USP9X localizes along the length of the ciliary axoneme, indicating that its loss of function could indeed disrupt cilium-regulated processes. Absence of dysregulated ciliary parameters in affected female-derived fibroblasts, however, points toward spatiotemporal specificity of ciliary USP9X (dys-)function

COVID-19 and tinnitus : an initiative to improve tinnitus care

OBJECTIVE : To investigate the effects of COVID-19 on individuals with tinnitus and their views to guide future tinnitus care. DESIGN : A mixed-methods cross-sectional research design. STUDY SAMPLE : An online survey was completed by 365 individuals with tinnitus from Australia and other countries. RESULTS : Tinnitus was reported to be more bothersome during the pandemic by 36% of respondents, whereas 59% reported no change and 5% reported less bothersome tinnitus. Nearly half of the respondents had received COVID-19 vaccination(s) and 12% of them reported more bothersome tinnitus while 2% developed tinnitus post-vaccination. Australian respondents spent less time in self-isolation or quarantine and saw fewer change in in-person social contact than respondents from other countries. More than 70% of respondents thought that tinnitus care services were insufficient both before and during the pandemic. Regarding their opinions on how to improve tinnitus care in the future, five themes including alleviation of condition, government policies, reduced barriers, self- and public-awareness, and hearing devices were identified. CONCLUSIONS : A majority of respondents did not perceive any change in tinnitus perception and one-third of respondents had worsened tinnitus during the pandemic. To improve tinnitus care, better awareness and more accessible resources and management are crucial.The Ida Institute.https://www.tandfonline.com/loi/iija202023-08-02hj2023Speech-Language Pathology and Audiolog

Inhibition of Upf2-Dependent Nonsense-Mediated Decay Leads to Behavioral and Neurophysiological Abnormalities by Activating the Immune Response

In humans, disruption of nonsense-mediated decay (NMD) has been associated with neurodevelopmental disorders (NDDs) such as autism spectrum disorder and intellectual disability. However, the mechanism by which deficient NMD leads to neurodevelopmental dysfunction remains unknown, preventing development of targeted therapies. Here we identified novel protein-coding UPF2 (UP-Frameshift 2) variants in humans with NDD, including speech and language deficits. In parallel, we found that mice lacking Upf2 in the forebrain (Upf2 fb-KO mice) show impaired NMD, memory deficits, abnormal long-term potentiation (LTP), and social and communication deficits. Surprisingly, Upf2 fb-KO mice exhibit elevated expression of immune genes and brain inflammation. More importantly, treatment with two FDA-approved anti-inflammatory drugs reduced brain inflammation, restored LTP and long-term memory, and reversed social and communication deficits. Collectively, our findings indicate that impaired UPF2-dependent NMD leads to neurodevelopmental dysfunction and suggest that anti-inflammatory agents may prove effective for treatment of disorders with impaired NMD

De Novo Loss-of-Function Mutations in USP9X Cause a Female-Specific Recognizable Syndrome with Developmental Delay and Congenital Malformations

Mutations in more than a hundred genes have been reported to cause X-linked recessive intellectual disability (ID) mainly in males. In contrast, the number of identified X-linked genes in which de novo mutations specifically cause ID in females is limited. Here, we report 17 females with de novo loss-of-function mutations in USP9X, encoding a highly conserved deubiquitinating enzyme. The females in our study have a specific phenotype that includes ID/developmental delay (DD), characteristic facial features, short stature, and distinct congenital malformations comprising choanal atresia, anal abnormalities, post-axial polydactyly, heart defects, hypomastia, cleft palate/bifid uvula, progressive scoliosis, and structural brain abnormalities. Four females from our cohort were identified by targeted genetic testing because their phenotype was suggestive for USP9X mutations. In several females, pigment changes along Blaschko lines and body asymmetry were observed, which is probably related to differential (escape from) X-inactivation between tissues. Expression studies on both mRNA and protein level in affected-female-derived fibroblasts showed significant reduction of USP9X level, confirming the loss-of-function effect of the identified mutations. Given that some features of affecte

Partial Loss of USP9X Function Leads to a Male Neurodevelopmental and Behavioral Disorder Converging on Transforming Growth Factor β Signaling

Background: The X-chromosome gene USP9X encodes a deubiquitylating enzyme that has been associated with neurodevelopmental disorders primarily in female subjects. USP9X escapes X inactivation, and in female subjects de novo heterozygous copy number loss or truncating mutations cause haploinsufficiency culminating in a recognizable syndrome with intellectual disability and signature brain and congenital abnormalities. In contrast, the involvement of USP9X in male neurodevelopmental disorders remains tentative. Methods: We used clinically recommended guidelines to collect and interrogate the pathogenicity of 44 USP9X variants associated with neurodevelopmental disorders in males. Functional studies in patient-derived cell lines and mice were used to determine mechanisms of pathology. Results: Twelve missense variants showed strong evidence of pathogenicity. We define a characteristic phenotype of the central nervous system (white matter disturbances, thin corpus callosum, and widened ventricles); global delay with significant alteration of speech, language, and behavior; hypotonia; joint hypermobility; visual system defects; and other common congenital and dysmorphic features. Comparison of in silico and phenotypical features align additional variants of unknown significance with likely pathogenicity. In support of partial loss-of-function mechanisms, using patient-derived cell lines, we show loss of only specific USP9X substrates that regulate neurodevelopmental signaling pathways and a united defect in transforming growth factor β signaling. In addition, we find correlates of the male phenotype in Usp9x brain-specific knockout mice, and further resolve loss of hippocampal-dependent learning and memory. Conclusions: Our data demonstrate the involvement of USP9X variants in a distinctive neurodevelopmental and behavioral syndrome in male subjects and identify plausible mechanisms of pathogenesis centered on disrupted transforming growth factor β signaling and hippocampal function

Partial Loss of USP9X Function Leads to a Male Neurodevelopmental and Behavioral Disorder Converging on Transforming Growth Factor β Signaling.

BackgroundThe X-chromosome gene USP9X encodes a deubiquitylating enzyme that has been associated with neurodevelopmental disorders primarily in female subjects. USP9X escapes X inactivation, and in female subjects de novo heterozygous copy number loss or truncating mutations cause haploinsufficiency culminating in a recognizable syndrome with intellectual disability and signature brain and congenital abnormalities. In contrast, the involvement of USP9X in male neurodevelopmental disorders remains tentative.MethodsWe used clinically recommended guidelines to collect and interrogate the pathogenicity of 44 USP9X variants associated with neurodevelopmental disorders in males. Functional studies in patient-derived cell lines and mice were used to determine mechanisms of pathology.ResultsTwelve missense variants showed strong evidence of pathogenicity. We define a characteristic phenotype of the central nervous system (white matter disturbances, thin corpus callosum, and widened ventricles); global delay with significant alteration of speech, language, and behavior; hypotonia; joint hypermobility; visual system defects; and other common congenital and dysmorphic features. Comparison of in silico and phenotypical features align additional variants of unknown significance with likely pathogenicity. In support of partial loss-of-function mechanisms, using patient-derived cell lines, we show loss of only specific USP9X substrates that regulate neurodevelopmental signaling pathways and a united defect in transforming growth factor β signaling. In addition, we find correlates of the male phenotype in Usp9x brain-specific knockout mice, and further resolve loss of hippocampal-dependent learning and memory.ConclusionsOur data demonstrate the involvement of USP9X variants in a distinctive neurodevelopmental and behavioral syndrome in male subjects and identify plausible mechanisms of pathogenesis centered on disrupted transforming growth factor β signaling and hippocampal function